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I" Q ,' "J ' x... ‘\ '/,, > r x 1,- _ “—- A \- a s I )' ~ _ .1, I “at \ \ ‘u ,-’. .> 5 " - '~—\.. Major professor r /-I r 4 l “I; MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 PLACE IN REFURN BOX to remove this checkout from your record. TO AVOID FINE return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE DETERMINING NUTRIENT- AND PREDA’I‘OR-LIMI'I‘ATION OF SYNECHOCOCCUS POPULATION SIZE USING CELLULAR rRNA CONCENTRATIONS By Paul Wesley Lepp A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree Of DOCTOR OF PHILOSOPHY Department of Microbiology 1997 no sul cor nun bod (fisu indx 8103 concc decre SP. SII CYChc- ofzbc “nun 5mm ABSTRACT DETERMINING NUTRIENT— AND PREDATOR-LIMITATION OF S YNECHOCOC C US POPULATION SIZE USING CELLULAR rRNA CONCENTRATIONS By Paul Wesley Lepp Cyanobacteria of the genus Synechococcus are important contributors to primary productivity, community composition and trOphic interactions in marine environments. Despite their importance, the factors controlling the size of Synechococcus populations are not well understood. Substantial evidence exists to suggest that synechococci are often substrate limited. However, the evidence does not preclude the possibility of predator control Of synechococci population size. The goal of this research was to distinguish nutrient-limitation from predator-limitation of the size Of S ynechococcus populations in both laboratory cultures and the environment. Nutrient- or predator-limitation was distinguished based on a plot of population size versus cellular 16S rRNA; which was indicative of specific growth rate. Light-limited batch cultures of both Synechococcus spp. strain PCC 6301 and WH 8103 exhibited a positive correlation between specific growth rate and the cellular concentrations of both RNA and DNA. The cellular concentration Of nucleic acids decreased during light periods and increased during dark periods in both Synechococcus sp. strain PCC 6301 and Synechococcus sp. strain WH 8103 entrained by diel cycles. The cyclical variation in cellular nucleic acids concentrations during diel cycles was a function of the timing Of cell division. Predator-prey models were applied to a chemostat culture of Synechococcus sp. strain PCC 6301 prey and a Tetrahymena pynformis predator. In the absence of predation, Synechococcus responded to increases in nutrients by increasing equilibrium population size, without a concurrent increase in growth rate. Addition of the predator increased the $ch1 detrc cxan mod grov vets thos 3W heat specific growth rate of the Synechococcus population approximately 10-fold while decreasing the size of the Synechococcus population by one-seventh. The cellular l6S rRNA concentrations and size of Synechococcus populations were examined at Oligotrophic and eutrophic stations in the Gulf of Mexico. Prey—dependent models predicted that a nutrient-limited population would be larger and have slower specific growth rates than a population limited by predation. The distribution of population size versus specific growth conformed to the predictions Of prey-dependent models better than those of ratio-dependent models. A significant portion of the synechococci examined appeared to be limited in population size by predation, in agreement with the reports of heavy grazing Of pelagic Synechococcus populations. To I It w To my wife, Cheryl, who endured years of separation in order to make this work possible. It would not have been possible to complete this work without her love, understanding and patience. iv my t inva Boh stim Brat ACKNOWLEDGMENTS I would like to express my thanks and gratitude to my mentor Tom Schmidt, who has taught me as much about life as about science. I would also like to extend my thanks to my committee: Richard Lenski, John Breznak and Donna Koslowsky have provided invaluable guidance during the course of this research. Thanks also go to Brendan Bohannan who provided much Of the inspiration for this work, as well as, invaluable and stimulating discusssion. Finally, I would like to thank the members Of the lab: Dr. Bonnie Bratina, Brad Stevenson and Dan Buckley have provided the support and laughs that make an undertaking such as this possible. i This research was supported by the US. Environmental Protection Agency (CR822014-Ol-0). TABLE OF CONTENTS LIST OF TABLES ............................................................................. viii LIST OF FIGURES .............................................................................. ix CHAPTER 1 GENERAL INTRODUCTION ............................................................. 1 Introduction and Specific Aims .................................................... 2 Background and Significance ............................................................. 3 Synechococcus spp. Physiology and Ecology ......................... 3 Prey-dependent models of population control ......................... 5 Alternative models and predictions ........................................... 8 CHAPTER 2 CONCENTRATIONS OF NUCLEIC ACIDS OF S YNECHOCOCC US SP. STRAIN PCC 6301 DURING GROWTH IN CONTINUOUS LIGHT AND LIGHT:DARK CYCLES ..................................................................... 1 8 Abstract .............................................................................. 1 9 Introduction .............................................................................. 20 Materials and Methods ............................................................ 21 Results .............................................................................. 23 Discussion .............................................................................. 3 1 References .............................................................................. 4 1 CHAPTER 3 CONCENTRATIONS OF NUCLEIC ACIDS OF SYNECHOCOCC US SP. STRAIN WH 8103 DURING GROWTH IN CONTINUOUS LIGHT AND LIGHT :DARK CYCLES ..................................................................... 46 Abstract .............................................................................. 47 Introduction .............................................................................. 47 Materials and Methods ............................................................ 49 Results .............................................................................. 5 1 Discussion .............................................................................. 55 References .............................................................................. 64 CHAPTER 4 USE OF 16S rRNA PROBES TO DETERMINE NUTRIENT-LIMITATION OR PREDATOR-LIMITATION OF THE SIZE OFS YNECHOCOC C US PCC 6301 POPULATIONS .............................................................................. 67 Abstract .............................................................................. 68 Introduction .............................................................................. 68 Materials and Methods ............................................................ 70 Results .............................................................................. 73 Discussion .............................................................................. 84 References .............................................................................. 95 CHAPTER 5 EVIDENCE OF PREDATOR-LIMITATION S YNECHOCOC C US POPULATION SIZE IN THE GULF OF MEXICO USING l6S rRNA PROBES 102 Abstract ............................................................................ 1 03 Introduction ............................................................................ 103 Materials and Methods .......................................................... 105 Results and Discussion .......................................................... 1 10 References ............................................................................ 1 24 CHAPTER 6 SUMMARY AND DISCUSSION .......................................................... 128 References ............................................................................ l 34 vii Table Table Table Table Table Table Table [0 N Table LIST OF TABLES Table 2.1 - Physiological parameters of Synechococcus sp. strain PCC 6301 in light-limited batch culture ........................................................ 25 Table 3.1 - Physiological parameters of Synechococcus sp. strain WH 8103 in light-limited batch culture ........................................................ 53 Table 4.1 - Physiological parameters of Synechococcussp. strain PCC 6301 grown in phosphate-limited chemostat ............................................ 76 Table 4.2 - Physiological parameters of Synechococcus sp. strain PCC 6301 in the presence and absence of T. pyriformis predation ......................... 78 Table 5.1 - Synechococci population density for October 27 to November 11, 1993 ...112 Table 5.2 - Synechococci population density for March 14 to March 20, 1993 ......... 112 Table 5.3 - Synechococci population density for November 19 to November 22, 1993 .......................................................................... 113 Table 5.4 - Synechococci population density for February 16 to February 21, 1993 ...1 l3 viii Figur Figur F1 gur Figur Figur. Figur. Figuri Figun Figure Flgme FigUIe FiEUre FiEUre LIST OF FIGURES Figure 1.1 - Predicted relationship between Synechococcus equilibrium population size based on potential nutrients and trophic interactions .................... 7 Figure 1.2 - Model of equilibrium population size and specific growth for predator—prey interactions ....................................................... 9 Figure 1.3 - Alternative relationships between Synechococcus equilibrium population size and specific growth rate (It) ................................. 10 Figure 2.1 - Total cellular RNA, as determined by orcinol, from light-limited batch cultures and phophate-limited chemostat cultures, as a function of growth rate .................................................................... 24 Figure 2.2 - Cellular 168 rRN A concentrations Of Synechococcus sp. strain PCC 6301 from phosphate-limited chemostats ........................................... 27 Figure 2.3 - Cell density of Synechococcus sp. strain PCC 6301 during diel growth ................................................................................ 28 Figure 2.4 - Cellular RNA concentrations of Synechococcus sp. strain PCC 6301 grown under a 12 h light: 12 h dark (shaded) diel cycle .................... 29 Figure 2.5 - Cellular DNA concentrations Of Synechococcus sp. strain PCC 6301 grown under a 12 h light: 12 h dark (shaded area) diel cycle .............. 30 Figure 3.1 - Concentrations of nucleic acids of Synechococcus sp. strain WH 8103 from light-limited batch cultures ............................................... 52 Figure 3.2 - Cell density Of Synechococcus sp. strain WH 8103 during diel growth ....56 Figure 3.3 - Cellular RNA concenuations of Synechococcus sp. strain WH 8103 during diel growth .............................................................. 57 Figure 3.4 - Cellular DNA concentrations of Synechococcus sp. strain WH 8103 during diel growth .............................................................. 58 Figure 4.1 - Kinetics of phosphate utilization by Synechococcus sp. strain PCC 6301 ............................................................ 74 Figur Figur Figur Figui Figure 4.2 - Synechococcus sp. strain PCC 6301 mean population size in chemostats supplied by reserviors containing 5 M, 10 ILM and 15 11M KzPO4 ............................................................... 77 Figure 4.3 - Synechococcus sp. strain PCC 6301 population size with and without predation ................................................................ 79 Figure 4.4 - Tetrahymena pyriformis grazing Synechococcus sp. strain PCC 6301 ..... 81 Figure 4.5 - Cellular 168 rRNA concentrations of Synechococcus sp. strain PCC 6301 in the presence and absence of predation by T. pyriformis ...82 Figure 5.1 - Sampling sites within the Gulf Of Mexico ..................................... 106 Figure 5.2 - Estimated growth rate of synechococci determined by the dilution method of Landry and Hasset ................................................ 115 Figure 5.3 - Cellular 16S rRNA concentration of synechococci populations along a nutrient gradient in the Gulf of Mexico ................................... 117 Figure 5.4 - Depth profile of synechococci populations .................................... 121 Figure 5.5 - Depth profile of station Tlf ...................................................... 122 Chapter 1 GENERAL INTRODUCTION Inlrt appro produ and a and n Despi SUUCI unden detem in deti interat Popul; Nutrie HIOdeT and ; intera rates measl Whicl to 5).! fTOm Introduction and Specific Aims Cyanobacteria of the genus Synechococcus are reported to contribute from approximately 2% of total primary productivity in coastal waters up to 46% of total primary productivity in pelagic waters. Synechococci (30, 54) have proven to be both ubiquitous and abundant by epifluorescence microscopy (5, 9, 30, 54, 55), flow cytometry (7, 34) and nucleic acid sequence analysis (18, 47), since their discovery in marine environments. Despite their significant contribution to total primary production and marine community structure, little is known about the mechanisms controlling their population size. An understanding of the mechanisms which control Synechococcus populations is essential in determining the rate of energy transfer to other trophic levels (21), the role of synechococci in determining microzooplankton composition and the construction of models of trophic interactions. The goal this research was to determine whether the size of Synechococcus populations in the environment was primarily nutrient-limited or predator-limited. Nutrient-limitation was distinguished from predator-limitation of population size by modeling predator-prey interactions in chemostats containing a Synechococcus sp. prey and a T etrahymena pynfonnis predator. Prey—dependent models of predator-prey interactions predicted that predator-limited populations would have higher specific growth rates than nutrient-limited populations. The relative differences in growth rates was measured using oligonucleotide probes complementary to 16S rRNA, the concentration of which was correlated with specific growth rates. Finally, this same approach was applied to synechococci populations in the Gulf of Mexico in order to distinguish nutrient-limitation from predator-limitation of population size in situ. Bar COII wit] nan fror and S yn that isol Rid] Cyc Cnv Background and Significance Synechococcus physiology and ecology. Picoplankton are defined as organisms from 0.2 um to 2 pm in size (48). These organisms are respOnsible for a large portion of the photosynthetic activity in the open ocean (l6). Picophytoplankton communities are often numerical dominated by members of the genus Synechococcus (5, 7, 55). Populations of Synechococcus range from 1x103 cells ml'1 to 1x106 cells ml1 within the euphotic zone (5, 7, 9, 45, 54, 55). Beyond mere abundance, the ubiquitous nature of Synechococcus is evident from counts by epifluoresence microscopy of samples from the southern Pacific (55), the subartic Pacific (11), the tropical Atlantic (9, 54, 55) and the northern Atlantic (5, 21). Cloned 16S rRNA sequences have revealed Synechococcus spp. to be abundant in bOth the Pacific (47) and the Atlantic ( 19); results that are supported by flow cytometry (34). Members of the genus Synechococcus are distinguished from other cyanobacteria by their unicellular, coccobacilli morphology, transverse binary fission in one plane and the absence of a polysaccharide sheath (46). Marine Synechococcus spp. can be divided into two distinct groups. The first group lacks phycoerythrin and does not have an elevated salt requirement. This halotolerant group has been found within the continental shelf margin but never in the open ocean. The second group contains phycoerythrin as its primary light harvesting pigment and possesses an elevated salt requirement. This group has been isolated on and off the continental shelf (55). Synechococcus is capable of fixing 10 fg C cell'lh‘1 (9, 40, 45), principally via the reductive pentose phosphate pathway (20). This reduction is tightly coupled to a diel light cycle (23, 44, 45) and probably occurs within carboxysomes (51). It is believed that up to half of the photosynthates produced are released, typically as glycolate, into the environment and capable of supporting multiple trophic levels (16). This is supported by the fact that the growth rate is saturated at light intensities of approximately 50 uEm‘ZS'l and photosynthesis at approximately 100 uEm'25'1 (21, 40, 45). The photosynthetic capacity of Synechococcus is often saturated by oceanic surface irradiance that can exceed 1000 uEm'23'1 on cloudless days. Characteristic of cyanobacteria and chloroplasts, reducing equivalents are provided by photosystems I and II (20). Although Synechococcus contains chlorophyll a, its primary light harvesting pigments are phycocyanin, phycoerythrin, allophycocyanin and allophycocyanin B (20). These accessory pigments, sequestered in phycobilisomes arranged along the outside of parallel stacks of thylakoid membranes, results in a light harvesting complex capable of supporting a maximum growth rate ”max at 50 ILEm45'1 (40) compared with approximately 100 uEm'25'1 for eukaryotic algae (20). The low light level at which maximal growth occurs is believed to contribute to the competitive edge that allows Synechococcus to dominate many marine phytoplankton communities (20). In addition to light, Synechococcus exhibits a high affinity for nitrate K, = 30 “M (15, 22), requires less than 0.1 nM manganese and less than 0.1 pM zinc (4). Synechococci also benefit from their small size approximately 1 um x 0.6 um (55), which results in a surfacezvolume ratio that aids substrate uptake ( 16). High substrate affinity and small size make Synechococcus extremely efficient at nutrient uptake and a key link in micronutrient recycling and trOphic transfer in pelagic systems (29). Should the Synechococcus population become nitrogen limited, a number of diazotrophic Synechococcus have been reported (25, 28) which could quickly displace non-nitrogen fixing species. Despite the advantages in Oligotrophic environments afforded by small size and high affinity for scarce resources, there is substantial support in the literature for adopting Sme , Fee+ conc [red P0PL phyt Inod. Inucl Popu and] Inode the position that Synechococcus is often substrate limited. Reports indicate that Synechococcus may at times be limited for light (38), trace metals (11, 39), in particular Fe2+ (24, 36, 37), nitrogen (22, 41), and phosphorus (31, 32, 56). However, the evidence for substrate limitation. of Synechococcus is far from conclusive and does not preclude the possibility of predator control. One of the foremost predictions of ecological theory is that predation can be a major factor in controlling population size (26, 27, 33, 42). An inverse correlation has been found between phytoplankton biomass and grazer density in lakes (41) and rivers (57). One would expect to find similar forces at work upon the Synechococcus in marine environments where flagellates and protozoa are known predators (8, 14, 16, 31, 32, 43). The reported in situ grazing coefficient for flagellates and protozoan specific to Synechococcus range from 0 to 1.7 d'1 (6, 8). Grazing pressure can reportedly reduce the population of Synechococcus by 30 to 50% (6, 8). Another possible control mechanism is parasitism by Synechococcus— specific bacteriophage, which can be considered predation for the purposes of mathematical models. Viruses have been reported to reduce phytoplankton primary productivity by as much as 78% (29, 50). However, more recent work has demonstrated that natural populations of Synechococcus are largely phage resistant (53). The effects of predation and nutrient limitation on Synechococcus populations can be predicted by mathematical models of predator-prey interactions, such as a prey-dependent model. Prey-dependent model of population control. In the absence of predators or free of predator control, bacterial populations tend to reach an equilibrium population density that is dependent on the concentration of the limiting substrate. Such a population is referred to as being controlled by bottom-up forces. This situation is analogous to a chemostat in which the specific growth rate is set by the flow of the limiting nutrient through the system (17). Wbe desc indi Syst defi the 3 p013 Sub: ther the 1 arm" thes 33m fTQn— Wate According to traditional predator-prey theory the composition of a food chain is dramatically altered by the addition of a predator species and can be mathematically described by the coupled differential equations, first described by Lotka (35) and Volterra (52): dN/dt=uN-qN-gNP 1.1 dP/dt = egNP - mP 1.2 where N and P are the size of the prey and predator populations at time t. The term It describes the specific growth rate of the prey species. The term q describes the loss of individual organisms, which may be due to inherent mortality or efflux of the prey from the system. The predator grazing coefficient is described by g. The trophic function eg defines the conversion of consumed prey into predator biomass and m the mortality rate of the predator. The most significant result of this addition is the reduction of the equilibrium prey population, referred to as top-down control (26, 27, 33, 42). The end result is that with the addition of each trophic level the bottom trophic level is alternately limited by available substrate or predation, depending the number of links in the chain Figure 1.1. By definition, the size of an equilibrium population is unchanging with time and therefore the specific growth rate must equal the rate at which individuals are removed from the system (26, 27, 33, 42). Loss of individuals within natural pelagic populations may be attributed to several causes: inherent mortality, emigration, sinking and predation. Of these causes, it is probable that only predation has a significant impact on marine Synechococcus. It is assumed for the purposes of this model that the rate of emigration from surface water is compensated for by the rate of irnrrrigration from the surrounding water mass. Likewise, the 2.5 cm (1'1 sinking rate of Synechococcus makes loss by this A7: cam 5.52:2...— EES..=:E~ Figi (Ad Equilibrium Population Size (N) nutrient-limited predator-limited l 1 L 1 l n l Nutrients Figure 1.1 - Predicted relationship between Synechococcus equilibrium population size based on potential nutrients and trophic interactions (Adapted from 27). ma} (49) dom tropl wom‘ mort incre woul plotte Specii contn Vertic Thus, limiter 31mm: sigmfi V01 [En means negligible due to the high degree of mixing of surface water (16). Synechococcus may be lost by the sinking of “sea-snow” that originates from zooplankton fecal pellets (49). However, this loss may be viewed as the result of predation. Within an environment dominated by Synechococcus and absent a predator, the mortality rate within a single trophic level would be due to the small loss by sinking and inherent mortality. Tire specific growth rate of Synechococcus would be constant and equal to the rate of sinking loss and inherent mortality even while the equilibrimn population density increases along a nutrient gradient. Thus, a plot of the equilibrium population size versus the specific growth rate It would result in a vertical line Figure 1.2. The appearance of a predator would increase the mortality rate of the Synechococcus prey, at equilibrium, and would be paralleled by an increase in the specific growth rate. Therefore, the introduction of a second trophic level would result in a horizontal line if the equilibrium Synechococcus population density was plotted against its specific growth rate Figure 1.2. Adding a third trophic level, a predator specific to the Synechococcus predator, would release S ynechococcus from predator control. The Synechococcus population again becomes substrate limited and results in a vertical plot of equilibrium S ynechococcus population size versus specific growth rate. Thus, vertical and horizontal lines should be indicative of substrate limitation or predator limitation, respectively Figure 1.2. Although prey-dependent models are widely accepted, alternative models of predator-prey interactions exist. These alternative models differ significantly from prey—dependent models in the predicted outcome of trophic interactions. Alternative models and predictions. An implicit assumption of the Lotka- Volterra model is that predator-prey interactions are random and analogous the principle of mass action in chemical systems (45). This analogy is one of the reasons that Lotka- Volterra type models have recently come into contention (45). It has been argued that natural systems are heterogeneous both spatially and in terms of the temporal scales of predator-prey interactions and reproduction. As a result, natural systems cannot be mode mode modeled on the homogenous systems characterized by mass action. Ratio-dependent models (1-3) of predator-prey interactions have been proposed to provide a mathematical nutrient control Ion size (N) lat Predator control I rrum popu Equ Specific growth rate "m" Figure 1.2 - Model of equilibrium population size and specific growth for predator—prey interactions. 10 "nutrient dominated" "Non-linear predation" Population Size (N) rrum r. I "predator dominated" . I . . I ”max ‘ ' f' Equil Specific growth rate (u) Figure 1.3 - Alternative relationships between Synechococcus equilibrium population size and specific growth rate ()1) for raio-dependent interactions. descri densi where thestr organ CODVC 1.3 an and 1. an inc therej b)" a ! ETOWIL depen. Specif; conmt than 0 “innit i“Cheat medal: 11 description of the heterogeneity in natural systems. In ratioodependent models the prey - density and mortality rate is dependent on the ratio of prey to predators (NIP): dN/dt = IIN - qN - gN/PP 1.3 dP/dt = egN/PP - mP 1.4 where N and P are the size of prey and predator populations at time t. The term It describes the specific growth rate of the prey species. The term q describes the loss of individual organisms, which may be due to inherent mortality or efflux of the prey from the system. The predator grazing coefficient is described by g. The trophic function eg defines the conversion of consumed prey into predator and m the mortality rate of the predator. The predictions of the interactions between trophic levels derived from equations 1.3 and 1.4 differ significantly from those predicted by equations 1.1 and 1.2 Figures 1.1 and 1.2. According to ratio-dependent models, an increase in primary productivity leads to an increase in the equilibrium population size at all trophic levels Figure 1.3. Furthermore, there is no lower productivity limit on the number of trophic leVels that may be supported by a system. A plot of the equilibrium Synechococcus population versus the specific growth rate may result in one of several alternatives relationships predicted by ratio- dependent models. A linear correlation between the equilibrium population density and specific growth rate would suggest that both substrate concentration and predation contribute to the equilibrium population size and specific growth rate. A slope of greater than one >1 would indicate that nutrient input has a relatively greater effect upon the equilibrium population size than predation. Likewise, a slope of less than one <1 would indicate the opposite. A curvilinear increase in consumption rate would be the result of predator pressure giving way to substrate limitation 1 (Figure 1.3). hete proc beca flagt wou mod mod the l micr to de plan] cone: in res In ch dOCUI “Ulric Specif Specif fatioi chapte a"d ti 12 The implicit assumption of the ratio-dependent model is that temporal or spatial heterogeneity exists within the system. It is unlikely that temporal heterogeneity could be produced by differences in generation times between Synechococcus and its predators because such differences are small. The estimated 1 to 2 divisions per day (13) of flagellates is comparable to the reported replication rate of Synechococcus (4, 10). This would exclude temporal heterogeneity as justification for application of a ratio-dependent model to this system. The other possible basis for the application of the ratio-dependent model is spatial heterogeneity. Spatial heterogeneity is principally the result of refugia for the prey population. One possible refugia for Synechococcus is the abundant marine microaggregates known as “sea-snow” (49). However, recent phylogenetic analysis failed to detect Synechococcus within these microaggregates (12). Thus, it is unlikely that the planktonic Synechococcus use microaggregates as refugia. Synechococci populations both in the laboratory and the environment were analyzed in the context of the afore mentioned models. In chapters 2 and 3 the nucleic acids concentrations of a freshwater cyanobacterium and a marine cyanobacterium were studied in response to changes in specific growth rate and during a 12 h light: 12 h dark diel cycle. In chapter 4 oligonucleotide probes specific for cyanobacteria] 16S rRNA were used to document the changes in specific growth rate of synechococci populations in response to nutrient-limitation or predator-limitation. In chapter 5 synechococci population size and specific growth within the Gulf of Mexico were examined, using oligonucleotide probes specific for cyanobacteria] 16S rRNA, in light of the predictions of prey-dependent and ratio-dependent models. Finally, chapter 6 discusses how the results of the previous chapters apply in a broad context to predator-prey interactions within the microbial world and the future directions such techniques and models may take. 13 References l. Arditi, R., and L. R. Ginzburg. 1989. Coupling in predator-prey dynamics: ratio-dependence. J. Theor. Biol. 139:311-326. 2. Arditi, R., N. Perrin, and H. Saiah. 1991. Functional responses and heterogeneities: an experimental test with cladocerans. OIKOS. 60:69-75. 3. Arditi, R., and H. Saiah. 1992. Emperical evidence of the role of heterogeneity in ratio-dependent consumption. Ecology. 73(5): 1544-1551. 4. Brand, L. E., W. G. Sunda, and R. R. L. Guillard. 1983. Limitation of marine phytoplankton reproduction rates by zinc, manganese and iron. Lirnnol. Oceanogr. 28:1182-1198. 5. Campbell, L., and E. J. Carpenter. 1986. Diel patterns of cell division in marine Synechococcus spp. (cyanobacteria): use of the frequency of dividing cells technique to measure growth rate. Mar. Ecol. Prog. 32:139-148. 6. Campbell, L., and E. J. Carpenter. 1986. Estimating the grazing pressure of heterotrophic nanoplankton on Synechococcus spp. using the sea water dilution and selective inhibitor techniques. Mar. Ecol. Prog. 33: 121-129. 7. Campbell, L., and D. Vaulot. 1993. Photosynthetic picoplankton community structure in the subtropical north Pacific Ocean near Hawaii (station ALOHA). Deep-Sea Research. 40(10):2043-2060. 8. Caron, D. A., E. L. Lim, G. Miceli, and J. B. Waterbury. 1991. Grazing and utlization of chroocoid cyanobacteria and heterotrophic bacteria by protozoa in laboratory cutlures and a coastal plankton community. Mar. Ecol. Prog. 76:205-217. 9. Carpenter, E. J., and L. Campbell. 1988. Diel pattern of cell division and growth rates of Synechococcus spp. in Long Island Sound. Mairne Ecology Progress Series. 47:179-183. 10. Chisholm, S. W., R. J. Olson, E. R. letter, R. Goericke, J. B. Waterbury, and N. A. Welschmeyer. 1988. A novel free-living prochlorophyte abundant in the oceanic euphotic zone. Nature. 334:340-343. 11. Coale, K. H. 1991. Effects of iron, manganese, copper, and zinc enrichments on productivity and biomass in the subarctic Pacific. Lirnnol. Oceanogr. 36(8): 1851-1864. 12. DeLong, E. F., D. G. Franks, and A. L. Alldredge. 1993. Phylogenetic diversity of aggregate-attached vs. free-living marine bacterial assemblages. Lirnnol. Oceanogr. 38(5):924-934. 13. Eccleston-Parry, J. D., and B. S. C. Leadbeater. 1994. A comparison of the growth kinetics of six marine heterotrophic nanoflagellates fed with one bacterial species. Mar. Ecol. Prog. 105:167-177. 14 14. Ferrier-Pages, C., and F. Rassoulzadegan. 1994. Seasonal impact of the microzooplankton on pico- and nanoplankton growth rates in the northwest Mediteranean Sea. Mar. Ecol. Prog. 108:283-294. 15. Flores, E., M. G. Guerrero, and M. Losada. 1983. Photosynthetic nature of nitrate uptake andreduction in the cyanobacterium Anacystis nidulans. Biochim. Biophys. Acta. 722:408-416. 16. Fogg, G. E. 1986. Picoplankton. Proc. R. Soc. Lond. 228:1-30. 17. Gerhardt, P., and S. W. Drew. 1994. Liquid Culture, p. 231-236. In P. Gerhardt, R. G. E. Murray, W. A. Wood, and N. R. Krieg (ed.), Methods for General and Molecular Bacteriology. American Society for Microbiology, Washington D. C. 18. Giovannoni, S. J., T. B. Britschgi, C. L. Moyer, and K. G. Field. 1990. Genetic diversity in Sargasso Sea bacterioplankton. Nature. 354:60-63. l9. Giovannoni, S. J., S. Turner, G. J. Olsen, S. Barns, D. J. Lane, and N. Pace. 1988. Evolutionary relationships among the cyanobacteria and green chloroplasts. J. Bacteriol. 170(8):3584-3592. 20. Glover, H. E. 1985. The physiology and ecology of the marine cyanobacterial genus Synechococcus, p. 49-107, Advances in Aquatic Microbiology, vol. 3. Academic Press Inc., London. 21. Glover, H. E., L. Campbell, and B. B. Prezelin. 1986. Contribution of Synechococcus spp. to size-fractioned primary productivity in three water masses in the Northwest Atlantic Ocean. Mar. Biol. 91:193-203. 22. Glover, H. E., B. B. Prezelin, L. Campbell, M. Wyman, and C. Garside. 1988. A nitrate-dependent Synechococcus bloom in surface Sargasso Sea water. Nature. 331:161-163. 23. Glover, H. E., and A. E. Smith. 1988. Diel patterns of carbon incorporation into biochemical constituents of Synechococcus spp. and larger algae in the northwest Atlantic Ocean. Mar. Biol. 97:259-267. 24. Greene, R. M., Z. S. Kolber, D. G. Swift, N. W. Tindale, and P. G. Falkowski. 1994. Physiological limitation of phytoplankton photosynthesis in the eastern equatorial Pacific determined from variablity in the quantum yeild of fluorescene. Lirnnol. Oceanogr. 39(5):1061-1074. 25. Grobbelaar, N., and T.-C. Huang. 1992. Effect of oxygen and temperature on the induction of a circadian nitrogenase activity rythm in Synechococcus RF-l. J. Plant Physiol. 140:391-394. 26. Hairston, N. G., F. E. Smith, and L. B. Slobodkin. 1960. Community structure, population control and competition. The American Naturalist. 154(879):421- 425. 27. Hansson, L.-A. 1992. The role of food chain composition and nutrient availability in shaping algal biomass development. Ecology. 73(1):241-247. aeroi. 29. . occur 104: 15 28. Ikemoto, H., and A. Mitsui. 1994. Diazotrophic synchronous growth of a marine unicellular cyanobacterium, Synechococcus sp. strain Miami BG 043511, under aerobic and microaerobic/anaerobic conditions. Anal. Biochem. 140:2153-2158. 29. Jiang, S. C., and J. H. Paul. 1994. Seasonal and diel abundance of viruses and occurrence of lysogeny/bacteriocinogeny in the marine environment. Mar. Ecol. Prog. 104: 163-172. 30. Johnson, P. W., and J. M. Sieberth. 1979. Chroococcoid cyanobacteria in the sea: a ubiquotous and diverse phototrophic biomass. Limnol. Oceanogr. 24:928—935. 31. Kivi, K., S. Kaitala, H. Kuosa, J. Kuparinen, E. Leskinen, R. Lignell, B. Marcussen, and T. Tamminen. 1993. Nutrient limitation and grazing control of the Baltic plankton community during annual succession. Limnol. Oceanogr. 38(5):893-905. 32. Kuuppo-Leinikki, P., R. Autio, S. Hallfors, H. Kuosa, J. Kuparinen, and R. Pajuniemi. 1994. Trophic interactions and carbon flow between picoplankton and protozoa in pelagic enclosures manipulated with nutrients and a top predator. Mar. Ecol. Prog. 107:89-102. 33. Leibold, M. A. 1989. Resource edibility and the effects of predators and productivity on th eoutcome of trophic interactions. The American Naturalist. 134(6):922- 949. 34. Li, W. K. W. 1994. Primary production of prochlorophytes, cyanobacteria, and eucaryotic ultraphytoplankton: measurements from flow cytometric sorting. Limnol. Oceanogr. 39(1): 169-175. 35. Lotka, A. J. 1925. Elements of physical biology. Williams and Wilkins, Baltimore, MD. 36. Martin, J. H., K. H. Coale, K. S. Johnson, S. E. Fitzwater, R. M. Gordon, S. J. Tanner, C. N. Hunter, V. A. Elrod, J. L. Nowicki, T. L. Coley, R. T. Barber, S. Lindley, A. J. Watson, K. V. Scoy, C. S. Law, M. I. Liddicoat, R. Ling, T. Stanton, J. Stockel, C. Collins, A. Anderson, R. Bidigare, M. Ondrusek, M. Latasa, F. J. Millero, K. Lee, W. Yao, J. Z. Zhang, G. Friederich, C. Sakamoto, R. Chavez, K. Buck, Z. Kolber, R. Greene, P. Falkowski, S. W. Chisholm, F. Hoge, R. Swift, J. Yungel, S. Turner, P. Nightingale, A. Hatton, P. Liss, and N. W. Tindale. 1994. Testing the iron hypothesis in ecosystems of the equatorial Pacific Ocean. Nature. 371:123-129. 37. Martin, J. H., R. M. Gordon, and S. E. Fitzwater. 1991. The case for iron. Limnol. Oceanogr. 36(8):1793-1802. 38. Mitchell, B. G., E. A. Brody, O. Holm-Hansen, C. McClain, and J. Bishop. 1991. Light limitation of phytoplankton biomass and macronutrient utilization in the Southem Ocean. Limnol. Oceanogr. 36(8): 1662-1677. 39. Morel, F. M. M., R. J. M. Hudson, and N. M. Price. 1991. Limitation of productivity by trace metals in the sea. Limnol. Oceanogr. 36(8):1742-1755. l979_ 16 40. Morris, 1., and H. E. Glover. 1981. Physiology of photosynthesis by marine coccid cyanobacteria - some ecological implications. Limnol. Oceanogr. 26:957-961. 41. Moss, B., S. McGowan, and L. Carvalho. 1994. Determination of phytoplankton crops by top-down and bottom-up mechanisms in a group of English lakes, the West Midland meres. Limnol. Oceanogr. 39(5): 1020-1029. 42. Oksanen, L., S. D. Fretwell, J. Arruda, and P. Niemela. 1981. Exploitation ecosystems in gradients of primary productivity. The American Naturalist. 118(2):240-261. 43. Peters, F. 1994. Prediction of planktonic protisan grazing rates. Limnol. Oceanogr. 39(1):195-206. 44. Prezelin, B. B., H. E. Glover, and L. Campbell. 1987. Effects of light intensity and nutrient availability on diel patterns of cell metabolism and growth in populations of Synechococcus spp. Mar. Biol. 95:469-480. 45. Prezelin, B. B., M. Putt, and H. E. Glover. 1986. Diurnal patterns in photosynthetic capacity and depth-dependent photosynthesis-irradiance relationships in Synechococcus spp. and larger phytoplankton in three water masses in the Northwest Atlantic Ocean. Mar. Biol. 91:205-217. 46. Rippka, R., J. Derruilles, J. B. Waterbury, M. Herdman, and R. Y. Stanier. 1979. Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 111(1-61). 47. Schmidt, T. M., E. F. DeLong, and N. R. Pace. 1991. Analysis of a marine picoplankton community by 16S rRN A gene cloning and sequencing. J. Bacteriol. 173(14):437l-4378. 48. Sieberth, J. M., V. Smetace, and J. Lenz. 1978. Pelagic ecosystem structure: heterotrophic compartments of the plankton and their relationship to plankton size fractions. Limnol. Oceanogr. 23: 1256-1263. 49. Silver, M. W., and A. L. Alldredge. 1981. Bathypelagic marine snow: deep- sea algal and detrital community. J. Mar. Res. 39:501-530. 50. Suttle, C. A., A. M. Chan, and M. T. Cottrell. 1990. Infection of phytoplankton by viruses and reduction of primary productivity. Nature. 347 :467-469. 51. Tabita, F. R. 1987. Carbon dioxide fixation and its regulation in cyanobacteria, p. 95-117. In P. Fay and C. Van Baalen (ed.), The Cyanobacteria. Elsevier, NY. 52. Volterra, V. 1928. Variations and fluctuations of the number of individuals in animal species living together. . 53. Waterbury, J. B., and F. W. Valois. 1993. Resistance to co-occurring phages enables marine Synechococcus communities to coexist with cyanophages abundant in seawater. Appl. Environ. Microbiol. S9(10):3393-3399. 54. Waterbury, J. B., S. W. Watson, R. R. L. Guillard, and L. E. Brand. 1979. Widespread occurence of a unicellular, marine, planktonic, cyanobacterium. Nature. 277:293-294. 55. 1986. Synec 56. betwe Sci 4 57. V SLI'UCIU 17 55. Waterbury, J. B., S. W. Watson, F. W. Valois, and D. G. Franks. 1986. Biological and ecological characterization of the marine unicellular cyanobacterium Synechococcus. Can. J. Fish. Aquat. Sci. 214:71-120. 56. Watson, 8., E. McCauley, and J. S. Downing. 1992. Sigmoid relationships between phosphorus, algal biomass, and algal community structure. Can. J. Fish. Aquat. Sci. 49:2605-2610. 57. Wootton, J. T., and M. E. Power. 1993. Productivity, consumers, and the structure of a river food chain. Proc. Natl. Acad. Sci. USA. 90: 1384-1387. COP Chapter 2 CONCENTRATIONS OF NUCLEIC ACIDS OF S YNECHOCOCC US SP. STRAIN PCC 6301 DURING GROWTH IN CONTINUOUS LIGHT AND LIGHT:DARK CYCLES l8 PCC cadunt batch t. cellula COIlCCI —-_ regula durin g — ___ a resul transcr actitvi grown ConSta oeuu] a COUCer the h}. Cellulr which conCCF concer 19 Abstract Cellular nucleic acids concentrations were determined for Synechococcus sp. strain PCC 6301 grown in either light-limited batch cultures, phosphate-limited chemostat cultures or batch cultures entrained by 12 h light: 12 h dark (diel) cycles. Both light-limited batch cultures and phosphate-limited chemostat cultures exhibited an exponential increase in cellular RNA concentrations, cellular 16S rRN A concentrations and cellular DNA concentrations that correlated with increases in growth rate. The ratio of RNA to DNA remained constant with increases in growth which suggests a lack of transcriptional regulation of ribosomal RNA operons. Cellular nucleic acids concentrations decreased during light periods and increased during dark periods in cultures entrained by a diel cycle; a result that was unexpected in light of experiments demonstrating a circadian increase in transcriptional activity during the subjective light periods and a decrease in transcriptional actitvity during subjective dark periods. The mean cellular RNA concentration during diel growth was comparable to the cellular RNA concentration at the same growth rate under constant illumination. Cellular 168 rRNA concentrations were closely correlated with cellular DNA concentrations during a diel cycle. The correlation between cellular DNA concentration and cellular l6S rRN A concentration, during a diel cycle, is consistent with the hypothesis that cellular 16S rRN A concentration is not transcriptionally regulated. Cellular 168 rRNA concentration also correlated with ribosomal RNA operon copy number which suggests that gene-dosage is the primary mechanism regulating cellular l6S rRNA concentrations in synechococci. The cyclical variation in cellular nucleic acids concentration during diel cycles appears to be a function of the timing of cell division. hetero growtf (43, 4 one-th cellula compl. Cyanol period under ‘ Cycle, (10), n ofdna Cl'anot cellular CUiture CODCen IRVA . 20 Introduction Cellular nucleic acids concentration is proportional to growth rate in a number of heterotrophic bacteria (32, 33, 55). Nucleic acids concentration is also proportional to growth rate in both filamentous and unicellular cyanobacteria under constant illumination (43, 45). The nucleic acids content of bacteria is predominately composed of rRNA and one-third of ribosomal rRNA (w/w) is composed of 168 rRNA. The correlation between cellular 16S rRNA concentrations and growth rate presents the possibility of using probes complementary to 168 rRN A to determine in situ growth rates. The concentration and synthesis of nucleic acids have typically been studied in cyanobacteria grown under constant illumination followed by a single dark incubation period (3, 17, 18, 22, 24, 46, 60, 63). Growth under constant illumination, however, may not accurately reflect the physiological state of synechococci populations growing under the diel cycles found in the environment (16). Cyanobacteria entrained by a diel cycle, for example, exhibit circadian rhythms in cell division (48, 62), amino acid uptake (10), nitrogenase activity (21, 27, 28), the expression of psbA (35-37) and the expression of dnaK (10). Circadian rhythms are not found in continuously illuminated cultures of cyanobacteria. In this study, we demonstrate that increases in cellular RNA concentrations and cellular DNA concentrations conelate with increases in growth rate in light-limited batch cultures and phosphate-limited chemostat cultures. Increases in cellular 16S rRNA concentrations also correlate with increases in growth rate. Cellular RNA, cellular l6S rRNA and cellular DNA concentrations, however, fluctuated in a regular pattern over the course of a diel cycle. (ATC . S)? was V at leaK Cg mt of40 Were 5 rate fn Vinela miiinta culmrE 21 Materials and Methods Bacterial strains and culture conditions. Synechococcus sp. strain PCC 6301 (ATCC 27144) was maintained at room temperature (RT) on BG-ll (1) agar at 86uEm'2s. 1. Synechococcus sp. strain PCC 6301 batch cultures were grown at 37°C. Growth rate was varied by changing the photon fluence rate from 15 ttEm-zs.1 to 64.4 IrEm'zs'1 in light- limited batch cultures. Diel cultures were entrained on a 12 h light: 12 h dark diel cycle for at least seven generations prior to the start of each diel experiment. Chemostat cultures of Synechococcus sp. strain PCC 6301 were grown on modified Cg medium (29). Chemostats were illuminated at 162 ItEm'zs'l at 37°C and aerated at a rate of 40 ml-min" with 3% CO2 (balance air). Phosphate-limited growth was measured at three different reservoir concentrations. Phosphate concentrations within the reservoirs were 5 M, 10 ILM and 15 M KzHPO‘. Growth rate was varied by changing the dilution rate from 20 nil-min.l to 365 nil-minl in a 250 ml Bellco Spinner flask (Bellco Glass Inc., Vineland, NJ). The culture was considered to have reached a stable equilibrium after maintaining the same optical density at 750 nm and 600 nm for three volume changes of the culture vessel. Cell density was determined by microscopic direct counts (34) in a Petrohoff—Hausser chamber (Hausser Scientific Partnership, Horsham, PA) or using a particle counter and channeler (Coulter Electronics Inc., Miami, FL). Growth rate was defined as the reciprocal of the generation time, given in hours. Chemical determination of RNA and DNA. Synechococcus sp. strain PCC 6301 cells were harvested by centrifugation of 10 ml of log-phase culture at 16,000 xg for 25 min at 4°C. Cellular RNA concentration was measured by the orcinol assay (13), using adenosine monophosphate as a standard. Cellular DNA concentration was measured by the diphe WCI‘C conce on th differl of det 6301 Chem nuclei comp; acids. glassu baked (7); ti (ESChg PIP, Pufifie NH.,o. addjfio and int (7) Wax 0f163 22 diphenylamine assay (13), using deoxyribose as a standard. Cellular RNA concentrations were estimated to be twice that of the AMP standard based on the approximately 55% G+C ratio of Synechococcus PCC 6301 16S rRNA and 23S rRNA (19, 41). Cellular DNA concentrations were calculated as 4.84 times the deoxyribose standard concentration based on the 52-53% G+C ratio of the Synechococcus PCC 6301 genome (12) and the difference between the average molecular weight of a nucleotide and the molecular weight of deoxyribose. Nucleic acids isolation and hybridization. Synechococcus sp. strain PCC 6301 cells were harvested by centrifugation of 1.5 ml log-phase batch culture or stable chemostat culture at 16,000 xg for 25 min at 4°C. Cells were disrupted nwchanically and nucleic acids isolated (61). The RNA extraction efficiency was 65.7i2.3 % based on the comparison between whole cell orcinol assays and orcinol assays of extracted nucleic acids. RNase contamination was reduced by using either virgin polypropylene or glassware treated with diethylpyrocarbonate (Sigma Chemical Co., St. Louis, MO) and baked at 240°C for 3 h. Nucleic acids were hybridized with a cyanobacterial-specific oligonucleotide probe (7); this probe was complementary to the small subunit rRNA at positions 360-376 (Escherichia coli numbering). The cyanobacterial-specific probe was 5’ end-labeled with y—32P-ATP (56) to a specific activity of ca. 0.5 ItCi-pmol'l probe. End-labeled probes were purified on TSK-DEAE columns (Supleco, Bellefonte, PA) equilibrated with 50 mM NH,OAc (7). Nucleic acids from batch cultures or chemostat cultures were denatured by the addition of phosphate-buffered gluteraldehyde [pH 7.0] to a final concentration of 1.5% and incubated at RT for 15 min. Purified Synechococcus sp. strain PCC 6301 16S rRNA (7) was denatured in the same manner and a standard curve constructed from 1 ng to 75 ng of 16S rRNA. Escherichia coli (E. coli) nucleic acids were denatured in the same manner and u sampi (3'1le 1 Bcdfc and E (Strat. manu: hybrid polyt.r contai: and or. Sample CA). D if fete: Sp. Stra:| 15 “Er reSpecti Cellular I IDCreaSe 23 and used to determine non-specific binding of the probe. Nucleic acids from experimental samples, 16S rRNA standards and E. coli were applied to MagnaCharge nylon membranes (Micron Separation Inc., Westbourgh, MA) using a slot-blot apparatus (Millipore, Bedford, MA). Approximately 1 ug, 500 ng and 250 ng from each experimental sample and E. coli were applied to nylon membranes. Nucleic acids were UV-crosslinked (Stratalinker 1800, Stratagene Inc., La Jolla, CA) to nylon membranes according to die manufacturer’s instructions. Membranes were prehybridized for 30 min at 37°C in 10 ml hybridization buffer [6X SET (56), 0.5% SDS, 1X Denhardt’s solution (56), 100 [J.g-ml’l poly(A)], followed by hybridization for 14—16 h at 37°C in 10 ml hybridization buffer containing lxlO‘5 CPM-ml'l 32P-radiolabeled probe. Membranes were washed twice at RT and once at 48°C for 30 rrrin each in wash buffer [2X SET, 0.5% SDS]. Standards and samples were quantified using a radioanalytical image system (AMBIS Inc., San Diego, CA). Results Cellular RNA and DNA concentrations of light-limited batch cultures. Differential growth rates were established for light-limited batch cultures of Synechococcus sp. strain PCC 6301 by varying photon fluence rates. Continuous photon fluence rates of 15 uBm'zs" to 64.4 ItEm‘zs" resulted in grth rates of 0.008 b" to 0.051 h", respectively. Cellular RNA and DNA concentrations were determined colorimetrically. Cellular RNA concentrations increased from 23.91222 fg-ce11.l to 45.5:l:0.9 fg-cell'l with increases in growth rate (Figure 2.1; Table 1). Cellular DNA concentrations farm: <2: .83. 24 60 50 Total RNA (fg-cell") 0.02 0.04 0.06 0.08 0.10 Specific Growth Rate (h'l) Figure 2.1 - Total cellular RNA, as determined by orcinol, from light-limited batch cu tures (O) and phosphate-limited chemostat cultures (I), as a function of growth rate (r'2 = 0.92). Error bars represent standard errors (n = 3). 36 H nmN n .9.“ H 538 memo 00m. 2 an: . n: In .55 e: 53 A3 , n3 .. . . _ EN . R; . . EN echefi _ . n: 5.2 I was . . has: none: 92 NS . 2d .213: 22.8 w; H wdm a; H _.Om rash: £13815 M 12927:. . ...AHéB _. we A :La... . we <2: .. ...e. :3 .42: 3...... <2: .38. .2330 :83 958:-sz 5 58 DU."— 593 dm Sooeoeaouzam he flouofiuam ano—ommbfi - flu 035—. were Cellu 19.7: CODCE 26 increased from ll.4:l:0.5 fg-cell'l to 18.6i-l.l fg-cell‘I with increasing growth rate (Table 2.1). The ratio of RNA to DNA remained a constant 2.4:t0.3 over a range of growth rates in Synechococcus sp. strain PCC 6301 (Table 2.1). Cellular RNA and 168 rRNA concentrations of phosphate-limited chemostat cultures. Growth rates were varied from 0.005 h'1 to 0.09 h'1 in each of three chemostats fed by reservoirs containing 15 uM, 10 M or 5 11M phosphate. Phosphate limitation does not preferentially restrict ribonucleic acid pools, but results in a proportional loss from all macromolecular pools with decreases in growth rate (20). Cellular RNA concentrations increased exponentially from 24.0:t1.6 fg-cell'l to 53.0:4.8 fg-cell'l with increases in growth rate (Figure 2.1). Cellular 16S rRNA concentrations were determined using 32P-radiolabeled, cyanobacterial-specific, oligonucleotide probes. Cellular 16S rRNA concentrations increased exponentially from 4.62119 fg-cell" to 19.710.45 fg-cell" with increasing growth rate (Figure 2.2); regardless of the phosphate concentration within the reservoir. Cellular RNA, 16$ rRNA and DNA concentrations of diel batch cultures. Batch cultures of Synechococcus sp. strain PCC 6301 were entrained in a 12 h light: 12 h dark diel cycle at 64.4 uEm'zsq. Entrained cultures had a growth rate of 0.03 b" (Figure 2.3) over the course of a single diel cycle (24 h). The growth rate was 0.06 h" during the light periods of a diel cycle because cell division was arrested in the absence of light. Cell division resumed upon reexposure to light. Entrained cultures did not exhibit a noticeable lag in growth upon being reexposed to light. Cellular RNA concentrations gradually decreased from approximately 40.8:t4.0 fg-cell'l to 32.7:l:1.l fg-cell'l during light periods and gradually returned to 38.0:l:1.4 fg-cell‘l fg-cell'l during subsequent dark periods (Figure 2.4A). The trend observed in cellular RNA concentrations was observed in cellular 16S rRNA concentrations as well. Cellular 16S RNA concentrations gradually hut-naiaiOEIiE (Zion U.u\l ills-II-nrrrh 27 Cellular 16S rRNA (fg ocell") 0.00 0.02 0.04 0.06 0.08 Growth Rate (h'l) Figure 2.2 - Cellular 16S rRNA concentrations of Synechococcus PCC 6301 from phosphate-limited chemostats. Phosphate concentrations of nutrient reservoirs were 15 M (O), 10 M (A) and 5 11M (0). Dashed lines indicate 95% confidence intervals (p < 0.001; r2 = 0.67). A—I-i l-nll I4\I\ 28 1.2 a" E 1.0- O m . = 8 a 0.8- Q :1 . 3% _ g2 0.6- 0 Q . 0 I-I g 0.4- 5 U . 0.2 0 6 12 18 24 , 30 36 Time (h) Figure 2.3 - Cell density of Synechococcus sp. strain PCC 6301 during diel growth. Each day consisted of 12 h light: 12 h dark (shaded area). Error bars represent standard errors (n=6). F igu grou- 3000: Stand --tIl-|\ M1 A {r“' -—'\ Total RNA (fg-cell") l6S rRNA (fg-cell") Time (b) Figure 2.4 - Cellular RNA concentrations of Synechococcus sp. strain PCC 6301 grown under a 12 h light: 12 h dark (shaded area) diel cycle. (A) Cellular RNA concentrations. (B) Cellular 168 rRNA concentrations. Error bars represent standard error (n=6). €30.45 <75 33:5 Figl "Dd-t err0 30 Cellular DNA (fgccell'l) Time (h) Figure 2.5 - Cellular DNA concentrations of Synechococcus sp. strain PCC 6301 grown under a 12 h light: 12 h dark (shaded area) diel cycle. Error bars represent standard error (n=6). cont: 3 get genor peso 61pm tune isco- incn: cont betw have acii cFan 31 decreased from approximately 14.9i6.2 fg-cell'1 to 7.5i1.7 fg-cell'l during light periods and gradually returned to 13.8i2.2 fg-cell'l during subsequent dark periods (Figure 2.48). The trend exhibited by cellular RNA concentrations and cellular 16S rRNA concentrations was also observed in cellular DNA concentrations (Figure 2.5). Cellular DNA concentrations gradually decreased from 10.7i0.3 fg-cell-l, corresponding to approximately 3 genomes (23, 25), to a low of 7.9:t0.2 fg-cell", corresponding to approximately 2 genomes, during light periods before returning to 10.9i0.4 fg-cell.l during subsequent dark periods. Discussion Light-limited, batch cultures of Synechococcus sp. strain PCC 6301 displayed an exponential increase in both cellular RNA concentrations and cellular DNA concentrations that correlated with increases in growth rate. The increase in cellular RNA concentrations is comparable to previous reports (45) of an approximately 28.7 fg-cell'l to 44.9 fg-cell'l increase in cellular RNA concentrations over the same range of growth rates in continuously illuminated, light-limited batch cultures. The exponential relationships between RNA, DNA and growth rate observed in Synechococcus sp. strain PCC 6301 have also been observed in Anabaena variabilis (43). The correlated increase in nucleic acids with increased growth rate appears to be a general physiological feature of cyanobacteria, as well as heterotrophic bacteria (32, 33, 55). The cellular DNA concentrations in this study are approximately 2.4 times the values (4.9 fg-cell'l to 7.8 fg-cell'l) reported previously for light-limited batch cultures (45). The apparent discrepancy between the two experiments may be attributed to previous studies reporting the deoxyribose concentration, calculated from the standard, as a DNA concentration when in fac‘ of gen incr ope: in h cent of g (45) rRN Stabl cellu rR‘x‘. heter (33. ; grow been Smal induc ()pr 32 fact there is a 2.4 fold difference in molecular weight between deoxyribose and nucleotides of DNA. The cellular DNA concentrations in this study agrees with the number of genomes per cell previously reported (6) for Synechococcus sp. strain PCC 6301. i In heterotrophic bacteria, such as E. coli, the rate of transcription from rRNA operons increases with increases in growth rate (51). The increase in transcription rates from rRNA operons is reflected in a linear increase in the RNA/DN A ratio with increasing growth rate' in heterotrophic bacteria (14, 32, 49, 55). Unlike in heterotrophic bacteria, the ratio of cellular RNA concentration to cellular DNA concentration remained constant over a range of growth rates in Synechococcus sp. strain PCC 6301 (Table 1), as reported previously (45). The constant RNA/DNA ratio may indicate a lack of transcriptional regulation of the rRNA Operons in Synechococcus sp. strain PCC 6301. A lack of transcriptional control of stable rRN As is also indicated by the constant ratio of cellular rRN A concentrations to cellular tRNA concentrations in Synechococcus sp. strain PCC 6301 (44). The constant rRNA/tRN A ratio in Synechococcus sp. strain PCC 6301 contrasts with the situation in heterotrophic bacteria where the rRN A/tRN A ratio increases with increasing growth rate (33, 55). The rRNA/tRNA ratio increased from approximately 1.8 to 4.5 with increases in growth rate in Salmonella typhimurium (33). Similar increases in rRNA/tRNA ratios have been observed in E. coli (55) and Bacillus subtilis ( 15); although these increases were smaller than those reported for S. typhimurium. It was originally argued (9, 44) that the constant ratio of rRNA to tRNA, the lack of induction or repression of key enzymes by their respective substrates, and the constant ratio of RNA to DNA are indicative of a general lack of transcriptional control in cyanobacteria. Transcriptional control has been demonstrated, however, for glycogen phosphorylase (58), glucose-6-phosphate dehydrogenase (58) and psbA (40) in cyanobacteria. Although not all gene expression lacks transcriptional control the constant RNA to DNA ratio observed in this study (Table 1) and by Mann and Carr (45) supports the conclusion that rRNA is not transcriptionally regulated in cyanobacteria. 33 Even though rRNA does not appear to be transcriptionally regulated, cellular RNA concentrations increased exponentially with increases in growth rate in phosphate-limited, chemostat cultures of Synechococcus sp. strain PCC 6301 (Figure 2.1). Cellular RNA concentrations have also been demonstrated to increase exponentially from approximately 20 fg-cell'l to 40 fg-cell'l over a range of dilution rates from 0.025 h'1 to 0.1 h'1 in light- linrited chemostats (54). Cellular RNA concentrations of 50—60 fgocell'l at a dilution rate of ' 0.09 h'1 have been reported for Synechococcus sp. strain PCC 6301 in CazI-limited chemostats at nonlimiting photon fluence rates (65). The cellular RNA concentration in Ca2*-limited chemostats is comparable to the cellular RNA concentration observed at a growth rate of 0.09 h‘1 in phosphate-limited chemostats (Figure 2.1). One exception to the correlation between cellular RNA concentration and growth rate, however, has been noted. Cellular RNA concentration was found to be independent of growth rate in Synechococcus sp. strain PCC 6301 grown in Mgz“-linrited chemostats (64). Synechococcus sp. strain PCC 6301 possessed a uniformly high concentration of RNA in Mgztlimited chemostats regardless of growth rate. Low Mg2+ concentrations were postulated to reduce ribosome efficiency which resulted in a reduction in growth rate. If ribosome efficiency had not been reduced by low Mg2+ concentrations the RNA concentration would have reflected growth rate. Cellular RNA concentrations, therefore, can be used to measure growth rate except in cases of Mg2+ limitation. The correlation between cellular RNA concentration and growth rate was also expected to apply to cellular rRNA concentrations because rRNA comprises a constant 75% of total RNA in continuously illuminated cultures of cyanobacteria (44). This hypothesis was tested using cyanobacterial-specific oligonucleotide probes complementary to the small subunit rRN A. Cellular 16S rRNA concentrations increased exponentially with increases in growth rate in phosphate-limited, chemostat cultures of Synechococcus sp. strain PCC 6301 (Figure 2.2). The conelation between cellular 16S rRNA concentrations and growth 34 rate presents the possibility of using group-specific probes complementary to 168 rRNA to determine in situ growth rates. Determining the in situ growth rate of cyanobacteria by using 16S rRNA probes requires an understanding of the diel variations of cellular RNA concentrations. The variation in cellular RNA concentrations was examined in batch cultures of Synechococcus sp. strain PCC 6301 entrained in a 12 h light: 12 h dark diel cycle and possessing a diel: growth rate of 0.043 b". Cellular RNA concentrations gradually decreased during the light periods of a diel cycle (Figure 2.4A). The decrease in cellular RNA concentrations does not appear to be due to a decrease in rates of RNA synthesis. High rates of incorporation of 3H- and l‘C-uracil (18, 22, 38, 58) indicate that RNA synthesis rates do not decrease in the light. Continuous rates of RNA synthesis are also indicated by the accumulation of rbcL gene transcripts (encoding the large subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase) in Synechococcus sp. strain PCC 8801 (l 1) and transcripts of the psbAl gene (encoding the D1 protein of P811) in Synechococcus sp. strain PCC 7942 (36) in the light. The decrease in cellular RNA concentrations appears to be the result of cell division. Cell division occurred only upon exposure to light in Synechococcus sp. strain PCC 6301 (Figure 2.3). During these light periods the rate of cell division exceeded the cells ability to double its cellular RNA content and therefore RNA concentrations decreased on a per cell basis. Cellular RNA concentrations decreased during the first 3 h of dark periods. This decrease in cellular RNA concentrations does not appear to be due to cell division because cell division is arrested during dark periods. The transient decrease in cellular RNA concentrations during the first 3 h of darkness coincides with decreases in the light-specific transcripts of the psbAI gene (36) and the rbcL gene (1 l) which offset the increase in 16S rRNA concentrations during the same period (Figure 2.4B). Cellular RNA concentrations increase approximately 33% over the remaining nine hours of darkness. The 33% increase in cellular RNA concentrations is similar to the 22% increase observed by Herdman er d 35 (24). The increase in cellular RNA concentrations coincides with increase in a dark- specific transcript of unknown function (63), transcripts of the nitrogen fixing gene nifl-I (11), transcripts encoding glycogen phosphorylase and glucose-6-phosphate dehydrogenase (58), and ribosomal RNAs (53, 58, 63). Incorporation of 3H- and '4C- uracil into both stable and unstable RNA species indicates that RNA continues to be synthesized throughout the dark period (17, 18, 22, 38, 53, 58). The majority of dark l‘C-' uracil incorporation was into RNA, with little incorporation into nucleotide pools (22). The small amount of l“C-uracil incorporation into nucleotide pools suggests a rapid turnover within nucleotide pools which is also indicative of continuous RNA synthesis. The small amount of l‘C-uracil incorporation into nucleotide pools in Synechococcus sp. strain PCC 6301 is unlike the incorporation in E. coli (47) where up to half of the I“C-uracil accumulates in nucleotide pools. The mean cellular RNA concentration during diel growth was comparable to the cellular RNA concentration at the same growth rate under constant illumination. The decrease in cellular RNA concentrations was accompanied by a gradual decrease in cellular 16S rRNA concentrations during the light period of a diel cycle (Figure 2.4B). Like cellular RNA concentrations, the drop in cellular 16S rRNA concentrations appears to be due to cell division. Cellular 168 rRNA concentrations doubled during the subsequent dark period. The steady increase in 16S rRN A concentrations has previously been observed in synchronized cultures of Synechococcus sp. strain PCC 6301 placed in the dark (17, 53, 58, 63). A rise in 16S rRNA concentrations during the night was also observed in in situ assemblages of synechococci (39). This assemblage of synechococci exhibited a marked decline in 168 rRNA during the first 4.5 h of darkness which was due to an increase in population density. The accumulation of 16S rRNA in the dark implies de novo synthesis of ribosomal RNAs (l7, 18, 58) without concurrent cell division. The net result of these two factors is an increase in 168 rRNA concentrations on a per cell basis. Cellular 16S rRNA 36 concentrations increased by approximately 6 fg-cell‘I during the course of a dark period. Cellular 168 rRNA comprises approximately one-third of ribosomal RNA. The approximately 6 fg-cell'I increase in cellular l6S rRNA concentration would have resulted in a 18 fg-cell‘l increase in ribosomal RNA. The calculated 18 fg-cell'l increase in cellular rRNA concentrations is in agreement with the observed increase of approximately 18 fg-cell’l in cellular RNA concentrations and accounts for the majority of the 33% increase in: cellular RNA concentrations. Cellular DNA concentrations gradually decreased from approximately 3 genomes to 2 genomes, during light periods, before returning to 3 genomes during subsequent dark periods (Figure 2.5). Like cellular RNA concentrations, the decrease in cellular DNA concentrations was due to the increase in population size. The average of 3 genomes observed is consistent with the 3 genomes-cell‘l reported by Binder and Chisholm (6) for continuously illuminated Synechococcus sp. strain PCC 6301 with a growth rate (0.07 h") similar to the growth rate during light periods in our diel experiments. Cell division can be synchronized in cyanobacteria by a period of dark incubation. Synchronized cultures of Synechococcus sp. strain PCC 6301 have shown continuous incorporation of radiolabeled thymine for up to 8 h after reentering the dark (60). The observed increase in cellular DNA concentrations seems to be dependent on the phase of the cell cycle of the culture upon reentering the dark (46). Synchronous cultures of Synechococcus sp. strain PCC 6301 have shown increases of 10-19% in cellular DNA concentrations within the first 4 h of dark incubation before decreasing to a plateau maintained over the next 12-23 h (46). The continuous, dark DNA synthesis observed in the entrained cultures of Synechococcus sp. strain PCC 6301 used in this study contrasts with the lack of dark DNA synthesis in asynchronous cultures of Synechococcus sp. strain WH 8101 (2) and Synechococcus sp. strain PCC 6301 synchronized by a single dark incubation period (22, 46). It would appear that entrained cultures of cyanobacteria possess greater resources for the synthesis 37 of DNA during the dark than asynchronously growing cultures or cultures synchronized by a single dark period. One possible explanation for the increase in cellular DNA concentrations is the initiation of genome replication during the dark. Replication has been demonstrated by the continuous incorporation of radiolabeled thymine into synchronized cultures of Synechococcus sp. strain PCC 6301 for up to 8 h after reentering the dark (60). The initiation of DNA replication is presumed to be either inhibited (16, 24) or aborted (46) in the absence of light. The initiation of genome replication is, by inference, a light-dependent reaction. Cellular nucleic acids concentrations were measured during diel growth by two sampling regimes in our study. The first regime briefly exposed cultures to light (<10 min) during sampling in the dark portion of the diel cycle. This regime raised concerns that brief exposure to light may have allowed a portion of the population to initiate genome replication during the dark. This concern was addressed by a second sampling regime in which all culture manipulations were performed in the dark during the dark phase of the diel cycle. Both regimes produced similar cellular DNA concentrations and the same trend of increasing cellular DNA concentration during the dark. These results demonstrated that brief exposure to light does not significantly perturb cell cycle events. A second possible explanation for the increase in cellular DNA concentrations is the initiation of genome replication shortly before entering the dark and continued DNA synthesis throughout the dark period of a die] cycle. Continuous DNA synthesis throughout the dark period would imply a genome replication time (C) in excess of 12 h. A replication time of 12 his significantly longer than the 65 min C period estimated by Mann and Carr (45) from shift-up experiments with asynchronous cultures of Synechococcus sp. strain PCC 6301. A difference in C periods would indicate that genome replication times vary between light and dark periods or with growth rate. Variations in genome replication times are not without precedence in the literature (4, 5). Genome replication times have been reported to vary between 60 nrin and 4 h for cultures of Synechococcus sp. strain 38 PCC 6301 (24, 46, 60). DNA synthesis has been reported to “run-out” within 3 h of placing a synchronous culture of rapidly growing Synechococcus sp. strain PCC 6301 in the dark (6). Synchronous cultures of Synechococcus sp. strain PCC 6301 with generation times of 8 h were reported to have DNA synthesis periods of up to 8 h (3). The long replication time reported by Asato and Folsome would be consistent with our observations of long C period in slowly growing cultures of Synechococcus sp. strain PCC 6301. At present we cannot distinguish between a slow DNA synthesis rate and initiation of DNA synthesis during the dark. It is unlikely that the increase in cellular DNA concentrations can be explained by an increase in plasmid copy number. Synechococcus sp. strain PCC 6301 contains two cryptic plasmids of approximately 48.9 kb and 7.9 kb (42, 57, 66). The approximately 2.8 fg-cell'l increase in cellular DNA would require the cell to cany more than 53 copies of the largest plasmid, an event that seems improbable. The pattern of decreasing cellular nucleic acids concentration during light periods and increasing cellular nucleic acids concentrations during dark periods is in part a consequence of the dynanrics of the growth rate of the cyanobacteria population. Population density increased exponentially when exposed to light, but was arrested immediately upon entering the dark phase of a diel cycle (Figure 2.3). The immediate arrest of cell division has also been observed in Synechococcus sp. strain PCC 6301 (22, 46), Synechococcus sp. strain WH 8101 (2) and Oscillator-la agardhii (67) in the absence of light. The immediate anest of cell division contrasts with the increase in population size observed in Synechococcus sp. strain WH 8101 and Synechococcus sp. strain WH 7803 during the first 3 h of darkness (5, 6, 68). This contrast may be explained by the longer generation times of Synechococcus sp. strain WH 8101 and Synechococcus sp. strain WH 7803 in these experiments. Variability in the period of cell division (D) has been documented (8, 26) and is expected to be longer in slowly growing cells. A protracted D period may have allowed cell division that was initiated before entering the dark period to continue to completion in 39 the dark in slowly growing Synechococcus WH 8101 and Synechococcus WH 7803. The extended period of cell division would have resulted in an increase in cell density during the first few hours of darkness. Cell counts demonstrated that Synechococcus sp. strain PCC 6301 does not undergo reductive cell division during the dark such as the reductive division exhibited by heterotrophs during starvation (52). Furthermore, based on the constant optical density at 600 nm and 750 nm (data not shown) biomass did not appear to decrease as might be expected of heterotrohic bacteria during a downshift in nutrients. Population density increased without the noticeable 30 min to 4 h lag time that has previously been observed in Synechococcus sp. strain PCC 6301 (6, 24) and in marine Synechococcus sp. strain PCC 6301 (2, 68) emerging into the light from the dark. The lag period, however, may have been shorter than the sampling period (3 h) used in this study. The cellular nucleic acids concentration of cyanobacteria correlates with grth rate under conditions of continuous illumination. This correlation is true regardless of whether growth rate is limited by available nutrients or light. Superficially the periodic absence of a cyanobacterium’s primary energy source (light) may be considered analogous to carbon-starvation in heterotrophic bacteria. Heterotrophic bacteria, such as E. coli, quickly degrade ribosomal RN As when faced with starvation conditions (30, 31). Cyanobacteria, however, continue to accumulate ribosomal RNA despite being confronted by starvation conditions. Degradation of ribosomes would be detrimental in the long run for a photoautotroph faced with periodic, short-term starvation. The ability to retain high concentrations of active ribosomes allows Synechococcus sp. strain PCC 6301 to quickly resume exponential growth upon being reexposed to light (Figure 2.3). The cyclical rise and fall of cellular RNA concentrations appear to be a consequence of initiation of genome replication and rRNA synthesis. Synechococcus sp. strain PCC 6301 contains two ribosomal RNA operons per genomes (rm) (19, 50) and therefore contains approximately 4 rRNA operons-cell.l at the beginning of the dark period and 6 40 rRNA operonscell" at the end of the dark period. Although the cellular l6S rRNA concentration doubles by the end of the dark period, 16S rRNA molecules per rRNA operon only increase from approximately 2100 16S rRNA molecules per operon to 2780 168 rRNA molecules per operon. The transcription rate of rRNA operons, therefore, is nearly invariant based on the ratio of 168 rRNA molecules to 168 rRNA operons within the cell. This result supports the hypothesis that rRNAs are not transcriptionally regulated and that the correlation between cellular RNA concentrations and growth rate is due to gene dosage effects. Gene dosage effects have also been postulated to account for the observed correlation between growth rate and cellular ribulose- l ,5-bisphosphate carboxylase/oxygenase concentrations (59). The implication of this hypothesis is that the growth rate is regulated or restricted by the DNA content of the cell. Cellular rRNA concentrations appear to be influenced primarily by genome copy number and gene dosage effects (50). Genome copy number and gene dosage effects may be one mechanism responsible for the expression of circadian rhythms in prokaryotes. 41 References 1. Allen, M. M. 1968. Simple conditions for growth of unicellular blue-green algae on plates. J. Phycol. 4: 1-4. 2. Armbrust, E. V., J. D. Bowen, R. J. Olson, and S. W. Chisholm. 1989. Effect of light on the cell cycle of a marine Synechococcus strain. Appl. Environ. Microbiol. 55(2):425-432. 3. Asato, Y., and C. E. Folsome. 1970. Temporal mapping of the blue-green alga, Anacystis nidulans. Genetics. 65:407-419. 4. Binder, B. J. 1997. Cell cycle regulation in marine Synechococcus sp (cyanobacteria) during light- and nitrogen-limited growth, abst. 100A, p. 100, ASLO '97 Program and Abstracts: Current and Emerging Issues in Aquatic Science. The American Society of Limnology and Oceanography Inc., Lawrence, KA. 5. Binder, B. J., and S. W. Chisholm. 1995. Cell cycle regulation in marine Synechococcus sp. Strains. Appl-Environ. Microbiol. 61(2):708-717. 6. Binder, B. J., and S. W. Chisholm. 1990. Relationship between DNA cycle and growth rate in Synechococcus sp. strain PCC 6301. J. Bacteriol. 172(5):2313-2319. 7. Bratina, B. J., M. Viebahn, and T. M. Schmidt. in press. Achieving specificity in nucleic acid hybridizations using nuclease S 1. Methods Mol. Cell. Biol. 8. Bremer, H., and G. Churchward. 1977. An examination of the Cooper- Helrnstetter theory of DNA replication in bacteria and underlying assumptions. J. Theor. Biol. 69:645-654. 9. Carr, N. G. 1973. Metabolic control and autotrophic physiology, p. 39-65. In N. G. Carr and, B. A. Whitton (ed.), The biology of Blue-green algae, vol. 9. University of California Press, Berkeley, CA. 10. Chen, T.-H., T.-L. Chen, L.-M. Hung, and T.-C. Huang. 1991. Circadian rhythm in amino acid uptake by Synechococcus RF-l. Plant. Physiol. 97 :55-59. 11. Chow, T.-J., and F. R. Tabita. 1994. Reciprocal light-dark transcriptional control of nif and rbc expression and ii ght-dependent posttranslational control of nitrogenase activity in Synechococcus sp. strain RF—l. J. Bacteriol. l76(20):6281-6285. 12. Craig, I. W., C. K. Leach, and N. G. Carr. 1969. Studies with deoxyribonucleic acid from blue-green algae. Arch. Microbiol. 65:218-227. 13. Daniels, L., R. S. Hanson, and J. A. Phillips. 1994. Chemical analysis, p. 512-554. In P. Gerhardt, R. G. E. Murray, W. A. Wood, and N. R. Krieg (eds.), Methods for General and Molecular Bacteriology. American Society for Microbiology, Washington DC. 14. Dennis, P. P., and H. Bremer. 1974. Macromolecular compostion during steady-state growth of Escherichia coli B/r. J. Bacteriol. 119:270-281. 42 15. Doi, R. H., and R. T. Igrarachi. 1964. Relation of ribonucleic acid composition to growth rate and dormancy in Bacillus subtilis. Nature. 203: 1092- 1094. 16. Doolittle, W. F. 1979. The cyanobacterial genome, its expression, and the control of that expression. p. 1-102, Advances in Microbial Physiology, vol. 20. American Society for Microbiology, Washington DC. 17. , Doolittle, W. F. 1972. Ribosomal ribonucleic acid synthesis and maturation in the blue-green alga Anacystis nidulans. J. Bacteriol. lll(2):3 16-324. 18. Doolittle, W. F., and R. A. Singer. 1974. Mutational analysis of dark endogenous metabolism in the blue-green bacterium Anacystis nidulans. J. Bacteriol. 119(3):677-683. 19. Douglas, S. E., and W. F. Doolittle. 1984. Complete nucleotide sequence of the 23S rRNA gene of the cyanobacterium, Anacystis nidulans. Nucleic Acids Res. 12(7):3373-3386. 20. Grillo, J. F., and J. Gibson. 1979. Regulation of phosphate accumulation in the unicellular cyanobacterium Synechococcus. J. Bacteriol. l40(2):508-517. 21. Grobbelaar, N., and T.-C. Huang. 1992. Effect of oxygen and temperature on the induction of a circadian nitrogenase activity rythm in Synechococcus RF-l. J. Plant Physiol. 140:391—394. 22. Hayashi, F., M. R. Ishida, and T. Kikuchl. 1969. Macromolecular synthesis in a blue-green alga, Anacystis nidulans, in dark and light phases. Ann. Repts. of Res. Reactor Inst. Kyoto Univ. 2:56-66. 23. Herdman, M., and N. G. Carr. 1974. Estimation of the genome size of blue- green algae from DNA renaturation rates. Arch. Microbiol. 99:251-254. 24. Herdman, M., B. M. Faulkner, and N. G. Carr. 1970. Synchronous growth and genome replication in the blue-green alga Anacystis nidulans. Arch. Microbiol. 73:238-249. 25. Herdman, M., M. Janvier, R. Rippka, and R. Stanier. 1979. Genome size of cyanobacteria. J. Gen. Microbiol. 111:73-85. 26. Holmes, M., M. Rickert, and O. Pierucci. 1980. Cell'division cycle of Bacillus subtilis: evidence of variability in period D. J. Bacteriol. 142:254-261. 27. Huang, T.-C., J. Tu, T.-J. Chow, and T.-H. Chen. 1990. Circadian rhythm of the prokaryote Synechococcus sp. RF-l. Plant. Physiol. 92:531-533. 28. Huang, T.-C., S.-T. Wang, and N. Grobbelaar. 1993. Circadian rhythm mutants of the prokaryotic Synechococcus RF- 1. Curr. Microbiol. 27 :249r254. 29. Ihlenfeldt, M. J. A., and J. Gibson. 1975. Phosphate utilization and alkaline phosphatase activity in Anacystis nidulans (Synechococcus). Arch. Microbiol. 102:23-28. 30. Kaplan, R., and D. Apirion. 1975. Decay of ribosomal ribonucleic acid in Escherichia coli cells starved for various nutrients. J. Biol. Chem. 250(8):3174-3178. 43 31. Kaplan, R., and D. Apirion. 1975. The fate of ribosomes in Escherichia coli cells starved for a carbon source. J. Biol. Chem. 250(5): 1854-1863. 32. Kerkhof, L., and B. B. Ward. 1993. Comparison of nucleic acid hybridization and fluorometry for measurement of the relationship between RNA/DNA ratio and grth rate in a marine bacterium. Appl. Environ. Microbiol. 59(5): 1303-1309. 33. Kjeldgaard, N. 0., and C. G. Kurland. 1963. The distribution of soluble and ribosomal RNA as a function of growth rate. J. Mol. Biol. 6:341-348. 34. Koch, A. L. 1994. Growth measurement, p. 248-277. In P. Gerhardt, R. G. E. Murray, W. A. Wood, and N. R. Krieg (ed.), Methods for General and Molecular Bacteriology. American Society for Microbiology, Washington DC. 35. Kondo, T., T. Mori, N. V. Lebedeva, S. Aoki, M. Ishiura, and S. S. Golden. 1997. Circadian rhythms in rapidly dividing cyanobacteria. Science. 275:224- 227. - 36. Kondo, T., C. A. Strayer, R. D. Kulkarni, W. Taylor, M. Ishiura, S. S. Golden, and C. H. Johnson. 1993. Circadian rhythms in prokaryotes: luciferase as a reporter of circadian gene expression in cyanobacteria. Proc. Natl. Acad. Sci. USA. 90:5672-5676. . 37. Kondo, T., N. F. Tsinoremas, S. S. Golden, C. H. Johnson, S. Kutsuna, and M. Ishiura. 1994. Circadian clock mutants of cyanobacteria. Science. 266:1233-1236. 38. Kramer, J. G. 1988. Ph. D. University of Maryland, College Park, MD. 39. Kramer, J. G., and F. L. Singleton. 1993. Measurement of rRNA variations in natural communities of microorganisms on the southeastern US. continental shelf. Appl. Environ. Microbiol. 59(8):2430-2436. 40. Kulkarni, R. D., M. R. Schaefer, and S. S. Golden. 1992. Transcriptional and posttranscriptional component of psbA response to high light intensity in Synechococcus sp. strain PCC 7942. J. Bacteriol. 174(11):3775-3781. 41 . Kumano, M., N. Tomioka, K. Shinozaki, and M. Sugiura. 1986. Analysis of the promoter region in the rrnA operon from Anacystis nidulans. Mol. Gen. Genet. 202:173-178. 42. Lau, R. H., and W. F. Doolittle. 1979. Covalently closed circular DNAs in closely related unicellular cyanobacteria. J. Bacteriol. 137 :648-652. 43. Leach, C. K., J. M. Old, and N. G. Carr. 1971. Aspects of macromolecular synthesis in the blue-green alga Anabaena variabilis. J. Gen. Microbiol. 68:xiv. 44. Mann, N., and N. G. Carr. 1973. A constant ratio of transfer to ribosomal ribonucleic acid in Anacystis nidulans grown with differing mean generation times. Biochemical Society Transactions. 1:702-704. 45. Mann, N., and N. G. Carr. 1974. Control of macromolecular composition and cell division in the blue-green alga Anacystis nidulans. J. Gen. Microbiol. 83:399-405. 44 46. Marine, G. T., and Y. Asato. 1986. Characterization of cell cycle events in the dark in Anacystis nidulans. J. Gen. Microbiol. 132:2123-2127. 47. McCarthy, B. J., and R. J. Britten. 1962. Biophysics Journal. 2:35. 48. Mori, T., B. Binder, and C. H. Johnson. 1996. Circadian gating of cell division in cyanobacteria growing with average doubling times of less than 24 hours. Proc. Natl. Acad. Sci. USA. 93:10183-10188. 49. Neidhardt, F. C., and B. Magasanik. 1960. Studies on the role of ribonucleic acid in the growth of bacteria. Biochim. Biophys. Acta. 42:99-116. 50. Nichols, J. M., I. J. Foulds, D. H. Crouch, and N. G. Carr. 1982. The diversity of cyanobacterial genomes with respect to ribosomal RNA cistrons. J. Gen. Microbiol. 128:2736-2746. 51. Nomura, M., R. Course, and G. Baughman. 1984. Regulation of the synthesis of ribosomes and ribosomal components. Ann. Rev. Biochem. 53:75-117. 52. Novitsky, J. A., and R. Y. Morita. 1976. Morphological characterization of small cells resulting from nutrient starvation of a psychophilic marine vibrio. Appl. Environ. Microbiol. 32:617-662. 53. Palfi, Z., and G. Suranyi. 1989. Heavy metal ion-induced changes of ribosomal RNA synthesis in Synechococcus sp. strain PCC 6301, a cyanobacterium. Acta Biochimia et Biophysic Hungarie. 24(4):343-354. 54. Parrott, L. M., and J. H. Slater. 1980. The DNA, RNA and protein composition of the cyanobacterium Anacytis nidulans grown in light- and carbon dioxide- limited chemostats. Arch. Microbiol. 127:53-58. 55. Rosset, R., J. Julien, and R. Monier. 1966. Ribonucleic acid composition of bacteria as a function of growth rate. J. Mol. Biol. 18:308-320. 56. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning; a laboratory manual, 2 ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 57. Simon, R. D. 1978. Survey of extrachromosomal DNA found in filamentous cyanobacteria. J. Bacteriol. 136:414-418. 58. Singer, R. A., and W. F. Doolittle. 1975. Control of gene expression in blue- green algae. Nature. 253:650-651. 59. Slater, J. H. 1975. The control of carbon dioxide assimilation and ribulose 1,5- diphosphate carboxylase activity in Anacystis nidulans grown in a light-limited chemostat. Arch. Microbiol. 103:45-49. 60. Ssymank, V., B. D. Kaushik, and H. Lorenzen. 1977. Synthesis of DNA during the cell cycle of synchronous Anacystis nidulans. Planta. 135:13-17. 61. Stahl, D., B. Flesher, H. R. Mansfield, and L. Montgomery. 1988. Use of phylogentically based hybridization probes for studies of ruminal microbial ecology. Appl. Environ. Microbiol. 54(5): 1079-1084. 45 62. Sweeney, B. M., and M. B. Brogese. 1989. A circadian rythm in cell division in a prokaryote, the cyanobacterium Synechococcus WH7 803. J. Phycol. 25: 183-186. 63. Tan, X., M. Verughese, and W. R. Widger. 1994. A light-repressed transcript found in Synechococcus PCC 7002 is similar to a chloroplast-specific small subunit ribosomal protein and to a transcription modulator protein associated with sigma 54. J. Biol. Chem. 269(33):20905-20912. 64. Utkilen, H. C. 1982. Magnesium-limited growth of the cyanobacterium Anacystis nidulans. J. Gen. Microbiol. 128: 1849-1862. 65. Utkilen, H. C., and T. Briseid. 1984. The response of Mg2+-limited Anacystis nidulans to alterations in photon fluence rate when grown in chemostats: a suitable system for studies of Mg2+-controlled cell division. J. Gen. Microbiol. 130:3079-3083. 66. van der Hondel, C. A. M. J. J., W. Keegstra, W. E. Borrias, and G. A. van Arkel. 1979. Homology of plasmids in strains of unicellular cyanobacteria. Plasmid. 2:323-333. 67. van Liere, L., L. R. Mur, C. E. Gibson, and M. Herdman. 1979. Growth and physiology of Oscillarotria agardhii Gomont cultivated in continuous culture with a light-dark cycle. Arch. Microbiol. 123:315-318. 68. Waterbury, J. B., S. W. Watson, F. W. Valois, and D. G. Franks. 1986. Biological and ecological characterization of the marine unicellular cyanobacterium Synechococcus. Can. J. Fish. Aquat. Sci. 214:71-120. Chapter 3 Concentrations of Nucleic acids of Synechococcus sp. strain WH 8103 During Growth in Continuous Light and Light:Dark Cycles 46 47 Abstract Light-limited batch cultures of Synechococcus sp. strain WH 8103 exhibited a positive correlation between specific growth rate and the cellular concentrations of both RNA and DNA. Like heterotrophic bacteria, the ratio of RNA to DNA increased with growth rate in Synechococcus sp. strain WH 8103. Cellular RNA and 16S rRNA concentrations generally decreased during light periods and increased during dark periods in cultures entrained by a die] cycle. There was a significant increase in total cellular RNA concentrations and cellular 16S rRNA concentrations during the last 3h of the dark period that may indicate an increase transcription rates. The trend in cellular DNA concentrations during a diel cycle differed from that observed in Synechococcus sp. strain PCC 6301. Cellular DNA concentrations of Synechococcus sp. strain WH 8103 increased during the first half of the dark period before decreasing to basal levels during the later half of the dark period. A similar trend was observed during the light period of a diel cycle. The variation in cellular nucleic acids concentration during diel cycles may be in part a function of the timing of cell division, in addition to transcriptional regulation. Introduction Since their discovery in marine environments, synechococci (19, 40) have proven to be both ubiquitous and abundant (15, 19, 26, 40, 41). It has also been recognized that marine synechococci are significant contributors to primary production within the world’ s oceans (16, 26, 41). There has been little investigation, however, in marine synechococci into the changes in cellular nucleic acids concentrations during diel cycles or accompanying changes in specific growth rates. Cellular nucleic acids concentrations are proportional to growth 48 rate in a number of heterotrophic bacteria (20, 21, 33) and both filamentous and unicellular cyanobacteria under constant illumination (23, 25, 28). Kramer (23) has demonstrated a correlation between total cellular RNA concentrations and specific growth rates in the marine cyanobacterium Synechococcus sp. strain WH 7803. Radiolabeled uracil incorporation demonstrated that stable RNA concentrations increased with growth rate in Synechococcus sp. strain WH 7803. The correlation between cellular 16S rRNA concentrations and growth rate presents the possibility of using probes complementary to 16S rRNA to determine in situ growth rates of marine synechococci, as has been done with sulfate-reducing bacteria in a biofilm (32). The concentration and synthesis of nucleic acids have typically been studied in cyanobacteria grown under constant illumination followed by a single dark incubation period (1, 13, 14, 17, 18, 29, 36, 38). The same is true of the incorporation studies of Kramer (23) in Synechococcus sp. strain WH 7803. Growth under constant illumination, however, may not accurately reflect the physiological state of synechococci populations growing under the diel cycles found in the environment (12). Another significant reason to study Synechococcus sp. strain WH 8103 is recent evidence by Binder and Chisholm (2) that synechococci display two distinct replication phenotypes. To date all studies of cellular RNA concentrations have been done with marine and freshwater synechococci exhibiting the asynchronous replication phenotype. These organisms contain even or odd numbers of complete genomes (eg, 3 or 4 genomesrcell"). The study of the variations in cellular RNA concentrations in Synechococcus sp. strain WH 8103 represents the first study of the variations in cellular RNA concentrations in a Synechococcus sp. exhibiting a synchronous replication phenotype (2). Organisms exhibiting a synchronous replication phenotype conform to the Helmstetter-Cooper model of genome replication (5, 9) and possess an even number of genomes. 49 In this study, we demonstrate that increases in cellular RNA concentrations and cellular DNA concentrations corelate with increases in growth rate in light-limited batch cultures of Synechococcus sp. strain WH 8103. Unlike Synechococcus sp. strain PCC 6301, the RNA/DNA ratio increased with growth rate in Synechococcus sp. strain WH 8103. It is argued that this increasing ratio is indicative of transcriptional control, primarily of the rRNA operons. The cellular concentration of total RNA and 168 rRNA in Synechococcus sp. strain WH 8103 displayed trends similar to those of Synechococcus sp. strain PCC 6301 during a diel cycle, except for an increase in last hours of the dark period that may also be indicative of up-regulation of transcription rates from the rRNA operons. The trends in cellular DNA concentrations in Synechococcus sp. strain WH 8103 during a diel cycle are also discussed in context of the trends observed in Synechococcus Sp. strain PCC 6301. Materials and Methods Bacterial strains and culture conditions. Synechococcus sp. strain WH 8103 (provided by John Waterbury, Woods Hole Oceanographic Institute, Woods Hole, MA) was maintained at RT on SN liquid media (41) at 86 [Him-28-1. Synechococcus sp. strain WH 8103 batch cultures were grown at 25°C. Growth rate was varied by changing the photon fluence rate from 15 uEm'z's.l to 53 ILEm'zs'l in light- limited batch cultures. Diel cultures were entrained on a 12 h light: 12 h dark diel cycle for . . . . -2 -1 at least seven generatlons prlor to the start of each drel expenment at 64.4 trEm S . Cell density was determined using a particle counter and channeler (Coulter Electronics Inc., Miami, FL). Cell volume was determined using a particle counter and 50 channelyzer (Coulter Electronics Inc.). Specific growth rates (11; h") of Synechococcus sp. strain WH 8103 in batch cultures were calculated from eq. 2.1. Chemical determination of RNA and DNA. Synechococcus Sp. strain WH 8103 cells were harvested by centrifugation of approximately 6x108 cells from log-phase cultures at 16,000 xg for 25 min at 4°C. Cellular RNA concentration was measured by the orcinol assay (10), using adenosine monophosphate as a standard. Cellular DNA concentration, was measured by the diphenylamine assay (10), using deoxyribose as a standard. Cellular RNA concentrations were estimated to be twice that of the AMP. Cellular DNA concentrations were calculated as 4.84 times the deoxyribose standard concentration based the difference between the average molecular weight of a nucleotide and the molecular weight of deoxyribose. . Nucleic acids isolation and hybridization. Synechococcus sp. strain WH 8103 cells were harvested by centrifugation of approximately 9x107 cells of log-phase batch culture at 16,000 xg for 25 nrin at 4°C. Cells were disrupted mechanically and nucleic acids isolated (37). RNase contamination was reduced by using either virgin polypropylene or glassware treated with diethylpyrocarbonate (Sigma Chemical Co., St. Louis, MO) and baked at 240°C for 3 h. Nucleic acids were hybridized with a cyanobacterial-specific oligonucleotide probe (4); this probe was complementary to the small subunit rRNA at positions 360-376 (Escherichia coli numbering). The cyanobacterial-specific probe was 5’ end-labeled with y—nP-ATP (34) to a specific activity of ca. 0.5 ttCi-pmol'l probe. End-labeled probes were purified on TSK-DEAE columns (Supleco, Bellefonte, PA) equilibrated with 50 mM NI-I4OAc (4). Nucleic acids from batch cultures or chemostat cultures were denatured by the addition of phosphate-buffered gluteraldehyde [pH 7.0] to a final concentration of 1.5 % 51 and incubated at RT for 15 min. Purified Synechococcus sp. strain WH 8103 168 rRNA (4) was denatured in the same manner and a Standard curve constructed from 1 ng to 75 ng of 16S rRNA. Escherichia coli (E. call) nucleic acids were denatured in the same manner and used to determine non-Specific binding of the probe. Nucleic acids from experimental samples, 16S rRNA standards and E. coli were applied to MagnaCharge nylon membranes (Micron Separation Inc., Westbourgh, MA) using a slot—blot apparatus (Millipore, Bedford, MA). Approximately 1 pg, 500 ng and 250 ng from each experimental sample and E. coli were applied to nylon membranes. Nucleic acids were UV-crosslinked (Stratalinker 1800, Stratagene Inc., La Jolla, CA) to nylon membranes according to the manufacturer’s instructions. Membranes were prehybridized for 30 min at 37°C in 10 ml hybridization buffer [6X SET (34), 0.5% SDS, 1X Denhardt’s solution (34), 100 rig-ml" poly(A)], followed by hybridization for 14-16 h at 37°C in 10 III] hybridization buffer containing 1x106 CPM«-nrl‘l 32P-radiolabeled probe. Membranes were washed twice at RT and once at 48°C for 30 min each in wash buffer [2X SET, 0.5% SDS]. Standards and samples were quantified using a radioanalytical image system (AMBIS Inc., San Diego, CA). Results Cellular RNA and DNA concentrations of light-limited batch cultures. Differential growth rates were established for light-limited batch cultures of Synechococcus sp. strain WH 8103 by varying photon fluence rates. Continuous photon fluence rates of 15 ILEm'zs'l to 53 IsEm'zs'l resulted in growth rates of 0.014 b" to 0.037 h". Cellular RNA and DNA concentrations were determined colorimetrically. Cellular RNA concentrations increased from 15611.2 fg-cell'l to 29.2il.2 fg-cellI with increases in growth rate RNA and DNA (fg-cell“) 52 10 .. 5 . + + ‘0’ ' 0 m 0.007 0.014 0.021 0.028 0.035 0.042 Specific Growth Rate (h'l) Figure 3.1 - Concentrations of nucleic acids of Synechococcus sp. strain WH 8103 from light-limited batch cultures. Error bars represent standard errors (n=3). 53 SR 83 emu me ..+. 3H 3 u Nam .H _. . ..ao.o. .. so... $2 N3 me u a: 3 n in e , as; us 83 m3 ‘ so h S: ‘ 3 u 3: age on. 82 m3 me u NS 3 h on: » Sod Assam: choose V 27525 :58 . .5 Eco . 5 _ as 0.1) increase in cellular 16S rRNA concentration compared to the chemostat supplied by a 15 11M phosphate reservoir (Fig. 4.5; Table 4.2). Based on previous results (Fig 2.2), these 16S rRNA concentrations corresponded to a growth rates of 0.016 b", 0.157 h", and 0.127 b", respectively. The increase in Synechococcus growth rate with the addition of T. pyrifarmis was tested by filtering an aliquot from the 15 11M PO4-chemostat through a 10 um filter to exclude the predator. The filtrate was added to 250 ml of 15 11M PO4-Cg medium and the growth rate of Synechococcus determined over a 2.5 h period. The growth rate, calculated from eq. 2.1, was 0.12 h". The calculated growth rate approaches the maximum Synechococcus growth rate on Cg medium (Fig. 4.1A) and confirms the increase in growth rates, due to predation, calculated from cellular l6S rRN A concentrations. Chemostat phosphate concentrations with and without predation. In the absence of predation the chemostat phosphate concentration was calculated to be 0.1 mM phosphate. This concentration increased to 8.6i0.2 mM phosphate upon introduction of the T. pyrrfarmis predator (Table 4.2). Increasing the phosphate concentration from 15 mM to 30 mM within the reservoir resulted in a corresponding increase in phosphate concentration to 21 .3:l:2.1 mM within the chemostat, indicating the increased phosphate concentration was not utilized by the cyanobacteria for growth. 84 Discussion Growth of a single trophic level. Developments in ecological theory and models have lead many to the conclusion that naturally occurring populations are limited in Size by either available nutrients or predation (32, 33, 49, 62). These two mechanisms can be mathematically modeled by coupled differential equations first proposed by Lotka and Volterra (51, 81): dN/dt = [J.N - qN- gNP (4.4) dP/dt = egNP - mP (4.5) where N and P are the prey and predator populations at time t. The term It describes the specific growth rate of the prey species. The term q describes the loss of individual organisms, which may be due to inherent mortality or efflux of the prey from the system. The predator grazing coefficient is described by g. The trophic function (eg) defines the conversion of consumed prey into predator and m the mortality rate of the predator. Based on the traditional prey-dependent models (eqs. 4.4 and 4.5), there are two possible outcomes to increases in substrate concentration: 1) The prey population (N) increases in size, 2) The prey population (N) maintains the same density while the predator population (P) increases. Which of these outcomes prevails depends upon the number of trophic interactions within the system (24, 32, 33, 49, 62). Under odd numbered trOphic levels the controlling factor in prey population density is the availability of limiting substrate. In the simplest case of a single trophic level the community consists solely of the prey population (P = 0), reducing equations 4.4 and 4.5 to: 85 dN/dt = uN- qN (4.6) Since the system is assumed to be in long term equilibrium dN/dt becomes zero (dN/dt = 0). The cellular growth rate of the individuals comprising the population is balanced by the loss of individuals from the system: Therefore, while an increase in available nutrients results in a increase in the equilibrium population size, the specific growth rate remains constant. In the absence of predation or free of predator control, bacterial populations tend to reach an equilibrium population dependent on the concentration of the limiting substrate. Such a population, typical of a food chain containing an odd number of links, is controlled by bottom-up forces. This situation is analogous to a chemostat in which the Specific growth rate is set by the flow of the limiting nutrient through the system, but population size is set by the concentration of the limiting substrate (58, 61). Before testing this hypothesis in chemostat cultures of Synechococcus Sp. strain PCC 6301 it was necessary to determine the parameters governing phosphate-limited growth of Synechococcus sp. strain PCC 6301. Synechococcus sp. strain PCC 6301 was grown in batch culture supplemented with 0.25 M to 20 M KQI-IPO4 in order to establish the maximum growth rate and K, of the organism on Cg media. The K, was 0.55 M (Fig 4.1B), in agreement with Grill and Gibson (31), and the maximum specific growth rate approached 0.13 h'1 (Fig. 4.1A). Using these parameters the phosphate concentration within phosphate-limited chemostats supporting the growth of Synechococcus sp. strain PCC 6301 was calculated. 86 The hypothesis that the concentration of available nutrients controlled population size was tested using nine Single-stage chemostats fed by reservoirs of Cg media containing 5 11M, 10 M, and 15 M phosphate. Dilution rates of approximately 0.005 h", 0.03 h", and 0.08 h‘I were established in all three chemostats. The phosphate concentration within each chemostat was calculated (eq. 4.1) to be approximately 0.02 M, 0.14 11M and 0.67 M at each dilution rate, irrespective of the phosphate concentration supplied by the reservoir (Table 4.1). As predicted for a microbial community consisting of a single or odd number of trophic levels, the community biomass increased with an increasing supply of limiting nutrient. The population size of Synechococcus sp. strain PCC 6301 increased by approximately 4x 107 cells-mll for each 5 [1M increase in phosphate supplied form the reservoir to each chemostat. The increase in population size was independent of the dilution rate of the chemostat (Figure 4.1). However, extensible properties such as cell volume and mass correlated with dilution rate (Table 4.1). Similar increases in population size have been observed in nutrient addition experiments with natural assemblages of bacterioplankton and phytoplankton. Bottle enrichments from the Widdel and Scotia seas Showed a sizable response by phytoplankton to iron additions, as measured by chlorophyll a concentrations (14). Kivi (43) observed a significant response of the phytoplankton population to NH4+ addition to in situ enclosures in the Baltic Sea. Nutrient limitation of phytoplankton population size has also been observed in a gradient of temperate lakes (56), the Mediterranean Ocean (78), and the Antarctic Ocean (15). Glover and colleagues (27) observed a bloom of synechococci concurrent with an increase in nitrate concentration, of unknown origin, in the Sargasso sea. Greene and colleagues (30) suggested that low intracellular iron concentrations limit photochemical 87 energy conversion efficiency, reducing both photosynthetic capability and growth rate. Such limitations were elegantly demonstrated in the IRONEX experiments in high-nitrate, low-chlorophyll (HNLC) waters off the coast of the Galapagos. A single in situ iron amendment Simulated a 4-fold increase in primary production, a 3-fold increase in chlorophyll a concentrations, and a 2-fold increase in phytoplankton biomass, composed predominately of synechococci (54). A similar response was observed during sustained iron inputs during IRONEX II (4). Control of population size by nutrient-limitation is not limited to photoautotrophic bacteria Pace found that heterotrophic bacteria were nutrient-limited in two lakes in Michigan (63). Billen and colleagues (5) also observed a linear correlation between increased biomass and increased bacterial production and concluded that in general assemblages of heterotrophic bacteria were nutrient-limited. However, Billen et al (5) observed that biomass and population size were not proportional over the range of environments examined because different size classes of bacteria dominate different environments. They also concluded that these nutrient-limited populations had low growth rates (0.001-0.1), comparable to those used in this study. Growth with two trophic levels. According to traditional predator-prey theory, the composition of a food chain is dramatically altered by the addition of a predator species. The most significant result of this addition is the reduction of the equilibrium prey population referred to as top-down control (24, 32, 33, 49, 62). In an even numbered food chain such as this the predator species exhibits substrate-limited growth. Increases in available nutrients are transferred up the trophic chain to the predator population without changing the prey equilibrium population size. 88 This result can be seen chemostat cultures of Synechococcus sp. Strain PCC 6301 to which a T. pyrifarmis predator had been added (Fig. 4.3). In the absence of predation Synechococcus sp. strain PCC 6301 reached an equilibrium population size of 1.4x108 cells-mll in chemostats supplied by a 15 11M phosphate reservoir (Table 4.2). The addition of T. pyrifarmis resulted in decrease of the Synechococcus sp. strain PCC 6301 prey population size to ca. 2x107 cells-ml" (Fig 4.3). The predator population reached an equilibrium population size of 25 predators-mil with a grazing rate (g) of 1.15x105 synechococci- ciliate"-h‘l (eq. 4.3). The population size of T. pyrifarmis is low compared to the greater than 1x103 ciliates-mll reported for ciliates raised on heterotrophic bacteria (10, 13, 79) and the 400 ciliates-mll for the algivorous Urotricha fitrcata (73). Although ciliate concentrations in this study were low for laboratory cultures, they were typical of those of freshwater pelagic environments (6) and lO—fold higher than typical concentrations of ciliates observed in marine pelagic environments (21). The low T. pyrifannis population was probably due to the low nutrient value of Synechococcus PCC 6301 reflected by the low 2.5x107 ciliate-cyanobacteriuml yield and a conversion efficiency of 0.4%. The yield and conversion efficiency of T. pyrifarmis raised on Synechococcus sp. strain PCC 6301 is low compared to the 2 to 54 % conversion efficiency reported for ciliates raised on heterotrophic bacteria (13, 69, 79). However, ciliates isolated from marine pelagic waters by Caron et al, exhibited cell yields that averaged 20% less when raised on synechococci compared to heterotrophic bacteria (10). The grazing rate of T. pyriformis on synechococci is approximately 200-fold higher than the grazing rate (ca. 500 vibrio-ciliate-h") reported for the smaller ciliate Uronema maimum (2). The grazing rate of T. pyrifarmis in this study is also significantly higher than the holotrichuous ciliate Euplates mutabilis, with an approximate volume of 2x105 um’, which has been reported to have grazing rates of 4.3 to 10.5x103 bacteria-ciliate"-h" 89 on a bacterial population of 5x10° to 30x10° bacteria-ml“ (79). The high grazing rates of T. pyrifarmis observed is most likely a product of the large size of T. pyrifarmis and the suspension feeding mode employed by the organism (19). A specific clearance rate of 5 ul-ciliate"-h" was calculated for each T. pyrifarmis by dividing the grazing rate of T. pyrifannis by the standing crop of cyanobacerial prey. T. pyrifarmis had an approximate body volume of 6.5x10‘ um3 (Fig. 4.4) and therefore cleared 1.05x10S body volumes-h". This high clearance rate agrees with the high clearance rates observed in pelagic ciliates (71), and indicates that ciliates are capable of supporting population growth at the very low prey densities typical of Oligotrophic systems (1x106 bacteria-ml") or on prey with low nutrient value, such as synechococci. Although, microflagellates are considered the major predator of. bacterioplankton (46), ciliates and cladocerans are also thought to contribute to bactivorous grazing, especially of cyanobacteria (10, 41, 71). Cyanobacteria with a mean cell volume of 0.5 um3 in situ are more likely to be effectively grazed by ciliates than smaller (0.07 um3) heterotrophic bacteria (19, 20, 71). Grazing coefficients of protozoan on synechococci have been estimated to range from 0 to 0.83 d" and estimated to consume 22-100% of synechococci production (8, 10). Unfortunately, prey-dependent models of predator-prey interactions could not be distinguished from ratio-dependent models of predator-prey interactions in the Synechococcus - T. pyrifarmis system used in this study because the low conversion efficiency and high grazing rate did not allow either population to respond to increases in available nutrients. Likewise, nutrients could not be lowered without the predator population going extinct from washout. 90 Macromolecular response under single and multiple trophic levels. The growth rate of the cyanobacteria prey population increased in order to maintain the population the under intense grazing pressure by the T. pyrifarmis predator population. The growth rate of the predator was governed by the dilution rate of the chemostat and ultimately by the flux of phosphate through both trophic levels. In order to maintain a prey population at equilibrium, the specific growth rate ([1) of the prey population increased in order to balance the increase in grazing pressure resulting from a larger predator population. Assuming that cellular losses (q) are negligible compared to the grazing rate of the predator population and the prey population is at equilibrium (dN/dt = 0), then the cellular growth rate of the Synechococcus prey population can be described by the equation: u=gP ms) the terms of which were defined earlier. One means of measuring changes in cellular growth rates is to monitor changes in macromolecular composition. Cellular nucleic acids concentration is proportional to growth rate in a number of heterotrophic bacteria (42, 44, 68). Nucleic acids concentration is also proportional to growth rate in both filamentous (47) and unicellular cyanobacteria (53) under constant illumination (Figure 2.1). The correlated increase in nucleic acids with increased growth rate appears to be a general physiological feature of cyanobacteria, as well as heterotrophic bacteria (42, 44, 68). The nucleic acids content of bacteria is predominately composed of rRNA and one-third of ribosomal rRNA (w/w) is composed of 16S rRN A. The correlation between cellular 16S rRNA concentrations and growth rate pemritted the use of cyanobacterial-specific probes complementary to 168 rRN A to determine in situ growth rates (Figure 2.2). 91 The increased grazing pressure imposed by introduction of T. pynformis on Synechococcus sp. strain PCC 6301 was accompanied be an increase in cellular 168 rRNA concentrations from 6.5 fg-cell'l to 27.5 fg-cell'I (Fig. 4.4). This 21 fg-cell'l increase corresponded to an increase in growth rate from 0.016 h" to 0.15 h" (Figure 2.2), and represents a roughly 85 fg-cell'l increase in total cellular RNA (52). The growth rate calculated from the cellular 16S rRNA concentration was near the maximal rate of 0.12- 0.14 h'1 (Figure 4.1A) for Synechococcus sp. strain PCC 6301 growing on Cg medium (36). The increase in specific growth rate of the Synechococcus population was also indicated by the increase in the phosphate concentration within the chemostat. The chemostat phosphate concentration increased from 0.1 |J.M to 8.6iO.2 [J.M upon the addition of the predator (Table 4.2). The 8 .6:t0.2 uM chemostat phosphate concentration was over lS-fold higher than the K, of Synechococcus sp. strain PCC 6301 (Figure 4.1B). The high chemostat phosphate concentration supports the argument that the specific growth rate of the Synechococcus population was not limited by available nutrients and was near the maximum rate for Cg medium. In order to confirm that the growth rate calculated from cellular 16S rRNA concentrations was near maximal, a subsample of the 15 M chemostat was filtered through a 10 um filter to remove the predator. The filtrate containing synechococci cells was used to inoculate Cg medium. The specific growth rate of the synechococci population was measured over a 2 h period, before any possible shift-up in growth rate could occur (53), and calculated to be 0.12 h" based on eq. 2.1 (Fig. 3.6; r2=0.989). The growth rate of autotrophic picoplankton was reported to increase from 0.08 h" and 0.2 h'1 due to protozoan grazing in the Mediten'anean Sea (21); values comparable to those observed in this chemostat. However, the authors attributed increased growth rate to 92 utilization of protozoan excretions rather than grazing. Grazing coefficients (g) of O to 0.035 h" have been reported for protozoan subsisting on synechococci along the Atlantic coast (8) ; results that are comparable to those observed in these chemostat experiments. Because the cyanobacterial population was growing near its maximum rate, any additional increase in available nutrients would not be expected to increase the population size of either the prey or the predator and consequently would not further increase the growth rate of the prey population. Increasing the available nutrient would result is an increase in the phosphate concentration within the chemostat that was unutilized by either population (77). The phosphate concentration was calculated to be 0.1 uM within chemostats containing only Synechococcus PCC 6301 and fed by a 15 M phosphate reservoir. The chemostat phosphate concentration increased to 8.6:tO.2 mM with the addition of the T. pyrifarmis predator. The chemostat phosphate concentration with Synechococcus sp. strain PCC 6301 growing near maximally can be calculated from a modification of an equation proposed by Thingstad (77), assuming no nutrient regeneration: Pc = Pr - (umN/YND) (4.9) where Pc is the chemostat phosphate concentration, Pr is the phosphate concentration of the reservoir, pm is the maximum growth rate of the synechococci, N is the synechococci population size, YN is the synechococci yield and D is the dilution rate. The phosphate concentration within the chemostat was expected to be approximately 2.1 uM based on eq. 4.9. The 6.5 uM difference in phosphate concentrations may be attributable to phosphate regeneration by T. pynformis. Increasing the reservoir phosphate concentration to 30 M 93 resulted in an increase in the chemostat phosphate concentration to 21.3i2.1 uM, indicating that the additional phosphate was unutilized by either predator or prey population because the Synechococcus prey population was already growing at um. Nutrient regeneration T. pynformis is capable of excreting or regenerating phosphate resulting from nucleic acid degradation (48). The expected concentration of phosphate within the predator—limited chemostat was 2.1 uM based on eq. 4.9. The actual phosphate concentration within the chemostat was ca. 8.6 [J.M, indicating that T. pyriformis may have been responsible for regeneration of up to 6.5 mM phosphate. Nutrient regeneration by protozoa appears to be a key component of bacterioplankton and phytoplankton growth and maintenance in pelagic systems (9, 21, 28, 35, 65). Hutchins (35), for example, demonstrated that ”Fe-labelled synechococci transferred iron to organisms >5 mm in size. The reciprocal experiment demonstrated that 5“Fe was returned to the <5 mm size fraction dominated by synechococci (35). Because not all of the available phosphate was utilized, additional organisms could have perhaps been supported in this system. The increase in available nutrients suggests that predation may led to an increase in diversity. Multilevel trophic responses Prey-dependent predator-prey models predict that the addition of a third trophic level results in the prey population (N1) being released from predator control and the 94 population once again comes under substrate limited control. Under these conditions the predator population (P2) comes under the control of its own predator (P3) and the population density remains the same over a range of increasing potential productivity or nutrient increases. A crucial difference between one and three level trophic chains is that the predator continues to graze upon the prey population in a three tiered trophic system. According to this scenario an equilibrium prey population is maintained by the cellular growth rate which is proportional to the ratio of the third trophic level population to the prey population, the term ran/eij being constant. ll, = (P3/N1Xm2P2/e12) (4-10) Because P3 is negligible compared to N1, the cellular growth rate is nearly constant across a range of increases in potential primary productivity. Similar to the ecosystem composed of a single trophic level, the cellular growth rate remains nearly constant while the prey population increases in size. The result is a “stair step” effect, where cellular growth rate remains constant while the population increases alternating with plateaus of increasing cellular growth rate and constant population size (fig. 1.2). The end result is that with the addition of each trophic level the bottom trophic level is alternately limited by available substrate or predator population, depending on whether there is an even or odd number of links in the chain (Fig. 4.6) (24, 32, 62). Examples of such a control mechanism were observed by Pace (63), who found that bacterioplankton population size was unaffected by manipulations of the top level predator, whereas the bacterioplankton predator was strongly affected by these manipulations. Protozoan and microzooplankton appear to be a link to higher trophic levels by grazing heterotrophic bacteria, cyanobacteria and eucaryotic algae (64, 71). The rate of protozoan predation of bacteria is reportedly sufficient to balance net bacterial production 95 and limit bacterial population size (86). Protozoan grazing is believed to strongly influence bacterial community composition ( 18). However, the transfer of microbial biomass to higher trophic levels remains equivocal (17, 72). This research demonstrates that group-specific probes complementary to 168 rRNA can be used to determine whether a particular population is controlled primarily by available resources (bottom-up control) or by predation (top-down control). 96 References 1. Allen, M. M. 1968. Simple conditions for growth of unicellular blue-green algae on plates. J. Phycol. 4:1-4. 2. Ashby, R. E. 1976. Long term variations in a protozoan chemostat culture. J. Exp. Mar. Biol. Ecol. 24:227-235. 3. Azam, F. and B. C. Cho. 1987. Bacterial utilization of organic matter in the sea, p. 261-281. In Fletcher (ed.), Ecology of Microbial Communities. Cambridge University Press, Cambridge, MA. 4. Behrenfeld, M. J., A. J. Bale, Z. S. Kolber, J. Aiken, and P. G. Falkowski. 1996. Confirmation of iron limitation of phytoplankton photosynthesis in the equatorial Pacific Ocean. Nature. 383:508-511. 5. Billen, G., P. Servais, and S. Becquevort. 1990. Dynamics of bacterioplankton in Oligotrophic and eutrophic aquatic environments: bottom-up or top— down control? Hydrobiologia. 207 :37-42. 6. Bloem, J. and M.-J. B. Bar-Gilissen. 1989. Bacterial activity and protooan grazing potential in a stratified lake. Limnol. Oceanogr. 34(2):297-309. 7. Bratina, B. J., M. Viebahn, and T. M. Schmidt. in press. Achieving specificity in nucleic acid hybridizations using nuclease 81. Methods Mol. Cell. Biol. 8. Campbell, L. and E. J. Carpenter. 1986. Estimating the grazing pressure of heterotrophic nanoplankton on Synechococcus spp. using the sea water dilution and selective inhibitor techniques. Mar. Ecol. Prog. 33:121-129. 9. Caron, D. A., J. C. Goldman, O. K. Andersen, and M. R. Bennett. 1985. Nuuient cycling in a microflagellate food chain. Mar. Ecol. Prog. 24:243-254. 10. Caron, D. A., E. L. Lim, G. Miceli, and J. B. Waterbury. 1991. Grazing and utlization of chroocoid cyanobacteria and heterotrophic bacteria by protozoa in laboratory cutlures and a coastal plankton community. Mar. Ecol. Prog. 76:205-217. 11. Coale, K. H. 1991. Effects of iron, manganese, copper, and zinc enrichments on productivity and biomass in the subarctic Pacific. Limnol. Oceanogr. 36(8): 1851-1864. 12. Coale, K. H., S. E. Fitzwater, R. M. Gordon, K. S. Johnson, and R. T. Barber. 1996. Control of community growth and export production by upwelled iron in the equatorial Pacific Ocean. Nature. 379:621-624. 13. Curtis, C. R. and A. Cockbum. 1971. Continuous monoxenic culture of Tetrahymena pyriformis. J. Gen. Microbiol. 66:95-108. l4. deBaar, H. J. W., A. G. J. Buma, R. F. Nolting, G. C. Cadee, G. Jacques, and P. J. Tr.eguer 1990. On iron limitation of the Southern Ocean. experimental observations in the Weddell and Scotia Seas. Marine Ecology Progress Series. 65:105-122. 97 15. deBaar, H. J. W., J. T. M. deJong, D. C. E. Bakker, B. M. Loscher, C. Veth, U. Bathmann, and V. Smetacek. 1995. Importance of iron for plankton blooms and carbon dioxide drawdown in the Southern Ocean. Nature. 373:412-415. 16. Ducklow, H. W. 1983. Production and fate of bacteria in the oceans. BioScience. 33(8):494-501. 17. Ducklow, H. W., D. A. Purdie, P. J. L. Williams, and J. M. Davies. 1986. Bacterioplankton: a sink for carbon in a coastal marine plankton community. Science. 232:865-867. 18. Epstein, S. S. and M. P. Shiaris. 1992. Size-selective grazing of coastal bacterioplankton by naturatl assemblages of pigmented flagellates, colorless flagellates, and ciliates. Microb. Ecol. 23:21 1-225. 19. Fenchel, T. 1980. Suspension feeding in ciliated protozoa: feeding rates and their ecological significance. Microb. Ecol. 6:13-25. 20. Fenchel, T. 1980. Suspension feeding in ciliated protozoa: functional response and particle size selection. Microb. Ecol. 6: 1-1 1. 21. Ferrier-Pages, C. and F. Rassoulzadegan. 1994. Seasonal impact of the microzooplankton on pico- and nanoplankton growth rates in the northwest Mediteranean Sea. Mar. Ecol. Prog. 108:283-294. 22. Fiske, C. H. and Y. Subbarow. 1925. The colorimetric determination of phosphorus. J. Biol. Chem. 66:375. 23. Fogg, G. E. 1986. Picoplankton. Proc. R. Soc. Lond. 228:1-30. 24. Fretwell, S. 1977. The regulation of plant communities by the food chains exploiting them. Perspectives in Biology and Medicine. Winter: 169-185. 25. Frost, B. W. 1996. Phytoplankton bloom on iron rations. Nature. 383:475-476. 26. Glover, H. E., L. Campbell, and B. B. Prezelin. 1986. Contribution of Synechococcus spp. to size-fractioned primary productivity in three water masses in the Northwest Atlantic Ocean. Mar. Biol. 91:193-203. 27. Glover, H. E., B. B. Prezelin, L. Campbell, M. Wyman, and C . Garside. 1988. A nitrate-dependent Synechococcus bloom in surface Sargasso Sea water. Nature. 331:161-163. 28. Goldman, J. G., D. A. Caron, and M. R. Dennett. 1987. Nutrient cycling in a microflagettate food chain: IV. Phytoplankton-microflagellate interactions. Mar. Ecol. Prog. 38:75-87. 29. Greenberg, A. E. 1995. Standard Methods for the Examination of Water and Wastewater, 19th ed. American Public Health Association Publications, New York, NY. 30. Greene, R. M., Z. S. Kolber, D. G. Swift, N. W. Tindale, and P. G . Falkowski. 1994. Physiological limitation of phytoplankton photosynthesis in the eastern equatorial Pacific determined from variablity in the quantum yeild of fluorescene. Limnol. Oceanogr. 39(5):1061-1074. 98 31. Grillo, J. F. and J. Gibson. 1979. Regulation of phosphate accumulation in the unicellular cyanobacterium Synechococcus. J. Bacteriol. 140(2):508-517. 32. Hairston, N. G., F. E. Smith, and L. B. Slobodkin. 1960. Community structure, population control and competition. The American Naturalist. 154(879):421- 425. 33. Hansson, L.-A. 1992. The role of food chain composition and nutrient availability in shaping algal biomass development. Ecology. 73(1):241-247. 34. Hennes, K. P., C. A. Suttle, and A. M. Chan. 1995. Fluorescently labeled virus probes show that natural virus populations can control the structure of marine microbial communities. Appl. Environ. Microbiol. 61(10):3623-3627. 35. Hutchins, D. A., G. R. DiTullio, and K. W. Bruland. 1993. Iron and regenerated production: evidence for biological iron recycling in two marine environments. Limnol. Oceanogr. 38(6): 1242-1255. 36. Ihlenfeldt, M. J. A. and J. Gibson. 1975. C02 fixation and its regulation in Anacystis nidulans (Synechococcus). Arch. Microbiol. 102:13-21. 37. Ihlenfeldt, M. J. A. and J. Gibson. 1975. Phosphate utilization and alkaline phosphatase activity in Anacystis nidulans (Synechococcus). Arch. Microbiol. 102:23-28. 38. Ikemoto, H. and A. Mitsui. 1994. Diazotrophic synchronous growth of a marine unicellular cyanobacterium, Synechococcus sp. strain Miami BG 043511, under aerobic and microaerobic/anaerobic conditions. Anal. Biochem. 140:2153-2158. 39. Iturriaga, R. and B. G. Mitchell. 1986. Chroococcoid cyanobacteria: a significant component in the food web dynamics of the open ocean. Mar. Ecol. Prog. 28:291-297. 40. Jiang, S. C. and J. H. Paul. 1994. Seasonal and diel abundance of viruses and occurrence of lysogeny/bacteriocinogeny in the marine environment. Mar. Ecol. Prog. 104: 163-172. 41. Johnson, P. W., H.-s. Xu, and J. M. Sieburth. 1982. The utilization of Chroococcoid cyanobacteria by marine protozooplankters but not by calanoid capepods. Annales de Institue de Oceanographic. 58(S):297-308. 42. Kerkhof, L. and B. B. Ward. 1993. Comparison of nucleic acid hybridization and fluorometry for measurement of the relationship between RNA/DN A ratio and growth rate in a marine bacterium. Appl. Environ. Microbiol. 59(5):1303-1309. 43. Kivi, K., S. Kaitala, H. Kuosa, J. Kuparinen, E. Leskinen, R. Lignell, B. Marcussen, and T. Tamminen. 1993. Nutrient limitation and grazing control of the Baltic plankton community during annual succession. Linmol. Oceanogr. 38(5):893-905. 44. Kjeldgaard, N. O. and C. G. Kurland. 1963. The distribution of soluble and ribosomal RNA as a function of growth rate. J. Mol. Biol. 6:341-348. 99 45. Koch, A. L. 1994. Growth measurement, p. 248-277. In P. Gerhardt, R. G. E. Murray, W. A. Wood, and N. R. Krieg (ed.), Methods for General and Molecular Bacteriology. American Society for Microbiology, Washington DC. 46. Kuuppo-Leinikki, P., R. Autio, S. Hallfors, H. Kuosa, J. Kuparinen, and R. Pajuniemi. 1994. Trophic interactions and carbon flow between picoplankton and protozoa in pelagic enclosures manipulated with nutrients and a top predator. Mar. Ecol. Prog. 107:89-102. 47. Leach, C. K., J. M. Old, and N. G. Carr. 1971. Aspects of macromolecular synthesis in the blue-green alga Anabaena variabilis. J. Gen. Microbiol. 68:xiv. 48. Leboy, P. S., S. G. Cline, and R. L. Conner. 1964. Phosphate, purines and pyrimidines as excretory products of Tetrahymena. J. Protozool. 11(2):217-222. 49. Leibold, 'M. A. 1989. Resource edibility and the effects of predators and productivity on the outcome of trophic interactions. The American Naturalist. 134(6):922- 949. 50. Li, W. K. W. 1994. Primary production of prochlorophytes, cyanobacteria, and eucaryotic ultraphytoplankton: measurements from flow cytometric sorting. Limnol. Oceanogr. 39(1):169-175. 51. Lotka, A. J. 1925. Elements of physical biology. Williams and Wilkins, Baltimore, NH). 52. Mann, N. and N. G. Carr. 1973. A constant ratio of transfer to ribosomal ribonucleic acid in Anacystis nidulans grown with differing mean generation times. Biochemical Society Transactions. 1:702-704. 53. Mann, N. and N. G. Carr. 1974. Control of macromolecular composition and cell division in the blue-green alga Anacystis nidulans. J. Gen. Microbiol. 83:399-405. 54. Martin, J. H., K. H. Coale, K. S. Johnson, S. E. Fitzwater, R. M. Gordon, S. J. Tanner, C. N. Hunter, V. A. Elrod, J. L. Nowicki, T. L. Coley, R. T. Barber, S. Lindley, A. J. Watson, K. V. Scoy, C. S. Law, M. I. Liddicoat, R. Ling, T. Stanton, J. Stockel, C. Collins, A. Anderson, R. Bidigare, M. Ondrusek, M. Latasa, F. J. Millero, K. Lee, W. Yao, J. Z. Zhang, G. Friederich, C. Sakamoto, R. Chavez, K. Buck, Z. Kolber, R. Greene, P. Falkowski, S. W. Chisholm, F. Hoge, 'R. Swift, J. Yungel, S. Turner, P. Nightingale, A. Hatton, P. Liss, and N. W. Tindale. 1994. Testing the iron hypothesis in ecosystems of the equatorial Pacific Ocean. Nature. 371:123-129. 55. Martin, J. H., R. M. Gordon, and S. E. Fitzwater. 1991. The case for iron. Limnol. Oceanogr. 36(8): 1793-1802. 56. Mazumder, A. 1994. Patterns of algal biomass in dominant odd- vs. even-link lake ecosystems. Ecology. 75(4): 1 141-1 149. 57. Mitchell, B. G., E. A. Brody, O. Holm-Hansen, C. McClain, and J. Bishop. 1991. Light limitation of phytoplankton biomass and macronutrient utilization in the Southern Ocean. Limnol. Oceanogr. 36(8): 1662-1677. 100 58. Monod, J. 1950. The growth of bacterial cultures. Ann. Inst. Pasteur Paris. 79:390-410. . 59. Morel, F. M. M., R. J. M. Hudson, and N. M. Price. 1991. Limitation of productivity by trace metals in the sea. Limnol. Oceanogr. 36(8):1742-1755. 60. Moss, B., S. McGowan, and L. Carvalho. 1994. Determination of phytoplankton crops by top-down and bottom-up mechanisms in a group of English lakes, the West Midland meres. Limnol. Oceanogr. 39(5): 1020-1029. 61. Novick, A. and L. Szilard. 1950. Experiments with the cheomstat on spontaneous mutations of bacteria. Proc. Natl. Acad. Sci. USA. 36:708-714. 62. Oksanen, L., S. D. Fretwell, J. Arruda, and P. Niemela. 1981. Exploitation ecosystems in gradients of primary productivity. The American Naturalist. 118(2):240-261. 63. Pace, M. L. and E. Funke. 1991. Regulation of planktonic microbial communities by nutrients and herbivores. Ecology. 72(3):904-914. 64. Pace, M. L., G. B. McManus, and S. E. G. Findlay. 1990. Planktonic community structure determines the fate of bacterial production in a terrnperate lake. Limnol. Oceanogr. 35(4):?95-808. 65. Pakulski, J. D., R. Benner, R. Amon, B. Eadie, and T. Whitledge. 1995. Community metabolism and nutrient cycling in the Mississippi River plume: evidence for intense nitrification at intermediate salinities. Mairne Ecology Progress Series. 117:193-206. 66. Pedros-Alio, C. (ed.). Toward an autecology of bacterioplankton. Springer- Verlag, New York. 67. Pomeroy, L. R. 1974. The ocean's food web: a changing paradigm. BioScience. 24:499-503. 68. Rosset, R., J. Julien, and R. Monier. 1966. Ribonucleic acid composition of bacteria as a function of growth rate. J. Mol. Biol. 18:308-320. 69. Rubin, H. A. and J. J. Lee. 1976. Informational energy flow as an aspect of the ecological efficiency of marine ciliates. J. Theor. Biol. 62:69-91. 70. Sambrook, J ., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning; a laboratory manual, 2 ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 71. Sherr, B. F. and E. B. Sherr. 1987. High rates of consumption of bacteria by pelagic ciliates. Nature. 325:710-711. 72. Sherr, B. F., E. B. Sherr, and L. J. Albright. 1987. Bacteria: link or sink? Science. 235:88. 73. Simek, K., J. Vrba, J. Pernthaler, T. Posch, P. Hartman, J. Nedoma, and R. Psenner. 1997. Morphological and compositional shifts in an experimental bacterial community influenced by protists with contrasting feeding modes. Appl. Environ. Microbiol. 63(2):587-595. 101 74. Stahl, D., B. Flesher, H. R. Mansfield, and L. Montgomery. 1988. Use of phylogentically based hybridization probes for studies of ruminal microbial ecology. Appl. Environ. Microbiol. 54(5):1079-1084. 75. Strathmann, R. R. 1967. Estimating the organic carbon content of phytoplankton from cell volume or plasma volume. Limnol. Oceanogr. 12:411-418. 76. Suttle, C. A., A. M. Chan, and M. T. Cottrell. 1990. Infection of phytoplankton by viruses and reduction of primary productivity. Nature. 347 :467-469. 77. Thingstad, T. F. and B. Pengerud. 1985. Fate and effect of allochthonous organic material in aquatic microbial ecosystems. An analysis based on chemostat theory. Mar. Ecol. Prog. 21:47-62. 78. Thingstad, T. F. and F. Rassoulzdegan. 1995. Nutrient limitations, microbial food webs, and 'biological C-pumps': suggested interactions in a P-lirnited Mediterranean. Mar. Ecol. Prog. 117:299-306. 79. Turley, C. M., R. C. Newell, and D. B. Robins. 1986. Survival strategies of two small marine ciliates and their role in regulating bacterial community structure under experimental conditions. Mar. Ecol. Prog. 33:59-70. 80. Vaulot, D., N. LeBot, D. Marie, and E. Fukai. 1996. Effect of phosphorus on the Synechococcus cell cycle in surface Mediterranean waters during summer. Appl. Environ. Microbiol. 62(7):2527-2533. 81. Volterra, V. 1931. Variations and fluctuations of the number of individuals in animal species living together. Arno, New York, NY. 82. Waterbury, J. B. and F. W. Valois. 1993. Resistance to co-occurring phages enables marine Synechococcus communities to coexist with cyanophages abundant in seawater. Appl. Environ. Microbiol. S9(10):3393-3399. 83. Waterbury, J. B., S. W. Watson, F. W. Valois, and D. G. Franks. 1986. Biological and ecological characterization of the marine unicellular cyanobacterium Synechococcus. Can. J. Fish. Aquat. Sci. 214:71-120. 84. Watson, 8., E. McCauley, and J. S. Downing. 1992. Sigmoid relationships between phosphorus, algal biomass, and algal community structure. Can. J. Fish. Aquat. Sci. 49:2605-2610. 85. Wootton, J. T. and M. E. Power. 1993. Productivity, consumers, and the structure of a river food chain. Proc. Natl. Acad. Sci. USA. 90: 1384-1387. 86. Wright, R. T. and R. B. Coffin. 1984. Factors affecting bacterioplankton density and productivity in salt marsh estuaries, p. 485-494. In M. J. Klug and C. A. Reddy (ed.), Current Perspectives in Microbial Ecology. American Society for Microbiology, Washington, D. C. Chapter 5 Evidence of predator-limited Synechococcus population size in the Gulf of Mexico based on indirect estimates of differences in growth rate in relation to population density 102 103 Abstract Synechococcus is a significant contributor to global CO2 fixation and often numerically dominates the phytoplankton component of a pelagic community. Predator- prey models predict that a nutrient-limited population will be larger and have slower specific growth rates than a population limited by predation. Since macromolecular composition is correlated with growth rate in synecococci it is possible to use specific oligonucleotide probes complementary to 16$ rRN A to determine the relative differences in cellular growth rate between two populations. Synechococci population size and specific growth rates were examined at a number of Oligotrophic and eutrophic stations in the Gulf of Mexico over a four year period. The distribution of population size versus specific growth conformed more closely to the predictions of simple predator-prey models than to those of ratio-dependent models. In addition, a significant portion of the synechococci examined appeared to be limited in population size by predation, in agreement with the reports of heavy grazing of pelagic synechococci populations. Introduction Cyanobacteria of the genus Synechococcus contribute from 2 to 46% of total primary productivity in coastal and oceanic waters (9, 23, 53, 42). Synechococcus population sizes range from 1x103 cells ml" to 1x10‘5 cells ml" within the euphotic zone (6, 9, 5, 53, 63, 62). Beyond mere abundance, the ubiquitous nature of Synechococcus is evident from counts by epifluoresence microscopy of samples from the southern Pacific (62), the subartic Pacific (11), the tropical Atlantic (63, 9, 62) and the northern Atlantic (23, 6). Cloned 16S rRNA sequences have revealed Synechococcus spp. to be abundant in both the Pacific (56) and the Atlantic (19); results that are supported by flow cytometry 104 (42). Due to its high abundance and ubiquity, Synechococcus is undoubtedly an important contributor to the global carbon cycle. The Louisiana-Texas shelf ecosystem is one region where anthropogenic inputs may have a profound effect on Synechococcus population size and dynamics. Nutrient loading from the Mississippi River into estuarine and coastal ocean environments have increased dramatically over the past half century. For example, nitrate concentrations in the lower Mississippi and carbon accumulation on the adjacent shelf have doubled since 1950 (47 ). A consequences of this nutrient-enhanced production is increased sedimentation of organic matter resulting in hypoxic zones in benthic environments (43). Despite the significant contribution of Synechococcus to total primary production and marine community structure, little is known about the mechanisms controlling their population size. An understanding of the mechanisms which control Synechococcus populations is the first step in determining the rate of energy transfer to other trophic levels (23), micronutrient recycling, the role of synechococci in determining microzooplankton composition (7 , 21) and the construction of future models of trOphic interactions. Synechococcus spp. may at times be limited for light (37), trace metals (11, 12, 38), in particular Fe2+ (4, 15, 18, 35, 36), nitrogen (17, 39), and phosphorus (23, 25, 48, 52). However, the evidence for nutrient-limitation of Synechococcus is far from conclusive and does not preclude the possibility of predator control. In situ grazing coefficients for flagellates and ciliates specific to Synechococcus range from 0 to 0.2 h.] (9, 13, 23, 25) and can reduce Synechococcus production by 30 to 100% (7, 9). Predator- prey models are capable of distinguishing nutrient-limitation from predator-limitation by plotting equilibrium Synechococcus population density and the specific growth rate (Figure 1.2). One means of measuring changes in cellular growth rates is to monitor changes in macromolecular composition. Nucleic acids concentration is correlated to growth rate in both heterotrophic bacteria (22, 24, 44) and cyanobacteria (29, 34). The correlation between cellular 16S rRNA concentrations and growth rate permits the use of 105 cyanobacterial-specific probes complementary to 16S rRNA to determine in situ growth rates (Chapters 1 and 2). To date, most investigations of microbial predator-prey interactions have been done on entire communities of heterotrophic bacteria. The trophic interactions of Synechococcus spp. is essential to understanding pelagic food webs (7). This work was designed to address the predator-prey interactions within a restricted group, the synechococci, and to demonstrate that 16S rRNA targetted probes can be used to distinguish top-down from bottom-up control for any microbial group for which a specific probe can be designed. Materials and Methods Sampling sites and protocol. Synechococcus spp. populations were sampled at 12 stations in the Gulf of Mexico off the coast of Louisiana (Figure 5.1). The decrease in salinity is indicative of the influence of the nutrient rich waters of the Mississippi River (65). Synechococci can be enumerated using epifluorescent counts following separation from other phycoerythrin containing organisms by size fractation of seawater samples. Seawater was initially collected, from a l m depth, through a 120 um nylon mesh using a pneumatic pump (Wilden Pump & Engineering Co., Colton, CA) operating at less than 15 psi. The collected seawater was prefiltered through a 10 um filter (Millipore Corp., Bedford, MA) before passing through a 1 pm filter (Micron Separations Inc., Westboro, MA). Aliquots (5 to 20 ml) of the final filtrate were concentrated on 0.22 pm black polycarbonate filters (Poretics Corp., Liverrnore, CA) for enumeration of the autofluorescent synechococci. Utilizing the autofluorescent properties of the Figure 5.1 - Sampling sites within the Gulf of Mexico. 107 photosynthetic pigments, the Synechococcus population size was determined by direct epifluorescent counts of 300-400 cells distributed among 20 to 30 random fields using a Zeiss 48.77.09 filter set . Synechococci cells from 5 to 25 L of 1 pm filtrate were collected onto either 0.22 mm, 113 mm dia. polycarbonate membranes filter (Millipore Corp., Bedford, MA) at <15 psi or onto 0.22 pm Gelrnan filtration cartridges (Gelrnan Sciences, Ann Arbor, MI). Polycarbonate filters or filter cartridges were sealed in either zip-lock bags or with parafilrn, immediately frozen and kept at -80°C until extracted. Station Tlf was sampled every 5 m to a depth of 80 m using a Niskin rosette sampler. Aliquots from station T If enumeration and nucleic acid isolation were not prefiltered through a 120 um mesh. Estimated rates of CO2 fixation. Surface seawater samples were collected as described above. Triplicate 250 ml light and dark samples of 120 um, 10 um and 1 urn filtrates were spiked with 5-20 uCi NaH["C]O3 (DuPont NEN, Boston, MA). Samples were incubated on board for 4-5 h at ca. 800 tlEm'zs'I and 20°C. Net primary productivity of the 120 um, 10 um and 1 pm filtrates were calculated from the change in cellular ”C relative to 1‘C incorporation from seawater (42). Estimated specific growth rate and grazing coefficients by dilution culturing. Seawater samples, from a 1 m depth, were passed through a 120 um nylon mesh, as described above. Several liters of the 120 um filtrate was filtered through a 0.2 pm filter (Micron Separations Inc., Westboro, MA) to remove microorganisms. Aliquots of the 120 108 um filtrate were mixed with the 0.2 pm filtrate in ratios of 1:4, 1:10 or left undiluted. Each aliquot was divideded into triplicate 500 ml subsamples. Samples were incubated for 24 h on deck in ambient light and cooled by running seawater. Synechococci cell density was determined by collection of 5 to 15 ml of each dilution subsample on black 0.22 pm polycarbonate filters and enumeration as described above. The rate of increase of synechococci population size for each dilution was calculated using the formula: r = u-g = (In N, - 1n N0)/I‘ (5.2) where u is specific growth rate, g is the mortality rate (due primarily to grazing), N0 is the population size at time 0 and N, is the population size at time T. Growth and grazing coefficients were determined by linear regression of the rate of increase in synechococci population size r at each dilution. Specific growth rate (k) was determined from the y- intercept and the grazing coefficient was equivalent to the slope of the regression. Nucleic acid isolation and probing. Nucleic acids were isolated from 113 um Millipore filters by adding 5 ml lysis buffer (40 mM EDTA [pH 8.0], 400 mM NaCl, 0.75 M Sucrose, 50 mM Tris-HCl [pH 8.0]) and 1 mg-r'nl'l lysozyme (Sigma Chemical Co., St. Louis, MO) and incubating for 20 min at 37°C. SDS (Sigma Chemical Co., St. Louis, MO) was added to 1% and 100 mg-ml’l Proteinase K (Sigma Chemical Co., St. Louis, MO) followed by a second 20 min incubation at 37°C. Filters were then incubated at 55°C for 10 min and gently agitated. The lysate was removed and the filters washed with 2 ml TE (10 mM Tris-HCl [pH 8.0], 1 mM EDTA [pH 8.0]) and combined with the lysate. Lysates where extracted with an equal 109 volume of phenol (0.1 M Tris-HCl [pH 8.0] saturated), followed by a phenolzchloroform extraction and a chloroform extraction. Nucleic acids were precipitated by the addition of 0.1 volume 3 M sodium acetate (Sigma Chemical Co., St. Louis, MO), 3 volumes 100% EtOH and centrifugation at 16,000 xg for 2 h. Nucleic acids were isolated from 0.22 um Gelrnan filtration cartridges (Gelrnan Sciences, Ann Arbor, MI) by sealing the ends of the cartridge with size 000 rubber stoppers, Duraseal (DiversifieDBiotech, Boston, MA) and teflon clamps (Cole-Farmer, Vernon Hills, IL). A 60 ml syringe and gasket was used to force 10 ml 1X SSC (cold) through the outlet port. The 1X SSC filtrate was collected through the cartridge air vent. Another 10 ml 1X SSC (cold) containing 1% SDS was added through the cartridge air vent and the cartridge incubated at 90°C for 30 min in a hybridization oven (Hoefer Scientific Inst., SF, CA). The lysate was collected through the air vent and combined with the previous filtrate. Another 10 ml 1X SSC was forced through the outlet and combined with the previous lysates. Lysates where extracted with an equal volume of phenol (0.1 M T ris-HCl [pH 8.0] saturated), followed by a phenolzchloroform extraction and a chloroform extraction. Nucleic acids were precipitated by the addition of 0.1 volume 3 M sodium acetate (Sigma Chemical Co., St. Louis, MO), 3 volumes 100% EtOH and centrifugation at 16,000 xg for 2 h. RNase contamination was reduced by using either virgin polypropylene or glassware treated with diethylpyrocarbonate (Sigma Chemical Co., St. Louis, MO) and baked at 240°C for 3 h. Nucleic acids were hybridized with a cyanobacterial-specific oligonucleotide probe (5); this probe was complementary to the small subunit rRNA at positions 360-376 (Escherichia coli numbering). The cyanobacterial-specific probe was 5’ end-labeled with g-32P-ATP y to a specific activity of ca. 0.5 mCi-pmol'l probe. End-labeled probes were 110 purified on TSK-DEAE columns (Supleco, Bellefonte, PA) equilibrated with 50 mM NH,OAc (5). Nucleic acids from batch cultures or chemostat cultures were denatured by the addition of phosphate-buffered gluteraldehyde [pH 7.0] to a final concentration of 1.5% and incubated at RT for 15 min. Purified Synechococcus sp. strain PCC 6301 16S rRNA (5) was denatured in the same manner and a standard curve constructed from 1 ng to 75 ng of 16S rRNA. Escherichia coli (E. coli) nucleic acids were denatured in the same manner and used to determine non-specific binding of the probe. Nucleic acids from experimental samples, 168 rRNA standards and E. coli were applied to MagnaCharge nylon membranes (Micron Separation Inc., Westbourgh, MA) using a slot-blot apparatus (Millipore, Bedford, MA). Approximately 1 mg, 500 ng and 250 ng from each experimental sample and E. coli were applied to nylon membranes. Nucleic acids were UV-crosslinked (Stratalinker 1800, Stratagene Inc., La Jolla, CA) to nylon membranes according to the manufacturer’s instructions. Membranes were prehybridized for 30 min at 37°C in 10 m1 hybridization buffer [6X SET (45), 0.5% SDS, 1X Denhardt’s solution (45), 100 mg-ml" poly(A)], followed by hybridization for 14-16 h at 37°C in 10 ml hybridization buffer containing 1x10° CPM-ml" 32P-radiolabeled probe. Membranes were washed twice at RT and once at 48°C for 30 min each in wash buffer [2X SET, 0.5% SDS]. Standards and samples were quantified using a radioanalytical image system (AMBIS Inc., San Diego, CA). Results and Discussion Synechococci population size along nutrient gradients Synechococci population density was determined by. epifluorescent counts of surface water samples (<3rn) taken along a nutrient gradient from the Gulf of Mexico (Figure 5.1). Synechococcus cell densities ranged from 2x102 cells-1111'l to 2x105 cells-ml" 111 (Tables 5.1-5.4). Synechococci cell densities in the surface waters of the Gulf of Mexico are comparable to those of previous reports (10, 16, 31, 32, 48-50). The 30 fold range in population densities in the Gulf of Mexico is comparable to both the seasonal range in cell densities observed in Woods Hole Harbor and along a 5400 mi north-south transect along the eastern coast of the Americas (50). It has been proposed that Synechococcus spp. are at times variously limited for light (37), trace metals (11, 12, 38), in particular Fe2+ (4, 15, 18, 35, 36), nitrogen (17, 39), and phosphorus (23, 25, 48, 52). The decrease in light transmittance moving from Oligotrophic stations, such as station 3, to the eutrophic stations heavily influenced by drainage from the Mississippi, such as station le, indicate that light is attenuated by silt and cell biomass from the Mississippi River. However, light attenuation does not appear to be a factor limiting synechococci population size since no correlation between percent transmittance and synechococci cell density was observed, results consistent with the findings of Lohrenz et al (33). Lorenz et al (33) were unable to find a correlation between the rate of carbon fixation in 10 pm seawater filterates and either salinity or in situ nitrate concentrations. Percent transmittance may also be used as an indicator of nutrient loading (6). The lack of correlation between percent transmittance and synechococci cell density is also an indication that synechococci population size is not nutrient-limited, a result consistent with the lack of conelation between primary production and nutrient loading noted by Lohrenz (33). Rates of carbon fixation It is commonly assumed that the specific growth rate of cyanobacteria is portional its rate of carbon fixation; and at least one method of determing specific growth rates of synechococci has been developed based on this assumption (50). However, recent evidence indicates that slow growing mats of cyanobacteria are capable of high rates of 112 H oooonomomquo \DHQ‘OVfl'Nmm r—l HH .82 .: configoz 8 ea c3800 c8 bacon 5:233 Eoeoonooiw n m 025,—. 113 Table 5.3 - Synechococci population density for November 19, 1994 to November 22, 1994. :fi ' rte ‘ Percent 'SyneChococci 16S rRNA Transmittance (10‘ cell-ml 1) (fg-cell") j 3 92.5 0.36 3.910.2 33 5 43.6 1.39 ll.4:tO.3 8 55.0 . 1.23 l9.5i2.9 Table 5.4 - Synechococci population density for February 16, 1996 to February 21, 1996. Percent 16S rRNA Transmittance (fg-cell") 0.2 0.0 1.5 12.7i1.1 36.0 4.1i0.8 45.3 12.5i1.4 35.3 1.610.2 4O . 1 4 . 1:0 . 8 56.5 1.0iO.l 62.0 0.1iO.1 62.3 0.8i0.l 114 carbon fixation in the absence of high growth rates (40). This assumption was tested by measuring rates of carbon fixation attributable to synechococci and attempting to correlate these rates with cellular l6S rRNA concentrations, which has been conelated with growth rate (Chapters 1 and 2). The maximum photosynthetic capacity (Pm) of Synechococcus spp. was determined by shipboard incubation at 25°C and ca. 800 mEm'zs'l in fall 1993, spring 1994, and fall 1994. Integrated sea-surface irradiance averaged 935 mEm'zs" from November 19 to November 20, 1993. Since mean surface irradiance was comparable to shipboard incubation irradiance, Pmax measurement are believed to be an accurate estimate of in situ photosynthetic rates. PM varied from 0.2 fg-cell" to 205.3 fg-cell’l (Tables 5.1- 5.4), rates comparable to in situ measurements from previous reports (16, 31, 50), further supporting the position that Pmax estimates were an accurate reflection of in situ surface photosynthetic rates by synechococci. There was no discernible correlation between photosynthetic rates and nutrient loading of a particular sampling site, in agreement with the findings of Lohrenz (33). Estimated growth rates by dilution culturing In the fall of 1994 synechococci growth rates were determined at stations 3, 5 and 8, using the dilution method of Hasset and Landry (27, 28). Synechococcus spp. cellular 16S rRN A was also determined for each station. Of the three station examined only two, stations 5 and 8, exhibited a significant increase in growth rate by diluting out predation pressure. Instantaneous growth rates were 0.026, 0.48 and 3.4 d’I at stations 3, 5 and 8, respectively (Figure 5.2). Grazing coefficients, presumably due to microflagellates (9) and ciliates (46), were 0.35 and 2.0 (1'l at stations 5 and 8. These grazing coefficients appeared to be capable of consuming approximately 75% of the daily net primary production of synechococci. Dilution culturing was not significantly correlated with growth rate at station 3, indicating that grazing was not a significant component of mortality within this population. 115 4 - L .9" "e 3" v 3 . A C6 x “E 2‘ e 1 x. CD ~5 1-l o G U . .8 ' + U I + o 0 I = I >: m . .1 U I I I I 1 I I j I I 00 02 04 0.6 08 10 1.2 Fraction Unfiltered Seawater Figure 5.2 - Estimated growth rate of Synechococci determined by the dilution method of Landry and Hasset (25). See text for growth rate and grazing coeffecients. November 1994 sampling of stations (I) 3, (O) 5 and (A) 8. 116 The growth rate at stations 3 and 5 are comparable to those reported Campbell and Carpenter (7)Iusing the Landry and Hasset technique. the growth rate at station 8 was markedly higher than the other two stations and comparable to the growth rates reported by Waterbury (50) using a similar approach. Synechococcus cellular 16S rRN A concentrations were determined all three stations (Table 5.3). Cellular 16S rRNA concentrations were significantly correlated with growth rates determined by the dilution culturing technique (r2=0.75, p<0.01). Cellular 168 rRNA concentrations and growth rates According to simple models of predator-prey interactions, population size is variously controlled by either available nutrients (bottom-up forces) or predation (top-down forces) (19, 20, 30, 41). Nutrient-limited populations are expected to possess relatively slow cellular growth rates (Figure 1.2), whereas predator-limited population sizes are expected to be small compared to nutrient-limited populations and to possess relatively high growth rates in order to maintain the population under grazing pressure (Figure 1.2). Relative difference in growth can be distinguished using group-specific rRNA probes because cellular RNA concentrations, in particular rRNA concentrations, reflect growth rate (22, 24, 44) (Figure 2.2). Synechococci cellular 16S rRNA concentrations were determined at each station and plotted versus population size at each station (Figure 5.3). The greater than 10-fold variation in 16S rRNA concentration in the environmental samples exceeded the 2 fold variation in 16S rRNA concentrations attributable to diel variations (Figures 2.4 and 3.3). The distribution of large slow growing populations versus small rapidly growing populations conforms to the predictions of simple prey-dependent models (14, 41) of predator-prey interactions (Figure 1.2), suggesting both resource-limitation and predator-limitation are responsible for controlling these populations at different times or locales. The eutrophic stations 1 and 5, sampled in the fall of 1993, had populations of 1x10’ce11s-ml'l or higher, and cellular 16S rRNA concentrations less 117 2.0- 1.5 - Synechecocci Population Size (105 cells-ml“) 4 1.0 - 4 0.5 - 3 2 3 0'5 0.5 tlf A6 52 tle 1 5't1 d A1 tlc A3 5 5”th 48 0.0 “I I I4 I I I I I ' I I I I I I I I I 0 5 10 15 20 Synechococci 16S rRNA (fg-cell") Figure 5.3 - Cellular 16S rRNA concentration of Synechococci populations in the Gulf of Mexico. Station colors correspond to the following sampling dates: (black) October-November 1993, (blue) March 1994, (red) November 1994 and (green) February 1996. Station numbers correspond to those in figure 5.1 and tables 5.1 through 5.4. 118 than 5 fg-cell", indicating slow growth rates (Figure 5.3; Table 5.1). Stations A6 and 8, sampled in the spring of 1994 and the fall of 1994, respectively, both possessed small synechococci population densities and greater than 10 fg-cell'l of 168 rRNA, indicating a rapidly growing population. A few stations, such as 4 and 5, exhibited considerable variation in both population size and 16S rRN A concentration. One explanation for this variation may be seasonal variation in nutrient inputs combined with seasonal variability in grazing intensity. A second explanation is the possiblity of “patchiness” in both nutrients and predator populations; although this explanation would require that the “patches” maintain their integrity over several days in order for the effects of added nutrients or predation to manifest themselves in the physiology of the relatively slow growing Synechococcus populations. The distribution of larger, slower growing synechococci populations and smaller, faster growing, however, are inconsistent with the predictions of ratio-dependent models (1-3) of predator-prey interactions. If synechococci population size conformed to the predictions of ratio-dependent models the expected distribution would also include relative larger, faster growing populations (Figure 1.3). The absence of these larger, growing synechococci populations would suggest that the effects of predation are not dampened in a ratio-dependent manner. The range of 16S rRNA concentrations observed in the Gulf of Mexico are similar to observed in pure cultures of Synechococcus PCC 6301 at specific growth rates of 0.007 to 0.08 h". These growth rates are comparable to estimates of rates of grazing on synechococci determined dilution and inhibition techniques, reported by Campbell and Carpenter (7). The high growth rates are consistent with heavy grazing limiting population size and primary production, as postulated by Lohrenz (33). There was no apparent correlation between nutrient loading and population size or growth rate. Large, slow growing populations of synechococci appeared in both eutrophic and Oligotrophic marine sites. This pattern may indicate patchiness in nutrients 119 accompanied by a non-steady state population. This pattern could also indicate patchiness in predator population size or predator populations alternately controlled by their own bottom-up and top-down forces. For example, mesozooplankton grazing may limit protozoan population size and therefore grazing intensity, just as protozoan are capable of limiting bacterial population size. Stations with large slowly growing synechococci populations may consist of an odd number of trophic levels. In an odd numbered trophic system the Synechococcus spp. predator population would be limited by its own predator. This second level of predation would release the synechococci population from predator control and return the population to a nutrient-limited status (14, 41). It appears that a significant number of synechococci populations in the shelf environment of the Gulf of Mexico, are controlled primarily by predation, regardless of whether the site is Oligotrophic or eutrophic. These results are consistent with the work of Carpenter and Campbell (7), who found grazing rate of 0 to 0.08 h'l of synechococci off the NY sound. It is also consistent with the predictions of Caron etal (9) that heterotrophic nanoflagellates (microflagellates) are the major predators of synechococci and capable of consuming 100% of the daily synechococci production. This contrasts with the results of recent experiments in the high-nitrogenzlow-chlorophyll waters off the coast of the Galapagos islands (35), where it was demonstrated that iron was the element limiting synechococci population size/production. Depth profile A depth profile of synechococci population size was conducted at station Tlf (28°01.03’N, 90°O7.23’W) in February of 1996 (Figure 5.4). The synechococci population was approximately 2.5x 104 cells-mlI to a depth of 20 m. The population peaked at 1.1x105 cells-ml" at 25 m. The population then declined with depths greater than 25 m and tended towards 0 at 80 m, the approximate bottom of the euphotic zone. The 120 distribution and cell density of synechococci is similar to the 20 m peak observed by Li (31) at a comparable latitude off the Moroccan coast and the 20 m peak observed at nertic front station P1 near Woods Hole Harbor, MA (16). In contrast the synechococci population peak at 25 m is deeper than the surface maximums typically observed by Waterbury et al (50) and much shallower than the approximately 100 m maximums observed at the Oligotrophic ALOHA station near Hawaii (8). Chlorophyll a concentration displayed the same trend as synechococci cell density, peaking at 0.32 mg-L'l at 25 m (Figure 5.5). This covariation between chlorophyll a and Synechococcus population size has been noted previously (8) in the vertical population of station ALOHA. The rapid decline in both cell density and chlorophyll a concentration after 25 m is typical of synechococci populations below the chlorophyll maximum (8). Synechococci populations typically peak at the chlorophyll maximum in oligou'ophic waters (8). The synechococci population peak, the chlorophyll a peak and rapid decrease in both below 25 m are evidence that a chlorophyll maximum exists at this station that is much shallower than previously reported for the Oligotrophic ALOHA station (8). Cellular 16S rRNA concentrations increased steadily for the first 20 m before peaking at 2.9 fg-cell" (Figure 5.4). The cellular 16S rRNA concentration fell to nearly zero at 25 m before increasing gradually to 1.7 fg-cell" and again decreased to low at 80 m. The high 16S rRNA concentrations near the surface indicate that surface populations possess a higher specific growth rate relative to populations at depth at this station. The high surface growth rates seen here are consistent with the high synechococci surface productivity reported by Waterbury et al. (51) in oligotrohic waters. Microzooplankton population size increased with depth to an approximate peak of 3000 cells-ml’l at 35 m before decreasing to undetectable levels at 80 m (Figure 5.5). Low synechococci 16S rRNA concentrations coincided with the maximum microzooplankton population. If microzooplankton grazing had a significant impact upon synechococci population size there should be a conelated increase in the growth rate and the cellular 16S rRNA concentration of the synechococci Depth (m) Depth (m) 121 Synechococci (104 cell-ml“) 0 2 4 6 8 10 12 0 . . 1 A "" 20 - I I-I-i . r-o-r III 40 .. I 60 u I 80 I—* 0 PH , B ._._. 20 - t——. n « I H—I 40 ' " 60 - r——-——o———1 30 .._I _ ' , U _ 0 1 2 3 -l 168 rRNA (fg-cell ) Figure 5.4 - Depth profile of Synechococcus populations. (A) Synechococcus population size. (B) Synechococcus cellular 16S rRNA concentrations. 122 Chlorophyll a (rig-L") 0 0.1 0.2 0.3 0.4 0 - l m- l n l n A I .‘I 20 - t—I—r A l-I-l E - H... V m a 40- - Q Q . 60 - I 80 I 0 III . - B 20 - W A t-—-I———r a - - 4 E. a 40 :- F—I—I Q G . 60 - l-——I—-t 80 'P fi I f I f I f 0 1 2 3 4 Small eukaryotes (103 cells-ml") Figure 5.5 - Depth profile of station tlf. (A) Chlorophyll a concentrations. (B) Small eukaryote population size. 123 population (Figure 2.2). The low cellular 16S rRNA concentrations coincident with the high microzooplankton population indicates that microzooplankton do not have a significant impact on synechococci population size. It appears that the population growth rate at this station is controlled by available nutrients (bottom-up forces). The larger and faster growing synechococci population, at 25 m within the depth profile, bounded by a very slow growing population is similar to previous observations (26, 53), although the maximum synechococci population in this study appears to be bounded on a single side by a low growth population. Wood (53) has interpreted this characteristic depth profile of maximum population growth bounded by negative population growth as evidence for bottom-up forces controlling synechococci populations in Oligotrophic waters, an interpretation consistent with the observations at this particular station. Synechococcus cellular 16S rRNA concentrations were consistent with prey- dependent models of predation and previous reports of high grazing coeffecients. These results do, however, appear to be inconsistent with the predicted outcome of ratio- dependent predation. Cellular 16S rRN A concentrations and the prediction of predator- limitation also conelated with growth rate and grazing coeffecients estimated from dilution cultures. There was no apparent correlation between 16S rRNA concentrations and rates of carbon fixation. This work demonstrates that 16S rRNA probes can be used to distinguish top-down from bottom-up control of any group for which a specific probe can be designed and address general ecological questions. 124 References 1. Arditi, R. and L. R. Ginzburg. 1989. Coupling in predator-prey dynamics: ratio-dependence. J. Theor. Biol. 139:311-326. 2. Arditi, R., N. Perrin, and H. Saiah. 1991. Functional responses and heterogeneities: an experimental test with cladocerans. OIKOS. 60:69-75. 3. Arditi, R. and H. Saiah. 1992. Empirical evidence of the role of heterogeneity in ratio-dependent consumption. Ecology. 73(5): 1544-155 1 . 4. Behrenfeld, M. J., A. J. Bale, Z. S. Kolber, J. Aiken, and P. G. Falkowski. 1996. Confirmation of iron limitation of phytoplankton photosynthesis in the equatorial Pacific Ocean. Nature. 383:508-511. 5. Bratina, B. J., M. Viebahn, and T. M. Schmidt. in press. Achieving specificity in nucleic acid hybridizations using nuclease 81. Methods Mol. Cell. Biol. 6. Bratkovich, A. and S. P. Dinnel. 1992. Presented at the Nutrient Enhanced Coastal Ocean Productivitiy, Luisiana Universities Marine Consortium. 7. Campbell, L. and E. J. Carpenter. 1986. Estimating the grazing pressure of heterotrophic nanoplankton on Synechococcus spp. using the sea water dilution and selective inhibitor techniques. Mar. Ecol. Prog. 33:121-129. 8. Campbell, L. and D. Vaulot. 1993. Photosynthetic picoplankton community structure in the subtropical north Pacific Ocean near Hawaii (station ALOHA). Deep-Sea Research. 40(10):2043-2060. 9. Caron, D. A., E. L. Lim, G. Miceli, and J. B. Waterbury. 1991. Grazing and utlization of chroocoid cyanobacteria and heterotrophic bacteria by protozoa in laboratory cutlures and a coastal plankton community. Mar. Ecol. Prog. 76:205-217. 10. Carpenter, S. R. and J. F. Kitchell. 1984. Plankton community structure and limnetic primary production. The American Naturalist. 124(2): 159-172. 11. Coale, K. H. 1991. Effects of iron, manganese, copper, and zinc enrichments on productivity and biomass in the subarctic Pacific. Limnol. Oceanbgr. 36(8):1851-1864. 12. Coale, K. H., S. E. Fitzwater, R. M. Gordon, K. S. Johnson, and R. T. Barber. 1996. Control of community growth and export production by upwelled iron in the equatorial Pacific Ocean. Nature. 379:621-624. 13. Ferrier-Pages, C. and F. Rassoulzadegan. 1994. Seasonal impact of the microzooplankton on pico- and nanoplankton growth rates in the northwest Mediteranean Sea. Mar. Ecol. Prog. 108:283-294. 14. Fretwell, S. 1977. The regulation of plant communities by the food chains exploiting them. Perspectives in Biology and Medicine. Winter: 169-185. 15. Frost, B. W. 1996. Phytoplankton bloom on iron rations. Nature. 383:475-476. 125 16. Glover, H. E., L. Campbell, and B. B. Prezelin. 1986. Contribution of Synechococcus spp. to size-fractioned primary productivity in three water masses in the Northwest Atlantic Ocean. Mar. Biol. 91:193-203. 17. Glover, H. E., B. B. Prezelin, L. Campbell, M. Wyman, and C. Garside. 1988. A nitrate-dependent Synechococcus bloom in surface Sargasso Sea water. Nature. 331:161-163. 18. Greene, R. M., Z. S. Kolber, D. G. Swift, N. W. Tindale, and P. G . Falkowski. 1994. Physiological limitation of phytoplankton photosynthesis in the eastern equatorial Pacific determined from variablity in the quantum yeild of fluorescene. Limnol. Oceanogr. 39(5): 1061-1074. 19. Hairston, N. G., F. E. Smith, and L. B. Slobodkin. 1960. Community structure, population control and competition. The American Naturalist. 154(879):421- 425. 20. Hansson, L.-A. 1992. The role of food chain composition and nutrient availability in shaping algal biomass development. Ecology. 73(1):241-247. 21. Iturriaga, R. and B. G. Mitchell. 1986. Chroococcoid cyanobacteria: a significant component in the food web dynamics of the open ocean. Mar. Ecol. Prog. 28:291-297. 22. Kerkhof, L. and B. B. Ward. 1993. Comparison of nucleic acid hybridization and fluorometry for measurement of the relationship between RN AIDNA ratio and growth rate in a marine bacterium. Appl. Environ. Microbiol. 59(5): 1303-1309. 23. Kivi, K., S. Kaitala, H. Kuosa, J. Kuparinen, E. Leskinen, R. Lignell, B. Marcussen, and T. Tamminen. 1993. Nutrient limitation and grazing control of the Baltic plankton community during annual succession. Limnol. Oceanogr. 38(5):893-905. 24. Kjeldgaard, N. O. and C. G. Kurland. 1963. The distribution of soluble and ribosomal RNA as a function of growth rate. J. Mol. Biol. 6:341-348. 25. Kuuppo-Leinikki, P., R. Autio, S. Hallfors, H. Kuosa, J. Kuparinen, and R. Pajuniemi. 1994. Trophic interactions and carbon flow between picoplankton and protozoa in pelagic enclosures manipulated with nutrients and a top predator. Mar. Ecol. Prog. 107:89-102. 26. Lande, R., W. K. W. Li, E. P. W. Horne, and A. M. Wood. 1989. Phytoplankton growth rates estimated from depth profiles of cell concentration and turbulent diffusion. Deep-Sea Research. 36(8):1141-1159. 27. Landry, M. R. 1993. Estimating rates of growth and grazing mortality of phytoplankton by the dilution method, p. 715-722. In P. F. Kemp, B. F. Sherr, E. B. Sherr, and J. J. Cole (ed.), Handbook of Methods in Aquatic Microbial Ecology, vol. 1. CRC Press, Inc., Boca Raton, FL. 28. Landry, M. R. and R. P. Hassett. 1982. Estimating the grazing impact of marine microzooplankton. Mar. Biol. 67:283-288. 126 29. Leach, C. K., J. M. Old, and N. G. Carr. 1971. Aspects of macromolecular synthesis in the blue-green alga Anabaena variabilis. J. Gen. Microbiol. 68:xiv. 30. Leibold, M. A. 1989. Resource edibility and the effects of predators and productivity on the outcome of trophic interactions. The American Naturalist. l34(6):922- 949. 31. Li, W. K. W. 1994. Primary production of prochlorophytes, cyanobacteria, and eucaryotic ultraphytoplankton: measurements from flow cytometric sorting. Limnol. Oceanogr. 39(1):]69-175. 32. Li, W. K. W. and A. M. Wood. 1988. Vertical distribution of North Atlantic ultr0phytoplankton: analysis by flow cytometry and epifluorescence microscopy. Deep- Sea Research. 35(9): 1615-1638. 33. Lohrenz, S. E., D. G. Redalge, G. L. Fahnenstiel, and G. A. Lang. 1992. Presented at the N OAA Nutrient Enhanced Coastal Ocean Productivity (TAMU-SG- 92-109), Louisiana Universities Marine Consortium, 1992. 34. Mann, N. and N. G. Carr. 1974. Control of macromolecular composition and cell division in the blue-green alga Anacystis nidulans. J. Gen. Microbiol. 83:399-405. 35. Martin, J. H., K. H. Coale, K. S. Johnson, S. E. Fitzwater, R. M. Gordon, S. J. Tanner, C. N. Hunter, V. A. Elrod, J. L. Nowicki, T. L. Coley, R. T. Barber, S. Lindley, A. J. Watson, K. V. Scoy, C. S. Law, M. I. Liddicoat, R. Ling, T. Stanton, J. Stockel, C. Collins, A. Anderson, R. Bidigare, M. Ondrusek, M. Latasa, F. J. Millero, K. Lee, W. Yao, J. Z. Zhang, G. Friederich, C. Sakamoto, R. Chavez, K. Buck, Z. Kolber, R. Greene, P. Falkowski, S. W. Chisholm, F. Hoge, R. Swift, J. Yungel, S. Turner, P. Nightingale, A. Hatton, P. Liss, and N. W. Tindale. 1994. gesting the iron hypothesis in ecosystems of the equatorial Pacific Ocean. Nature. 71:123-129. 36. Martin, J. H., R. M. Gordon, and S. E. Fitzwater. 1991. The case for iron. Limnol. Oceanogr. 36(8): 1793-1802. 37. Mitchell, B. G., E. A. Brody, O. Holm-Hansen, C. McClain, and J. Bishop. 1991. Light limitation of phytoplankton biomass and macronutrient utilization in the Southern Ocean. Limnol. Oceanogr. 36(8):1662-1677. 38. Morel, F. M. M., R. J. M. Hudson, and N. M. Price. 1991. Limitation of productivity by trace metals in the sea. Limnol. Oceanogr. 36(8): 1742-1755. 39. Moss, B., S. McGowan, and L. Carvalho. 1994. Determination of phytoplankton crops by top-down and bottom-up mechanisms in a group of English lakes, the West Midland meres. Limnol. Oceanogr. 39(5):1020-1029. 40. Nold, S. C. and D. M. Ward. 1996. Photosynthate partitioning and fermentation in hot spring microbial mat communities. Appl. Environ. Microbiol. 62(12):4598-4607. 41. Oksanen, L., S. D. Fretwell, J. Arruda, and P. Niemela. 1981. Exploitation ecosystems in gradients of primary productivity. The American Naturalist. l l8(2):240-261. 127 42. Parsons, T. R. and e. al. 1984. Photosynthesis as measured by the uptake of radioactive carbon, p. 115-120, A Manual of Chemical and Biological Methods for Seawater Analysis. Pergamon Press 43. Rabalais, N. N., R. E. Turner, W. J. W. Jr., and D. F. Boesch. 1991. A brief summary of hypoxia on the northern Gulf of Mexico continental shelf: 1985-1988., p. 33-47. In R. V. Tyson and T. H. Pearson (ed.), Modern and ancient continental shelf anoxia, vol. 58. Geological Society Special Publication 44. Rosset, R., J. Julien, and R. Monier. 1966. Ribonucleic acid composition of bacteria as a function of growth rate. J. Mol. Biol. 18:308-320. 45. Sambrook, J ., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning; a laboratory manual, 2 ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 46. Sherr, B. F. and E. B. Sherr. 1987. High rates of consumption of bacteria by pelagic ciliates. Nature. 325:710-71 1. 47 . Turner, R. E. and N. N. Rabalais. 1991. Changes in Mississippi River water quality this century. BioScience. 41: 140-147. 48. Vaulot, D., N. LeBot, D. Marie, and E. Fukai. 1996. Effect of phosphorus on the Synechococcus cell cycle in surface Meditenanean waters during summer. Appl. Environ. Microbiol. 62(7):2527-2533. 49. Waterbury, J. B., S. W. Watson, R. R. L. Guillard, and L. E. Brand. 1979. Widespread occurence of a unicellular, marine, planktonic, cyanobacterium. Nature. 277:293-294. 50. Waterbury, J. B., S. W. Watson, F. W. Valois, and D. G. Franks. 1986. Biological and ecological characterization of the marine unicellular cyanobacterium Synechococcus. Can. J. Fish. Aquat. Sci. 214:71-120. 51. Waterbury, J. B. and J. M. Willey. 1988. Isolation and growth of marine planktonic cyanobacteria, p. 100-105, Methods In Enzymology, vol. 167. Academic Press, Inc. 52. Watson, 8., E. McCauley, and J. S. Downing. 1992. Sigmoid relationships between phosphorus, algal biomass, and algal community structure. Can. J. Fish. Aquat. Sci. 49:2605-2610. 53. Wood, A. M. 1990. Presented at the Proc. 5th Int. Symp. Microbial Ecology, Japan.x Chapter 6 Summary and Conclusions 128 129 Over thirty years ago it was demonstrated that cellular nucleic acids concentrations were proportional to growth rate in Salmonella typhimurium and Escherichia coli (6, 12). A recent synthesis of the literature on the relationship between cellular nucleic acids concentrations and growth rate has revealed that nucleic acids concentrations are proportional to growth rate in all heterotrophic bacteria examined to date (5). Nucleic acids concentrations are also proportional to growth rate in both filamentous and unicellular cyanobacteria under constant illumination (7, 9). The work presented in this series of studies demonstrates that cellular nucleic acids concentrations are proportional to growth rate in the freshwater cyanobacterium Synechococcus sp. strain PCC 6301 and the marine cyanobacterium Synechococcus sp. strain WH 8103. Furthermore, this proportionality is maintained over a range of growth rates which are ecologically relevant. The nucleic acids content of bacteria is predominately composed of rRNA (6, 8) and one-third of ribosomal rRNA (w/w) is composed of 16S rRNA. The correlation between cellular l6S rRNA concentrations and growth rate presents the possibility of using probes complementary to 16$ rRN A to determine in situ growth rates. Probes complementary to 16S rRNA were used to demonstrate that 168 rRNA was proportional to growth rate in Synechococcus sp. strain PCC 6301 (Figure 2.2). This relationship was assumed to hold for Synechococcus sp. strain WH 8103, as well, a position supported by the fact that cellular 16S rRNA concentrations mirrored those of total cellular RNA concentrations during the course of a diel cycle (Figure 2.4). The constant RNA/DNA ratios of Synechococcus sp. strain PCC 6301 contrasts with the increasing RNA/DNA ratios of Synechococcus sp. strain WH 8103. The constant RNA/DNA ratio observed in Synechococcus sp. strain PCC 6301 has been used to argue that Synechococcus sp. strain PCC 6301 is not capable of transcriptional regulating the majority of its genes. This position is further supported by the fact that both total RNA and 16S rRN A concentrations mirror those of DNA concentrations during the course of diel growth (Figure 1.3 and 1.4). The possible lack of transcriptional regulation hints that 130 many of the observed features of circadian rhythms may be a result of gene dosage effects (10). . By implication then the increasing RNA/DNA ratios observed in Synechococcus sp. strain WH 8103 would indicate that this organism possesses the ability to transcriptionally regulate the majority of its genes, including the ribosomal RNAs. This position is supported by examination of the nucleic acids concentrations of the organism during diel growth. Although the trends are similar to those of Synechococcus sp. strain PCC 6301 there is one notable exception. During the final 3h of the dark period there was a large increase in both total cellular RNA concentrations and cellular l6S rRNA concentrations (Figure 2.2). This dramatic increase is even more remarkable in view of the fact that cellular DNA concentrations had dropped to a low equivalent to one genome'cell’l (Figure 2.3). The ability of Synechococcus sp. strain WH 8103 to dramatically increase cellular RNA concentrations from a single genome copy would indicate that the organism is capable of transcriptionally regulating its rrn operons. This evidence is also consistent with the interpretation that increasing RNA/DNA ratios indicate transcriptional control. Once it was established that 16S rRN A probes were capable of distinguishing relative difference in ecologically relevant growth rates, it was a natural progression to use this technique to address ecological questions. One such question was whether cellular 16S rRNA concentrations could be used as a proxy for cellular growth rate to distinguish between nutrient limitation and predator limitation of a cyanobacterial population. This question was addressed in laboratory experiments using a model system consisting of a chemostat population of a Synechococcus sp. strain PCC 6301. In the absence of predation Synechococcus sp. strain PCC 6301 equilibrium population size was dependent upon the concentration of available limiting nutrients, in this case phosphate, within the reservoir supplying the chemostat culture vessel (Figure 3.1). The population size remained largely unaffected by the specific growth of the individual organisms which was determined by the dilution rate of the chemostat and ultimately the flux of the limiting 131 nutrient through the system. Ecological theories of predator-prey interactions predicted that the addition of a predator would decrease the equilibrium prey population size while simultaneously increasing the specific growth rate of the individuals within the prey population. The addition of the protozoan predator T. pyrifarmis to a chemostat culture of Synechococcus sp. strain PCC 6301 resulted in a 7-fold decrease in the equilibrium population size of Synechococcus sp. strain PCC 6301. The grazing pressure imposed by T. pyriformis resulted in Synechococcus sp. strain PCC 6301 reaching its maximal specific growth rate (Figure 3.1), which was reflected in the high cellular concentrations of cellular 16S rRN A compared to the low cellular l6S rRNA concentrations observed in the absence of predation (Table 3.1; Figure 3.3). Based on prey-dependent models of predator-prey interaction, increases in available nutrients supplied by the reservoir should result in an increase in the equilibrium population size of the T. pyrifarmis predator, while the equilibrium population size the Synechococcus sp. strain PCC 6301 prey remains unchanged. However, the larger T. pyrifarmis population would result in increased grazing pressure forcing a compensatory increase in growth rate and therefore cellular 16S rRN A concenu'ations in the Synechococcus sp. strain PCC 6301 prey. Unfortunately, because the specific growth rate of the Synechococcus sp. strain PCC 6301 population was already at maximal, the increase in available limiting nutrients went unutilized (Table 3.2). It was therefore not possible to distinguish between ratio-dependent models of predator- prey interactions and prey-dependent models of predator-prey interactions using the particular predator-prey system from these studies. Nor was it possible to distinguish between ratio—dependent and prey-dependent models by reducing the supply of limiting nutrient without the already low T. pyrrformis predator population washing out of the system. Regardless of the short-comings of this system it was possible to demonstrate that 16S rRNA probes in combination with a knowledge of the equilibrium prey population size could be used to distinguish nutrient-limited prey populations from predator-limited populations. 132 Having demonstrated that predator-prey interactions conform to model predictions and that 16S rRNA probes could be used to distinguish nutrient-limited prey populations from predator-limited populations, these methods were applied to a nutrient gradient within the Gulf of Mexico. There is a well established nitrate and phosphate gradient where the Oligotrophic water of the Gulf of Mexico are influenced by nutrient loading fi'om the Mississippi River drainage system. If synechococci population size was limited by available nutrients it would be a reasonable expectation to find a decreasing synechococci population size moving from the near shore, eutrophic waters, heavily influenced by the Mississippi River, to the Oligotrophic water of the open ocean within the Gulf of Mexico. Conversely, if synechococci population sizes were limited by predation then one would expect an increase in growth rates but not population size , along the gradient, as available nutrients were passed up the trophic chain to an increasing predator population. Synechococci population sizes in the Gulf of Mexico were primarily small with a significant number of these populations containing high concentrations of 16S rRN A, indicative of rapid growth rates (Figure 4.1). It is significant that there were no populations in the upper right quadrant of figure 5.3, comprised of large quickly growing synechococci populations. Such populations would be predicted from ratio-dependent models of predator-prey interactions (1-3). Rather, the Gulf of Mexico environment appeared to be composed of a gradient between large, slow growing synechococci populations and small, fast growing synechococci populations. These results conform to the predictions of prey-dependent models of predator-prey interactions (4, 11) and appear to be inconsistent with predictions of ratio-dependent models of predator-prey interactions. Thus, it appears that some synechococci populations were nutrient-limited while others were predator-limited. There was no clear correlation between the trophic status of the local environment (Oligotrophic vs. eutrophic) and whether a synechococci population was nutrient-limited or predator limited. This could be a result of increasing number of trophic 133 levels along the gradient, resulting in alternate switching between nutrient-limitation and predator-limitation (4, ll). 134 References 1. Arditi, R., and L. R. Ginzburg. 1989. Coupling in predator-prey dynamics: ratio-dependence. J. Theor. Biol. 139:311-326. 2. Arditi, R., N. Perrin, and H. Saiah. 1991. Functional responses and heterogeneities: an experimental test with cladocerans. OIKOS. 60:69-75. 3. Arditi, R., and H. Saiah. 1992. Emperical evidence of the role of heterogeneity in ratio-dependent consumption. Ecology. 73(5): 1544- 1 55 1. 4. Fretwell, S. 1977. The regulation of plant communities by the food chains exploiting them. Perspectives in Biology and Medicine. Winter: 169-185. 5. Kerkhof, L., and B. B. Ward. 1993. Comparison of nucleic acid hybridization and fluorometry for measurement of the relationship between RNA/DNA ratio and growth rate in a marine bacterium. Appl. Environ. Microbiol. 59(5): 1303-1309. 6. Kjeldgaard, N. O., and C. G. Kurland. 1963. The distribution of soluble and ribosomal RNA as a function of growth rate. J. Mol. Biol. 6:341-348. 7. Leach, C. K., J. M. Old, and N. G. Carr. 1971. Aspects of macromolecular synthesis in the blue-green alga Anabaena variabilis. J. Gen. Microbiol. 68:xiv. 8. Mann, N., and N. G. Carr. 1973. A constant ratio of transfer to ribosomal ribonucleic acid in Anacystis nidulans grown with differing mean generation times. Biochemical Society Transactions. 1:702-704. 9. Mann, N ., and N. G. Carr. 1974. Control of macromolecular composition and cell division in the blue-green alga Anacystis nidulans. J. Gen. Microbiol. 83:399-405. 10. Nichols, J. M., I. J. Foulds, D. H. Crouch, and N. G. Carr. 1982. The diversity of cyanobacterial genomes with respect to ribosomal RNA cistrons. J. Gen. Microbiol. 128:2736-2746. 11. Oksanen, L., S. D. Fretwell, J. Arruda, and P. Niemela. 1981. Exploitation ecosystems in gradients of primary productivity. The American Naturalist. l l8(2):240-26l. 12. Rosset, R., J. Julien, and R. Monier. 1966. Ribonucleic acid composition of bacteria as a function of growth rate. J. Mol. Biol. 18:308-320.