1.1.31ka Michigan §tato . UnivcrfltY This is to certify that the thesis entitled STUDIES ON DISSIMILATORY REDUCTION OF NITRATE TO AMMONIUM BY SOIL CLOSTRIDIA presented by William Horton Caskey has been accepted towards fulfillment of the requirements for Ph.D. Microbiology degree in 4%ng flwta Date 9 NOV. 1978 0-7639 c l b Mar-O ‘1‘: i.- ..4- .‘w,-mu%— -—— A. l A STUDIES ON DISSIMILATORY REDUCTION OF NITRATE TO AMMONIUM BY SOIL CLOSTRIDIA BY William Horton Caskey A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1978 ABSTRACT STUDIES ON DISSIMILATORY REDUCTION ON NITRATE TO AMMONIA BY SOIL CLOSTRIDIA By William H. Caskey In some soils incubated anaerobically, reduction of N03— to NH4+ in excess of amounts attributable to assimilatory reduction has been observed. Fresh soil and soil which had been air-dried and stored for several years were amended with NO3 and glucose, acetate, or water and incubated anaerobically. Soils were either untreated or heat—shocked at 680 C for 1 hour prior to amendment. Ammonium appeared to be produced rapidly after the onset of anaerobiosis, and thereafter was incorporated 15 into organic matter. NH4+ plus 15N-organic matter production was observed in glucose-amended fresh soil in quantities up to 23.0% of added 15N03- in untreated samples and 32.0% in heat-shocked samples after 24 hours incubation. Extending incubation to 5 days resulted in N03- reduction to NH4+ equivalent to 42.9% and 54.9% of added N, respec- tively. The amount of N114+ produced in the air-dried soil was essen- tially the same as in the heat-shocked fresh soils. Heat treatment had no effect on NH4+ production in the air—dried soil. The activity of clostridia during the N03_ reduction was indicated by the absence of any effect exerted by heat-treatment, the production of H2 and C02, and higher numbers of anaerobic sporeforming bacteria relative to denitrify- ing bacteria in the air-dried soil. Also, the most common isolate William Horton Caskey capable of reducing N03- to NH4+ was a Clostridium spp. The addition of washed spores of a N03 -reducing bacterium isolated from the air-dried Kranzburg soil (ClostridiuleDHSZ) to glucose-amended Conover soil increased the formation of 1SNHXLN plus 15N-organic N four-fold, an accumulation equivalent to 83% of added 15N03.--N. - + The reduction of N03 to NH4+ in soils was not inhibited by NH4 or glutamine, which suggested the mechanism of reduction was dissimilatory. This conclusion is supported by studies with several Clostridium spp. isolated from the soils. The isolates were capable of reducing N03 to NH4+ and exhibited increased cell yields when N03 was included in the growth medium. In pure culture, Clostridium KDHSZ reduced N03- forming N114+ as the product. A 13% increase in cell yield was observed when the organ- ism was cultured in N03--containing media. The reduction of 13N03- 13NH4+ by resting cells was not inhibited by NH4+, glutamate, or gluta- mine. The formation of 13NH4+ from 13N03- was also unaffected by methionine sulfoximine (0.01 mM and 10 mM) and azaserine (1 mM). SO = 4 13N03', but $03= inhibited the reaction,. apparently at the level of N02 reduction. Conversely, N03 35803: to 358.. In fact, growth in NOB—- containing media caused a 10-fold increase in the ability of the cells to did not inhibit the reduction of exerted no influence on the reduction of to reduce $03. to 8:. Growth in the presence of SO only mildly 4 enhanced this $03II reducing capacity. 8048 was not reduced to SS. 3- of 0.5 mM and reduced N03- to + NH4 maximally at a rate of 1.5 pg N/hr-mg cells. Partially purified Clostridium KDHSZ exhibited a KS for N0 3 3- of 0.15 mM. Glutamine nitrate reductase from the organism catalyzed the reduction of NO to N02“. The nitrate reductase has a Km for N0 syntt synth but v gluta‘ activ to NE N0 Pathx William Horton Caskey synthetase was produced by the bacterium. The activity of the glutamine synthetase was only slightly inhibited by 0.01 mM methionine sulfoximine, but was inhibited 94% by 10 limethionine sulfoximine and 60% by 10 mM . glutamine. Glutamine synthetase exerted no control over either the activity or synthesis of the enzymes involved in the reduction of N03- to NH4+. The data presented are consistent with the hypothesis that N03- reduction to NH“+ in Clostridium KDHSZ occurs via a dissimilatory pathway. To furry little Magic, because Dr. Pace said I should. ii ACKNOWLEDGEMENTS I am grateful to my fellow members of the "Institute of Microbial Ecology," namely Mike Betlach, Mary Firestone, Tom Gamble, Joe Robinson, Scott Smith, and Dick Strayer. Their sharing of knowledge and open discussion of ideas were the most significant contributions to my educa- tional experience. Special thanks are extended to Dr. James Tiedje, my major professor, for his guidance and encouragement. He has established an atmosphere most conducive to research, an atmosphere of cooperation and indepen- dence. I am especially grateful to my wife Nancy, also a.more recent inductee into the "Institute." She served as a sounding board for ideas, provided moral support during my experimental slumps, and always was there. There can be no better proofreader anywhere. I acknowledge the assistance of my guidance committee, Drs. C. A. Reddy, Erik Goodman, and R. N. Costilow. I am indebted to Dr. Goodman for introducing me to the world of systems science. My appreciation is extended to Lillian McLean for typing this dissertation and all its revisions; and, especially, for meeting all the deadlines. I am grateful to the Department of Microbiology and Public Health for providing financial assistance throughout the course of my graduate study. This research was supported in part by USDA Regional Research Project NE-39, and by a grant from the National Science Foundation. iii TABLE OF CONTENTS LIST OF TABLES O I O C C O I O O O O O O O O O O 0 LIST OF FIGURES O O O O O O O O O O O I O I I O 0 CHAPTER I. CHAPTER II. CHAPTER III. APPENDIX A. APPENDIX B. INTRODUCTION AND EXPERIMENTAL OBJECTIVES EVIDENCE FOR CLOSTRIDIA AS AGENTS OF DISSIMILATORY REDUCTION OF NITRATE IN SOILS . . MATERIALS AND METHODS . . . . . . . RESULTS . . . . . . . . . . . . . . DISCUSSION . . . . . . . . . . . . . ACKNOWLEDGEMENT . . . . . . . . . . LITERATURE CITED . . . . . . . . . . THE REDUCTION OF NITRATE TO AMMONIUM CLOSTRIDIUM SPP. ISOLATED FROM SOIL MATERIALS AND METHODS . . . . . . . RESULTS . . . . . . . . . . . . . . DISCUSSION . . . . . . . . . . . . . ACKNOWLEDGEMENT . . . . . . . . . . LITERATURE CITED . . . . . . . . . . THE ABSENCE OF A REGULATORY FUNCTION FOR GLUTAMINE SYNTHETASE IN THE SYNTHESIS OF INVOLVED IN DENITRIFICATION . . . . ENZYMES NITRATE-STIMULATED MINERALIZATION OF AMMONIUM IN ANAEROBIC SOILS . . . . . . . . . iv Page vii 15 17 22 31 35 36 39 40 45 53 6O 61 64 73 Table LIST OF TABLES Page CHAPTER I The standard free energy change at+pH 7 for the reduction of N03 to N2 and to NHA . . . . . . . . 3 Observations of NH + as a product of N0 - reduction in excess of amounts attributable to assimilatory processes . . . . . . . . . . . . . . . . . . . . . 6 CHAPTER II Eff t of exposure of Pseudomonas fluorescens (rif ) in soil to 68° C for 1 hour . . . . . . . . 18 Accumulation of 15NH +¥N and 15N-organic matter in glucose-amended soils . . . . . . . . . . . . . 23 Efffgt of carbig source on the reduction of 15N03- to NH and N-organic matter and on the concurrent production of H and C02 in soils incubated anaerobically for 5 days . . . . . . . . 24 Population densities of denitrifying bacteria and sporeforming N03 -reducing bacteria in three soil samples 0 I O O O O O O O O I O O O O O O O O O O O 26 Reduction of N0 - to NH + by pure cultures of bacteria isolated from soils and growth yields in the presence and absence of N0 . . . . . . . . . 27 3 Reduction of 15NO - to 15NH + by Clostridium spores added to glucose-amended 8011 o o o o e o o o o o o 29 ngect of inhibitors on the reduction of 15NO - to NH4 and N-organic matter in heat-shocked (68° C for 1 hour) Kranzburg soil (freshly collected) . . . . . . . . . . . . . . . . . . . . . 30 Table Page CHAPTER III The reduction of N0 - to NH + by Clostridium KDHSZ in axenic culgure and its effect on cell yield 0 I I O O O O O D O I I O O I O O O O O O O O 48 ESfect of various compounds on the reduction of NH by resting cells of Clostridium KDHSZ grown in basal medium containing N03 . . . . . . . . . . 50 Effect of N0 - on the reduction of 35$0 = and 35 = 353= 4 $0 to 8 after growth with different electron acceptors . . . . . . . . . . . . . . . . . . . . . 52 Effect of inhibitors on activity of glutamine synthetase prepared from cell-free extracts of Clostridium KDHSZ O I O O O I I O O O O O O O O O O 54 Efffst of methionige sulfoximine on the reduction of N03 to NH4 by resting cells of Clostridium ”H82 0 O O O O O O O I O O O O O O O O O O O O O O 56 APPENDIX A Effect of inhibitors on activity of glutamine synthetase from Pseudomonas fluorescens . . . . . . 67 Effect of methionine sulfoximine on the rate of denitrification by resting cells of Pseudomonas fluorescens . . . . . . . . . . . . . . . . . . . . 69 APPENDIX B Production of total NH + and 15NH + from 15N0 _ after 5 days anaerobic incubation in response to N03- and carbon additions to three soils . . . . 75 vi Figure LIST OF FIGURES Page CHAPTER III Growth response of Clostridium KDHSZ to N0 - in the medium. Growth in presence (0-0) and absence (H) of 3.5 mM N0 for inoculum grown without NO -. Growth in presence of N0 (H for inoculum grown in N03 -containing medium . . . 46 The reduction of 13N0 - and 13N0 - tolaNH4+ by resting cells of CIostridiumo H82, by a resting cells in the presence of 3. 5 mM SO 9 and by autoclaved cells . . . . . . . . .3. . . . 51 Direct linear plot of Km estimation for NH for glutamine synthetase of Clostridium KDHSZ . . 55 APPENDIX A Dir ct linear plot (4) of Km estimation for NH for glutamine synthetase of Pseudomonas fluorescens (Strain 72) . . . . . . . . . . . . . 68 vii Chapter I INTRODUCTION AND EXPERIMENTAL OBJECTIVES Nitrates in soil face three possible fates; and, of these, only plant uptake is desirable. In a 10-year study with citrus, Pratt, et al (31) found that NO _ not used by the plant either leached through the 3 soil or was denitrified. The drainage characteristics of the soil are especially important in determining whether N03- will be lost through leaching or through denitrification (15). The former fate is character- istic of sandy, more porous soils, while the latter fate is generally encountered in finer-textured soils. Considerable nitrogen can be lost through leaching of nitrates effected by rainfall or irrigation, since the anions are mobile in soils. As much as 55 to 60% of the nitrate in drainage waters may originate from applied fertilizers (23). Denitrification has long been recognized as an agriculturally undesirable fate of nitrogen from soil (29). The amount of denitrifica- tion is generally obtained by subtracting the amounts of nitrogen removed by leaching, plant uptake, and immobilization from the fertilizer inputs. These values are at best no more reliable than the reliability of the other measurements, and errors of all the measurements are summed in the difference estimate. Allison (1) observed a nitrogen deficit that averaged 15% in a number of lysimeter studies and Broadbent and Clark (3) reported denitrification losses ranged from 10-30%. Measuring denitrification directly using N2 and N20 fluxes, Ralston, et a1. (32) calculated 45-65% of the fertilizer nitrogen in an irrigated soil column was lost via this pathway. From an analysis of a number of nitrogen balance studies, a value of 25% appears to be emerging as an average 1 estimate of nitrogen losses resulting from denitrification in agricul- tural soils. Even in the absence of accurate measurements, it is apparent that denitrification impoverishes the soil of the limiting nutrient for productivity and minimizing denitrification is an important agronomic concern. Denitrification is generally considered to be the major route of NO3- reduction under anaerobic conditions. However, several reported cases of NO3- reduction differing from the established pathway have appeared. Koike and Hattori (24) and Sdrensen (35) observed significant production of NH4+ from N03- in coastal sediments, leading Sdrensen (35) to conclude the rate of reduction of N03- to NH4+ was of the same magni- tude as denitrification. Since sediments are rich in reduced nitro- genous compounds, the formation of NH4+ appeared to be the product of an 3 . reports by Stanford, et al (36,37) presented evidence for the existence alternate dissimilatory pathway for the reduction of NO Recent of a similar pathway in soil. Jones (22) found NH4+ was the dominant product of N03 reduction by a mixed culture of bovine rumen bacteria and concluded the quantitative significance of denitrification as a 3 dissimilation in the rumen is small. The thermodynamic feasibility of the reduction of N03- to NH4+ is evidenced by the change in free energy for the reaction at pH 7 (Table pathway of N0 1). The free energy data have been calculated using the free energies of formation from the elements as tabulated by Thauer, et a1 (38), and do not include the formation or consumption of ATP. The equivalent value for the reduction of N03- to N2 is given for comparison. Delwiche (ll) constructed a similar table, but the values for the free energies were reported inconsistently. Considering only net free energy change Table 1. The standard free energy change at+pH 7 for the reduction of NO to N and to NH 3 2 4 0' AG (kcal/mole) Reaction H2 NO3 N0-+4H+"H++NH++3H0 -358 -1433 3 2 ‘ 4 2 ' ‘ - + 2N03 + 5H2 + 2H + N2 + 6H20 -53.6 -133.9 Calculated from Gibbs free energies of formation from the elements as tabulated by Thauer, et al (38). for the reduction of N03-, he concluded the most efficient reaction under conditions of limiting carbon source is denitrification. Con- 3 was limiting and carbon was abundant, the reduction of N03- to NH4+ would be more efficient. These predictions, of course, ignore the biological factors, but may be of use in predicting the versely, when N0 environments in which N03- reduction to NH4+ vs. N2 might occur. Indeed, evidence has been presented that both carbon availability and concentration of N03- control selection of denitrifying and nitrate- reducing flora (see Focht and Verstraeta (15) for a detailed discus- sion). Carbon-rich environments which contain limiting quantities of N03- include marine and deep freshwater sediments, the lower portions of the water column of eutrophic lakes, the rumen, silage, sewage sludge digesters, and stream beds below sewage plant discharge sites. In addition to these habitats, some soils which receive pulses of N03 and which become transiently anaerobic might also be expected to harbor significant numbers of bacteria capable of reducing N03- to NH4+. Certain agricultural soils and river deltas are examples. Primitive Earth was also a habitat that was rich in carbonaceous compounds and contained N03 (34). It seems logical that nitrate respiration (linked to oxidative phosphorylation) evolved from ferment- 3 electron sink, producing NH4+ as the product. Such a theory has been ative "prokaryotes" which had developed the ability to use NO as an proposed by Egami (13,14). His concept of the evolution of energy- yielding metabolism in its general form is: fermentation + fermentation with H2 release + inorganic types of fermentation + anaerobic respi- rations + oxygen respiration. Broda (4,5) has argued that N03 reduction + to NH4 occurred merely as an evolutionary sideline and that NO 3 respiration evolved from aerobic respiration. He cites the absence of N03- on primitive Earth prior to the existence of 02 in the atmosphere and the omission of photosynthesis in the evolutionary sequence as the weakest points in Egami's scheme. Thermochemical calculations (34) predicted that N03 could be formed in the absence of 02, and, indeed, N03- and N02- have been found in aqueous solutions in anoxic cavities of rocks [Sugawara, 1949, as cited by Egami (14)]. Although Egami did not consider photosynthesis in his scheme, it seems plausible that a photo- autotroph could have evolved from an organism that had developed the capacity for anaerobic respiration. The sequence proposed by Broda (5) did not allow for the presence of cytochromes in fermentative bacteria, a fact now established (16). An evolutionary branch for development of photosynthesis at this point would allow the similarities in electron transport involved in photosynthesis and oxygen respiration to be conserved. Observations of N114+ as the product of N03 reduction under condi- tions suggesting a dissimilatory mechanism are listed in Table 2. In addition to the pure cultures listed, Staphyloccus aureus (38) and a number of enteric bacteria including Escherichia coli (10,38), Proteus mirabilis, Citrobacter freundii, and Klebsiella aerogenes (38) reduce - + N02 to NH4 . + These observations of NH4 production from NO3 are particularly . + interesting, since NH4 , as a cation, is adsorbed to soil particles and, is therefore, less mobile than N03 . It is also readily assimilated by the soil organisms. The production of NH4+ under anaerobic conditions represents a portion of N03 which has been rendered unavailable as a substrate for denitrification. Therefore, N03- reduction to NH4+ Table 2. Observations of NH + as a product of NO - reduction in excess of amounts attributable to assimilatory processes. System Reference Natural Systems Flooded soil Well-drained soils Lake sediments Marine sediments Rumen Pure culture Clostridium perfringens Clostridium tertium Photobacterium fischeri Veillonella alcalescens Vibrio succinogenes Bacillus licheniformis Bacillus filaris and ‘B; polymzxa Selenomonas ruminantium Spirillum itersonii Candida boydinii MacRae, et a1 (25) Stanford, et al (36,37), Buresh, et a1 (6) Chen, et a1 (7) Koike and Hattori (24), Sdrensen (35) Jones (22) Woods (47), Hasan and Hall (18) Hasan and Hall (17) Prakash and Sadana (30) Inderlied and Delwiche (20) Wolin, et a1 (46) Verhoeven (42), Woldendorp (45) Illina and Khodakova (21) deVries, et a1 (43) Thauer, et al (38) Middelhoven, et a1 (26) represents a nitrogen-conserving process. An understanding of the etiology of the NH4+ production from NO3- and the factors controlling the pathway may lead to ultimate exploitation of the reaction to improve nitrogen economy in agriculture. Although considerable information has accumulated concerning N03- reduction to NH4+ by pure cultures, little use of the knowledge has been made to explain NH4+ production in natural systems. This is not sur- prising for soils, since most investigators have failed to detect NH4+ as a product of N03- reduction (2,28,44). Also, most of the observa- tions of N03- reduction to NH4+ by pure cultures arose incidentally during studies done for other purposes. To this author's knowledge, the only attempt to correlate pure culture experiments with soil studies was by Woldendorp (45) who introduced Bacillus licheniformis into soil after observing the organism reduced N03- to NH4+ in laboratory culture. 3 The observations of Stanford, et a1 (36) that up to 55% of added However, the organism failed to reduce N0 in soil. N03--N is reduced to NH4+-N emphasized the need for studies to identify the agents catalyzing the N03- reduction. The soils used by Stanford, et al (36,37) were air—dried and had been stored for several years. This suggested a shift may have occurred in the microflora favoring sporeforming bacteria over vegetative cells. The first major objective of this research was to test the hypothesis that Clostridium species 3t0 and/or Bacillus species were responsible for the reduction of NO NH4+ in soils. Clostridium species appeared to be largely responsible for the N03 reduction to NH4+ observed in the air-dried soils. One isolate, Clostridium KDHSZ, was particularly active and evidence indicated the N03- was reduced to NH4+ by a dissimilatory, rather than assimilatory process. The amounts of NH4+ produced were in excess of those needed for assimilation, and the formation of NH4+ occurred in presence of reduced nitrogenous compounds. The second objective of this study was 3 reduction was a dissimi- to provide evidence that the pathway of NO latory route. Considerable information is available regarding NO3 reduction to NH4+ by pure cultures of bacteria. Among the enteric bacteria, ATP generation is coupled to-N03- reduction to N02-, but not to the reduc- tion of N02- to NH4+ (38). The Clostridium species reported to reduce N03- to NH4+, do so in conjunction with their energy metabolism, but the increased ATP yield is attributed to the shift in the fermentation end products toward increased acetate production in the presence of NO -, thereby increasing the amount of substrate-level phosphorylation (17,18). An interesting discovery is the possession, by the majority of N03 - reducing anaerobes, of cytochromes, some of which are functional in N03- reduction (16,43). Electron transport appears to be mediated by ferredoxin in Clostridium perfringens (9). The nitrate reductase of Clostridium_perfringens has been purified to near homogeneity and was characterized as an iron-sulfur-molybdenum protein (8). Information regarding the regulation of the N03--reducing pathway in Clostridium species is conspicuously lacking. Inhibition by oxygen is a trait generally attributed to dissimilatory enzymes, although considerable variance in the tolerance of O2 exists for enzymes from different organisms. Use of this criterion, as well as the typical experiments involving inhibition of electron transport, would provide no useful information, since Clostridium spp. are obligately anaerobic and the presence of cytochromes has been established for only two species. Furthermore, both the dissimilatory and assimilatory nitrate reductases share common structures and kinetic parameters. The only clear distinc- tion is based on function. The best approach, it was decided, was to demonstrate the absence of regulatory features commonly attributed to the enzymes involved in assimilatory N03- reduction. These investiga- tions formed the third objective of this research. In assessing the regulatory characteristics of assimilatory nitrate reduction, the question of regulation by glutamine synthetase arose. Enzymes subject to nitrogen catabolite repression in Klebsiella aerogenes have been shown to be under positive control by glutamine synthetase (41). A review of the literature describing glutamine synthetase, its regulation, and its regulatory role, in enteric bacteria is not within the scope of this dissertation. This information has been critically examined in excellent reviews by Tyler (41) and Shapiro and Stadtman (33). Of interest relative to this study is the report by Tubb (39) that the assimilatory N02 reductase in Klebsiella pneumoniae may be controlled by glutamine synthetase. Newman and Cole (27), however, observed glutamine synthetase exerted no regulatory control over nitrite reductase in Escherichia coli. The glutamine synthetase of Gram posi- tive bacteria appears to be different, both immunologically and bio- chemically, from the Gram negative (or enteric-type) enzyme (40). Rather than its activity controlled by an adenylation-deadenylation reaction, the activity of the glutamine synthetase from Bacillus subtilis is regulated by alkylation of sulfhydryl groups on the surface of the enzyme (12). No involvement of Bacillus glutamine synthetase in regulation of other enzymes involved in nitrogen metabolism has been repor genou gluta simil There sough of po was t rn KDHSZ abilit It is redUCt helpfu the f0! exPloit 10 reported, but synthesis of the B; subtilis enzyme is apparently auto- genously regulated (41). Only a single reference to the existence of glutamine synthetase in a CloStridium (Clostridium pasteurianum) and its similarity to the enzyme found in Bacillus has been published (19). Therefore, the presence of glutamine synthetase in Clostridium KDHSZ was sought. A rudimentary characterization of the enzyme and an examination of possible regulatory effects exerted by it on the NO --reducing system 3 was to be accomplished. Some kinetic parameters describing NO3 reduction by Clostridium KDHSZ were estimated. A thorough investigation of the competitive ability of this organism, relative to denitrifying bacteria is needed. It is hoped the information about the ecology and physiology of NO3 reduction to NH4+ by Clostridium KDHSZ presented in this study will be helpful in understanding the reaction in nature and, perhaps, provide the foundation for research which will reveal approaches permitting exploitation of this nitrogen-conserving reaction. 10. 11. 12. 13. 14. LITERATURE CITED Allison, F. E. 1955. The enigma of soil nitrogen balance sheets. Adv. Agron. 7:213-250. Bremner, J. M., and K. Shaw. 1965. Denitrification in soil. I. Methods of investigation. J. Agric. Sci. 51:22-39. Broadbent, F. E., and F. Clark. 1965. Denitrification. In (W. V. Bartholomew and F. E. Clark, eds) Soil Nitrogen, pp. 344-359. Amer. Soc. Agron., Madison, Wis. Broda, E. 1977. The position of nitrate respiration in evolution. Origins of Life 8:773-774. Broda, E. 1975. The history of inorganic nitrogen in the bio- sphere. J. Mol. Evol. 7:87-100. Buresh, R. J., K. R. Reddy, and W. H. Patrick. 1976. Influence of carbon substrate on nitrate reduction. Agronomy Abstr., p. 135. Chen, R. L., D. R. Keeney, D. A. Graetz, and A. J. Holding. 1972. Denitrification and nitrate reduction in Wisconsin lake sediments. J. Environ. Qual. 1:158-162. Chiba, S., and M. Ishimoto. 1977. Studies on nitrate reductase of Clostridium perfringens. I. Purification, some properties, and effect of tungstate on its formation. J. Biochem. 82:1663-1671. Chiba, S., and M. Ishimoto. 1973. Ferredoxin-linked nitrate reductase from Clostridium perfringens. J. Biochem. 73:1315-1318. Coleman, K. J., B. M. Newman, A. J. Cornish-Bowden, and J. A. Cole. 1978. Nitrite reduction by bacteria, pp. 334-338. In Microbiology- 1978 (D. Schlessinger, ed), Amer. Soc. Microbiol., Washington, D.C. Delwiche, C. C. 1978. Biological production and utilization of N20. Pageoph 116:414-424. Deuel, T. F. 1971. Bacillus subtilis glutamine synthetase. Specific catalytic changes associated with limited sulfhydryl modification. J. Biol. Chem. 246:599-605. Egami, F. 1976. Comment on the position of nitrate respiration in metabolic evolution. Origins of Life 7:71-72. Egami, F. 1974. Inorganic types of fermentation and anaerobic respirations in the evolution of energy-yielding metabolism. Origins of Life 5:405-413. 11 15. 16. 17. 18. 19. 20. 210 22. 23. 24. 25. 26. 27. 12 Focht, D. D., and W. Verstraete. 1977. Biochemical ecology of nitrification and denitrification. Adv. Microbiol Ecology 1:135- 199. Hall, J. B. 1978. Nitrate-reducing bacteria, pp. 296-298. In Microbiology-1978 (D. Schlessinger, ed), Amer. Soc. Microbiol., Washington, D.C. Hasan, M., and J. B. Hall. 1977. Dissimilatory nitrate reduction in CloStridium tertium. Z. Allg. Mikrobiol. 17:501-506. Hasan, S. M., and J. B. Hall. 1975. The physiological function of nitrate reduction in Clostridium perfringens. J. Gen. Microbiol. 87:120-128. Hubbard, J. S., and E. R. Stadtman. 1967. Regulation of glutamine synthetase. II. Patterns of feedback inhibition in microorganisms. J. Bacteriol. 93:1045-1055. Inderlied, C. B., and E. A. Delwiche. 1973. Nitrate reduction and the growth of Veillonella alcalescens. J. Bacteriol. 114:1206- 1212. Ilina, T. K., and R. N. Khodakova. 1976. Chemistry of denitrifica- tion in sporeforming soil bacteria. Mikrobiologya 45:602-606. (English summary). Jones, G. A. 1972. Dissimilatory metabolism of nitrate by the rumen microbiota. Can. J. Microbiol. 18:1783-1787. Kohl, D. H., G. B. Shearer, and B. Commoner. 1971. Fertilizer nitrogen: Contribution to nitrate in surface water in a corn belt watershed. Science 174:1331-1334. Koike, I., and A. Hattori. 1978. Denitrification and ammonia formation in anaerobic coastal sediments. Appl. Environ. Microbiol. 35:278—282. MacRae, I. C., R. R. Ancajas, and S. Salandanan. 1968. The fate of nitrate nitrogen in some tropical soils following submergence. Soil Sci. 105:327-334. Middelhoven, W. J., J. Berends, C. Repelius, and A. J. M. vanAert. 1976. Excessive production of ammonium from nitrate by some methanol-assimilating yeast strains. Eur. J. Appl. Microbiol. 2:169-173. Newman, B. M., and J. A. Cole. 1977. Lack of a regulatory function for glutamine synthetase protein in the synthesis of glutamate dehydrogenase and nitrite reductase in Escherichia coli K12. J. Gen. Microbiol. 98:369-377. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 13 Nommik, H. 1956. Investigations on denitrification in soils. Acta Agri. Scand. 6:197-228. Payne, W. J. 1973. Reduction of nitrogenous oxides by micro- organisms. Bacterial. Rev. 37:409-452. Prakash, 0., and J. C. Sadana. 1973. Metabolism of nitrate in Achromobacter fischeri. Can. J. Microbiol. 19:15-25. Pratt, P. F., W. W. Jones, and V. E. Hunsaker. 1972. Nitrate in deep sail profiles in relation to fertilizer rates and leaching volume. J. Environ. Qual. 1:97-102. Ralston, D. E., M. Fried, and D. A. Goldhamer. 1976. Denitrifica- tion measured directly from nitrogen and nitrous oxide gas fluxes. Soil Sci. Soc. Am. J. 40:259-266. Shapiro, B. M., and E. R. Stadtman. 1970. The regulation of glutamine synthesis in microorganisms. Ann. Rev. Microbial. 24: 501-524. Shimizu, M. 1975. Molten earth and the origin of prebiological molecules. Origins of Life 6:15-21. Sérensen, J. 1978. Capacity for denitrification and reduction of nitrate to ammonia in a coastal marine sediment. Appl. Environ. Microbiol. 35:301-305. Stanford, C., J. O. Legg, S. Dzienia, and E. C. Simpson, Jr. 1975. Denitrification and associated nitrogen transformations in soils. Soil Sci. 120:147-152. Stanford, G., J. O. Legg, and T. E. Staley. 1975. Fate of 15N- labelled nitrate in soils under anaerobic conditions, pp. 667-673. In (E. R. Klein and P. D. Klein, eds) Proceedings of the Second International Conference on Stable Isotopes, Oak Brook, Ill. Thauer, R. K., K. Jungermann, and K. Decker. 1977. Energy conser- vation in chemotrophic anaerobic bacteria. Bacterial. Rev. 41:100-180. Tubb, R. S. 1974. Glutamine synthetase and ammonium regulation of nitragenase synthesis in Klebsiella. Nature 251:481-484. Tronick, S. R., J. E. Ciardi, and E. R. Stadtman. 1973. Compara- tive biochemical and immunological studies of bacterial glutamine synthetases. J. Bacterial. 115:858-868. Tyler, B. 1978. Regulation of the assimilation of nitrogen com- pounds. Ann. Rev. Microbiol. 47:1127-1162. 42. 43. 44. 45. 46. 47. 14 Verhoeven, W. 1950. On a spare-farming bacterium causing the swelling of cans containing cured ham. Antonie Van Leeuwenhoek J. Microbial. Seral. 16:269-281. deVries, W., W. M. C. vanWyck-Kapteyn, and S. K. H. Oosterhuis. 1974. The presence and function of cytochromes in Selenomonas ruminantium, Anaerovibrio lipolytica, and Veillonella alcalescens. J. Gen. Microbiol. 81:69-78. ' Wijler, J., and C. C. Delwiche. 1954. Investigations on the denitrifying process in soil. Plant and Soil 5:155-169. Woldendorp, J. W. 1965. Formation d'ammoniaque dans le sol an cours de la reduction des nitrates. Ann. Inst. Pasteur 109: 316- 327. (English summary) Wolin, M. J., E. A. Wolin, and N. J. Jacobs. 1961. Cytochrome- praducing anaerobic Vibrio, Vibrio succinogenes, sp. n. J. Bacteriol. 81:911-917. Woods, D. D. 1938. The reduction of nitrate to ammonia by Clostridium welchii. Biochem. J. 32:2000-2012. CHAPTER II EVIDENCE FOR CLOSTRIDIA AS AGENTS OF DISSIMILATORY REDUCTION OF NITRATE IN SOILS The putative concept that denitrification results in essentially quantitative conversion of the N03— removed to nitrogenous gases (N2 plus N20) has been challenged by Stanford, et al. (24,25). They re- + .. ported substantial amounts of 15NH4 production from 15NO3 in six widely differing soils after 4 hours of anaerobic incubation (24), and observed that the proportion of N03- reduced to NH4+ and organic N was correlated with available carbon (25). Although other investigators (2,19,27) using 15N03- failed to detect NH4+ as a reduction product in soils, MacRae, et a1. (16) observed that 4.5-39% of the added 15N03- was converted to organic N after 6 weeks in rice soils. In marine was 3 converted to ammonia and particulate organic N. S¢rensen (23) demon- sediments, Koike and Hattori (11) reported 20-70% of the added NO 3 was of the same magnitude as denitrification and suggested the process strated in marine sediments that the capacity for NO reduction to NH 4 is quantitatively important in marine sediments. These observations - + suggest that reduction of N03 to NH4 in excess of amounts attributable to assimilatory reduction may be a significant fate of NO3- in some - + soils under denitrifying conditions. Moreover, NO3 reduction to NH4 is thermodynamically favorable. The AG°' for the reduction of N03- to NH4+ is -l43.3 kcal/mole of NO3 ; for the reduction of N03- to N2, kcal/ mole. Considering only net free energy change for the reduction -l33.9 of N03-, Delwiche (7) concluded the most efficient reaction under conditions of limiting carbon source is denitrification. However, when 15 16 N03- was limiting and carbon was more abundant, the reduction of N03 to NH+ 4 would be more biologically advantageous. The dissimilatory reduction of N03- to NH4+ has been observed in lake sediments (15), in marine sediments (11,23), in the rumen (14), and in pure culture by bacteria (13,22,28) and by yeast (18). Among soil bacteria, the capacity for the reduction of N03- to NH4+ seems to reside primarily with sporeforming bacteria (12,30). The respiratory nature of this process was demonstrated with pure cultures of Clostridium perfringens by Hasan and Hall (10). The inclu- sion of N03- in the culture medium permitted increased growth yield of the bacterium and resulted in increased production of more-oxidized metabolites. The yield of acetate per mole of glucose was doubled, while the amounts of ethanol, butyrate, and hydrogen were reduced. The increase in acetate production reflected an increase in ATP synthesis from acetyl phosphate made possible by N03- replacing acetyl phosphate as the terminal electron acceptor. This increased capacity for substrate level phosphorylation accounted entirely for the increased cell yields observed in the presence of NO3-. A similar phenomenon has been described in Escherichia coli with N02- serving as electron acceptor (6). In both cases, the flow of electrons to NO 3- (or N02-) is not coupled to phos- phorylation, but merely serves as an electron sink increasing the proportion of metabolite molecules which can participate in substrate .1eve1 phosphorylation. The reduction of N03- to NH4+ in soils is particularly interesting because it conserves N. Because the high levels of NH4+ accumulation observed by Stanford, et a1. (25) occurred in air-dried soils, increased auztivity of sporeforming bacteria was considered the likely reason. 17 This study was designed to estimate the relative importance of this pathway in fresh and air-dried soils, to identify the organisms respon- sible for the reduction of N03- to NH4+, and to provide evidence for the dissimilatory rather than assimilatory nature of the reduction. MATERIALS AND METHODS Soil'Studies Two agricultural soils, a Kranzburg silt loam (udic haploboroll) from South Dakota and a Conover loam (udollic ochraqualf) from Michigan were used in this investigation. The Conover soil was freshly collected and had a pH of 6.8 and an organic carbon and Kjeldahl N content of 3.1% and 0.18%, respectively. The air-dried Kranzburg soil was one of the six studied by Stanford, et a1. (25) and has been characterized by Stanford and Smith (26). One Kranzburg sample had been air-dried and stored for several years. Another Kranzburg sample was freshly col- lected at the same site as the air-dried sample. Soil (5 g) was placed into Hungate tubes (Bellco Glass Co., Vineland, New Jersey), wetted with 0.5 ml sterile water and incubated aerobically overnight at 28° C. Duplicate series of tubes for each soil were prepared, one of which was heat-shocked (68° C for 1 hour) following pre-incubation. The adequacy of this treatment for significant killing of vegetative cells was verified (Table 1). Both series received 10 m1 sterile water containing 400 ug N03--N (as KN03) and carbon source as indicated. Carbon sources were glucose and acetate (40 mg C per 10 m1 NO3- solution) which were filter-sterilized before use. For each series, duplicate samples were prepared and chemical analyses were performed on 18 Table 1. Effe t of exposure of Pseudomonas fluorescens (rif ) in soil to 68 °C for 1 hr Population Treatment (#cells/g soil) 9 None 3.60 x 10 1 hr heat-shock 1.00 x 104 2 hr heat-shock 1.10 x 104 1 hr heat-shock, 4 incubated aerobically 1.60 x 10 Washed resting cells, starved for 8 hour, were added to Conover soil. Standard plate counts were made immediately and after 1 hour and 2 hour heat-shock using tryptic soy agar (Difco) supplemented with 0.1 % (w/v) KNO and 50 ug/ml each of rifampicin and cyclohexamide. Tge plates were incubated anaerobically in a Fretertype anaerobic glavebox for 48 hours. The origin and characterization of the rifampicin-resistant mutant is described by Smith (M. S. Smith, Ph.D. Thesis, Mich. State Univ., 1978). 19 the combined samples. All treatments, except additions of inhibitors, were repeated and results are reported as means of the two replicates. The tubes were capped under an atmosphere of 02-free Ar and incubated at. 28° C for up to 5 days. Following incubation, the soil slurry was centrifuged, and the soils were extracted with 10 ml 1N KCl and centri- fuged again. The supernatants were combined and NH4+-N was assayed by steam distillation as described by Bremner (1). Soil organic N, where determined, was assayed as NH4+-N after Kjeldahl digestion of the extracted soil. No attempt was made to directly measure gaseous losses. Samples containing low concentrations of N were diluted with unenriched NH4C1 prior to final distillation to yield samples containing at least 5 mg N for analysis in the mass spectrometer. Gas analyses for 002 were accomplished using a Carle Model 8515 gas chromatograph equipped with a microthermister detector and He as the carrier gas. Hydrogen was similarly determined, but Ar was the carrier gas. Nitrate was added as K15NO3 enriched to 56.75 atom percent 15N. Samples were prepared for 15N analysis by redistilling into HCl the distillates which had been collected and titrated as described above. These samples were evaporated to dryness, converted to N2 using the method of Porter and O'Deen (21), and analyzed by mass spectrometry in the laboratory of J. 0. Legg, USDA, Beltsville, Md. All N0 - not 3 reduced to NH4+ or organic matter presumably was denitrified as essen- tially no residual N03- was observed after 5 days incubation. In subsequent experiments using 15N, a single determination of combined samples was judged adequate because of the very low standard error observed in earlier replicated experiments. 20 The effect of various compounds on the reduction of N03- to NH4+ was determined by including the compound in the added NO3--glucose solution. The influence of general assimilatory inhibitors was deter- mined using heat-shocked, freshly collected Kranzburg soil. Inhibitors used were NH4+ (as NH4C1, 2.9 mM) and glutamine (1.5 mM) and L-methionine- DL-sulfoximine (0.02 mM and 20 mM) obtained from Sigma Chemical Co., St. Louis, MO. Population Densities and Pure Culture Studies The numbers of denitrifying bacteria and N0 --reducing sporeforming 3 bacteria were determined using a modified MPN procedure. The medium used was tryptic soy broth (Difco) prepared in 0.5% (w/v) concentration, supplemented with 3.5 mM KNO and 0.05% sodium thioglycollate. The 3 medium (TSBN) was dispensed in 9 m1 aliquots into Hungate tubes flushed with Ar using the anaerobic procedure of Bryant and Robinson (5). The tubes were sterilized at 121° C for 15 min and allowed to stand over- night before inoculation. A series of lO-fold dilutions of the soil incubation mixture was prepared in Hungate tubes containing sterile 0.85% NaCl. Suspended inocula of 1.0 m1 of each dilution were trans— ferred using syringes to each of 5 tubes of TSBN. The tubes were incubated for 1 week at 28° C and scored for the presence of denitrify- ing bacteria by analyzing for N20 (20). The tubes were then heat- shocked (68° C for 1 hour) and 0.1 ml from each tube was inoculated into a fresh tube containing the same medium. Growth, as evidenced by turbidity, supplemented by microscopic examination to determine cell morphology, was used as the criterion for the presence of sporeforming bacteria. 21 Pure cultures of sporeforming bacteria were obtained from TSBN Agar plates which had been streaked with aliquots of positive tubes from the MPN assay. TSBN Agar was prepared by adding 1.5% agar to TSBN and substituting 10-3M Ti(III) citrate (31) for sodium thioglycollate as the reductant. The isolates were examined for their ability to grow aerobically and for catalase production. The ability of each of the isolates to reduce N03— to NH4+ was determined by inoculating TSBN with 0.1 ml of an actively growing culture. Aliquots of the medium were assayed for NH4+ and N03- after maximum growth had occurred. Growth was monitored spectrophotometrically at 640 nm, and absorbance was converted to dry weight values using experimentally determined conversion factors. A spore suspension of Clostridium KDHSZ, isolated from the air- dried Kranzburg soil as described above, was prepared by growing the organism on the sporulation medium of Duncan and Strong (8). The presence of spores was confirmed microscopically. The spores were harvested by centrifugation, washed three times with distilled water, and resuspended in a small volume of water. The spore suspension was heat-shocked (68° C for 30 min), diluted and added to 5 g Conover soil in Hungate tubes at a concentration of approximately 2 x 107 spores/g soil. KlsNO3 and glucose were added as before. Incubation was under 02-free Ar. For comparison, two similar samples of Conover soil were prepared and amended with the glucose-N03- solution containing no spores. One of these soils was heat-shocked (68° C for 1 hour) before amendment. .All treatments were prepared in duplicate and incubation was at 28° C £01724 hours. The incubation mixtures were extracted and analyzed for 1‘SNH4+ and 15N-organic N as described. 22 RESULTS The accumulation of 15NH4+-N and 15N-organic matter as products of 15NO -reduction in the fresh Conover and the fresh and air-dried Kranzburg 3 soils amended with glucose is shown in Table 2. The proportion of N03- reduced to free NH4+ relative to that present as organic N is much higher after 24 hours of incubation when compared to that observed after - + 5 days incubation. After 5 days, most of the 15N03 reduced to NH4 had been incorporated into organic N. This suggests that NO3 was rapidly + reduced to NH4+, then converted to organic N. (Thus, reference to NH4 as the product of NO 3 and organic N unless otherwise stated.) Heat-shocking caused reduction should be understood to mean both free NH4+ greater accumulation of NH4+ and organic N in both the Conover and fresh Kranzburg soils after 24 hours incubation. The heat treatment had no effect on the accumulation of NH4+ plus organic N in the Conover soil and in the air-dried Kranzburg soil after 5 days incubation, but resulted in a higher concentration in the fresh Kranzburg soil. Since air-drying has the same effect as heat-shock, these data suggest that sporeforming bacteria are responsible for the reactions. The effect of energy source on the reduction of 15N03- to 15NH4+ is illustrated in Table 3. Glucose, which is readily utilized by many clostridia, greatly stimulated the amount of ISNH4+ produced as compared to the unamended soils. Acetate, which cannot be utilized as electron donor by clostridia, resulted in only a mild increase in the levels of 151m: observed. The amounts of C02 and H2 produced during the incubation correlated + ‘we11.with enhanced NH4 production. Both gases are produced copiously 23 .z oaamwuo now m.o mam ZI «m2 you some some you ~.o ma moomfium> mo mfimhamam Scum commasoaao Hanna mummcmum .muamawuaaxo mowmmaou ma momma mum mosHm> m .ZIoaawmuo can ZI+¢mz mo 83m ma 2 HmuOH * .ucoemcmam au uawun A.un H you w owov mmxoaanumo: uo moumouuaa nanuwm mums mHHom .Avowuv uocv Hfiom mo Baum Han AN Baum mn.omv ZII oz we ow mo coaufivvm mafiaoaaow U cam on whom n can mam H Ham kHHmoHnouamam mmumnaoaw mam amoonaw mm ammo w\o we w Sufi? woodman mums maaom + H.Ne H.mH m.o I I I maoz AhuvIuHoaoo IIIII on w waIIIII IIIII on w walllII *He \zms was \zmH I e . a ZmH vmvo< z z + :2 2na v vv< z zI+emz uaoaumoua Haom mo N owaawuo no N oaaawuo when m . man H c +mafiom macadamIamaooaw ow Houuma oaameOIZmH can ZI+ mama mo cowumaaaaoo mo mammamcm Baum voumaaoamo Hanna vumvcmum .mucmewuoaxw mammoomu mo momma mum 2 now mmaam> ma H .Avmfium uoav Hwom w you oumuoom Ho omooaam mm 0 we w mam ImOZmH w: om sows uaoSvcosm ou Rowan A.u£ H How u omov moxoonqumon no mmumouucs nonuwa mums mawom + o mum OOHH «com How mom o.e w.mH ¢.H moxoonqummm 0 0mm new mnem NmN Nam m.~ m.mH m.o oaoz Amuqufimv muonuamum o em mmq mama mm mHH n.m o.m~ m.m moxoonmlumom 0 me mma mac ma om m.H w.HN N.~ aaoz AnmonV wuonuamuM I I I I omH omm e.o q.om w.o moxoonqumom I I I I on no m.m «.0m m.o maoz uo>oaoo H: HmuOu Hagan w\2mH wnIIIIIIIII N N N N. N N . . m 00 z 00 m 00 oumuoo< mwooaao meanness: uaoaumouu a HHom ouaumo< omooaao mmmaosman mouaom aonumo coauoamoum mow .mmam n now mHHmownouomam meow soda mawam aw moo mam mm mo soauoavoua unouusocoo ago no can pagoda owameOIz mam «m2 ou Ioz mo cowuosmou ago so mouaom aonumo mo uoommmIIm manna ma + na na + 25 during the growth of C108tridium species, but not of Bacillus species. The addition of glucose to the soils tremendously increased the amounts of bath C0 and H2 produced, whereas the addition of acetate did not 2 increase CO2 or H2 production. Again, heat treatment increased the quantity of both gases in the glucose-amended fresh Kranzburg soil, but no effect was observed in the air-dried Kranzburg. The above data suggested that the reduction of N03- to NH4+ observed was the result of the activity of Clostridium spp. Further evidence that clostridia were responsible for the NH4+ production is reflected in the population densities of denitrifying and sporeforming- NO3- reducing bacteria shown in Table 4. The freshly collected Kranzburg soil, which produced more N114+ than the Conover soil in the first day of incubation, and the Conover soil contained essentially the same number of sporeforming N03--reducing bacteria. However, the ratio of the sporeforming bacteria to denitrifying bacteria was greater in the Kranzburg sample. These population differences are also reflected in the responses to heat-shock in the fresh soils. Assuming the heat- treatment affected the non-sporeforming population of each soil similarly, the differences between the ratios would be amplified. Thus, a greater reSponse to heat-treatment would be expected for the fresh Kranzburg soil. The lack of a response to heat-treatment in the air-dried Kranzburg sample suggested the entire population was shifted in favor of sporeform- ing bacteria, many of which were denitrifying bacteria. A total of 22 sporeforming NO3--reducing bacteria were isolated and the characteristics of representative isolates are shown in Table 5. Nine of the organisms were Bacillus spp., based on their ability to grow aerobically. The three Bacillus isolates listed are representative of 26 .Nm.o ummma um mp weapoMMHm maaam> Ham uaooo Hm>mH mocovamaoo Nnm man on mausoHMMMHm unmoHMfiawwm SHHMoHumaumum a .AmuoawauuwcavumuaoavouI moz mafiahommuommv ma afiumm + omo.o ea.n em.e Aseeieeev museueeee smo.o ce.n ms.s Aemmeev meannemes moo.o me.e Hem.m eeeoeou IIIIIIIIIHfiom w\nonaaa onIIII IIIII aaumm muawwfiuuaama muaosvmuI moz uamaummuu w Hfiom mafiauowoumam .maaaamm aaom mounu ca mauauomp wafioaooHIImoz waaauOMauonm can mwuauomn wawmmauuwaom mo mofiufimamv coauaaaaomllq manna 27 .aawumoamwuufiaov ..ufioma maafiaoaam ou aOHuoacou aumuuaa ..vmm .ufiz maoaumuawauom ..auom = .aafinoa m>anm muonmmaaum :H mauoauom omz m .asavma mommanooaaca a“ venomous qmz menu you monomuuao mmaam> 1Vzz + + H .mouauaao on» ma has aw vouooumm was Noz oz + T: «.3 m.- 36 36 .359 + + $me a m.mw w.HN o.o~ cm.o mm.o .vmm .qu I I mmznz Sufivfiuumoau w.a~ m.HN n.5H om.o Hm.o .mwm .ufiz I I Nmznz anamauumaao e.Hm m.ea ~.m~ en.o es.o .emm .uez I I mammqe asseseumoau n.0n N.©H e.HN so.o mm.o .mmm .uwz I I .0 com um zoo H pom zHHmoHnoummam ooumpaocH can HooHuv uoav HHom w\o we N.m nuHa voocoam mmB HHom H + o.~q m.eN c.0H H.¢H aoHumHHaHmmm :8 o.o~ ocHBHxOMHam mHooaBm manHnaH mcHaoHSumz w.mq m.mm e.m m.MH :oHumHHBHmmm :8 No.o oEHROMHam mHaoaam manHnaH oaHaOHnumz e.om m.nH m.m o.OH aoHuoaoou oumuuHa zHOumHHEHmmm mommouooam XE m.H maHEmusHo o.mm «.mH m.m o.mH GOHuoaomu mumuuH: zHOumHHaHmmm mommoummom 28 o.~ Hoemz «.mm q.oH o.HH e.m oaoz IIIII IIIIIHHHom w\Zm :IIIIIIIIII z omoo< z z 2I+qmz GOHuo< mo moo: aoHumuuamoaoo HoanHnaH mo N Hmuae oHamwuo use Kean oaxoonquma; aH Hmuuma OHamwuoIano Homuoonao zH nmmuwv HHom muanncmux Anson H mom 0 owov oz «0 :OHuoaomu osu co muaanHnaH mo uammMMIIn mHnMH mH 31 reduced N available to the bacteria. Methionine sulfoximine, an inhibi- + . tor of ammonia assimilation (3), stimulated NH4 production at both concentrations used (0.02 mM or 20 mM). DISCUSSION The present study confirms earlier observations (24,25) that NH4+ may be produced in significant amounts from NO3- in air-dried soil placed under anaerobic conditions. The amounts of N114+ produced varied significantly in the two unamended fresh soils, but the potential for NH4+ production was essentially equal in both soils as demonstrated by the similar quantities of NH4+ produced in the glucose-amended samples after 5 days incubation (Table 3). Free NH4+ generally has not been detected as a product of N03- reduction in soils. MacRae, et a1. (16) observed a significant portion of N03- incorporation into the organic N fraction without observing N114+ as an intermediate. However, the analyses were performed after 6 weeks incubation and the present study demonstrates that NH4+ was produced during the first 24 hours of anaerobiosis and by 5 days it had been virtually completely converted to organic N (Table 2). Stanford, et al. (24,25) observed the same sequence of events. Ammonia appeared after 4 hours incubation and increased for the first 24 hours. During this period there was a simultaneous increase of 15N in organic matter, suggesting NH4+ was an intermediate. Keeney, et a1. (15) observed a 15 relatively constant level of NH4+ during incubation of lake sediments following addition of 15NO3-. Although termed assimilatory reduction by the authors, the fate of the N03 in this sediment is not unlike that observed in soils. 32 Several genera of bacteria have been reported capable of reducing N03- to NH4+ (Chapter I). More recently, an Alcaligenes and a Micrococcus have been observed to produce NH + (M. K. Firestone, personal 4 communication). Although this study does not obviate the involvement of these bacteria, reduction of N03- to NH4+ in the soils studies appeared primarily to be the result of the activity of sporeforming NO3--reducing bacteria, principally Clostridium spp. Five facts support this thesis. First, heat-treatment (68° C for 1 hour) did not reduce the quantity of NH4+ produced in the Conover soil and the air-dried Kranzburg soil. In 4. 4 accumulation. Bacterial spores are not damaged by exposure to these the fresh Kranzburg soil, heat treatment stimulated the rate of NH temperatures; in fact, spore dormancy is broken and germination is induced. Vegetative cells, on the other hand, are generally destroyed by such harsh treatment (Table 1). Second, the amount of NH4+ was greatly increased by glucose, while acetate produced only a slight produced increase. Clostridium spp. are able to metabolize glucose, but not acetate, as an energy and carbon source. Third, copious production of CO2 and H2 in the glucose-amended samples also indicated a high level of activity by Clostridium spp., since Bacillus spp. do not produce large amounts of H2. There was no H2 detected in the acetate-amended samples, and only a small increase in CO2 evolution was observed. Fourth, the initial ratio of sporeforming NO3--reducing bacteria to denitrifying bacteria was greater in the soil which produced larger quantities of NH4+ in the first 24 hours. Finally, the most common isolate encountered 3- to NH4+. A number of organisms have been isolated previously which were was Clostridium, all but one of which reduced NO . + capable of reduCing N03 to NH4 under laboratory conditions. Some of 33 these could reduce N03- only as resting cells (29), whereas others reduced N03- to NH4+ during growth (9,10,13,22,30). But, none were shown to be active in a soil system. Woldendorp (28) observed that Bacillus licheniformis reduced N03- to NH4+ in pure culture, but the 3 when introduced into the rhizosphere of pea plants. Clostridium KDHSZ, however, did produce NH4+ from N03- when added back to soil (Table 6), indicating it can effectively compete with organism failed to reduce NO 3 This observation also provides additional support for the thesis that denitrifying bacteria for NO in anaerobic soils if carbon is available. clostridia are responsible for the reduction of N03- to NH4+ in soils. The process by which NO3 was reduced to NH4+ appeared to be a dissimilatory mechanism. Available carbon did limit the amount of N03- reduced to NH4+ (Table 3), and the increase in NH4+ produced following amendment with glucose could imply an assimilatory mechanism. However, all of the Clostridium strains isolated exhibited increased cell yields when N03- was present in the growth medium (Table 5), demonstrating the dissimilatory function of the reduction. As shown in Table 7, neither glutamine nor NH4+, both inhibitors of assimilatory NO3 reduction suppressed the reduction of N03_ to NH4+. When the extracellular concentration of NH + is less than 1 mM, 4 NH4+ is assimilated by enteric bacteria via the combined activity of glutamine synthetase and glutamate synthase (4) as shown in Equations 1 and 2. (a) Glutamate + N114+ + ATP + Glutamine + ADP + P i (l) (b) Glutamine + a-Ketoglutarate + NADPH + H+ + 2 Glutamate + NADP+ (2) 34 (a) glutamine synthetase (b) glutamate synthase In addition to this function, glutamine synthetase has been shown to regulate the synthesis of a number of enzymes responsible for supplying nitrogen to cells (17). Brenchley (3) observed with Klebsiella aerogenes that 0.01 mM methionine sulfoximine caused approximately 70% inhibition of glutamine synthetase activity. Increasing the concentration to 10 mM produced a complete inhibition of both glutamine synthetase and glutamate synthase. Therefore, if this assimilatory process were involved in the observed formation of NH4+, the quantity of N114+ produced would be much greater in the presence of methionine sulfoximine. The observed increase suggests that regulation of the enzymes reducing NO - to NH4+ may be 3 linked to this assimilatory mechanism, contradicting the evidence obtained using glutamine and NH4+ as effectors. However, glutamine synthetase has been shown to exert no effect on the reduction of N03 to + NH4 by pure cultures of Clostridium KDHSZ (Chapt. III). Thus, the presence of methionine sulfoximine was probably inhibiting the growth of other microorganisms in the soil, thereby reducing competition for both electron donor and acceptor. The increase in NH4+ accumulation, then, was the result of greater activity of the NH4+-producing bacteria and was unrelated to the character of the enzymes involved. All of the evidence presented above indicated the reduction of N03 to NH4+ occurred via a true dissimilatory process. This theory is supported additionally by previous reports that two Clostridium spp. - + reduce N03 to NH4 by a primitive form of anaerobic respiration (9,10). 35 Also, none of the effectors above exert any influence on the reduction of N03- to NH4+ by pure cultures of Clostridium KDHSZ (Chapt. III). + Because significant amounts of N03 can be reduced to NH4 by Clostridium spp., caution must be exercised in studies of denitrifica- tion using air-dried soils in which the ratio of sporeforming to non- sporeforming N03--reducing bacteria may have shifted. But, as indicated by the data in Table 2, the potential for NH4+ formation from N03- exists in fresh soils, too. Although other investigators (19,27) using 15NO3-have not detected NH4+, the lengthy incubation periods, as pointed out by Stanford, et a1. (25), could have made detection of the process difficult. Therefore, any short-term study of anaerobic nitrogen transformations in soil systems should include analysis for this fate of 3 O The observation that Clostridium KDHSZ, when supplied with an NO 3 with deni- trifying bacteria is encouraging. A greater understanding of the appropriate electron donor, successfully compete for NO 3 between Clostridium spp. and denitrifying bacteria could lead to develop- biochemical and ecological controls exerted on the competition for NO ment of management practices which conserve nitrogen in soils under denitrifying conditions. ACKNOWLEDGEMENT The author expresses appreciation to Joe Legg, Agricultural Research Station, Beltsville, Md., for his assistance in analysis of the 15N samples by mass spectrometry. Air-dried soil samples were kindly supplied by J. O. Legg and G. Stanford and the fresh Kranzburg sample, by R. F. Holt, North Central Soil Conservation Research Center, Morris, Minnesota. 10. 11. 12. LITERATURE CITED Bremner, J. M. 1965. Exchangeable ammonium, nitrate, and nitrite by steam-distillation methods. In C. A. Black (ed.) Methods of Soil Analysis. 2. Chemical and microbiological Properties. American Society of Agronomy, Madison, Wisconsin, pp. 1191-1206. Bremner, J. M., and K. Shaw. 1958. Denitrification in soil. 1. Methods of investigation. J. Agric. Sci. 51:22-39. Brenchley, J. E. 1973. Effects of methionine sulfoximine and methionine sulfone on glutamate synthesis in Klebsiella aerogenes. J. Bacteriol. 114:666-673. Brenchley, J. E., M. J. Prival, and B. Magasanik. 1973. Regula- tion of the synthesis of enzymes responsible for glutamate forma— tion in Klebsiella aerogenes. J. Biol. Chem. 248:6122-6128. Bryant, M. P., and I. M. Robinson. 1961. An improved nonselective culture medium for ruminal bacteria and its use in determining diurnal variation in numbers of bacteria in the rumen. J. Dairy Sci. 44:1446-1456. Coleman, K. J., B. M. Newman, A. J. Cornish-Bowden, and J. A. Cole. 1978. Nitrite reduction by bacteria. In David Schlessinger (ed.) Microbiology-1978, American Society for Microbiology, Washington, D.C., pp. 334-338. Delwiche, C. C. 1978. Biological production and utilization of N20. Pageoph 116:414-424. Duncan, C. L., and D. H. Strong. 1968. Improved medium for sporulation of Clostridium perfringens. Appl. Microbiol. 16:82- 89. Hasan, M., and J. B. Hall. 1977. Dissimilatory nitrate reduction in Clostridium tertium. Z. Allg. Mikrobiol. 17:501-506. Hasan, S. M., and J. B. Hall. 1975. The physiological function of nitrate reduction in Clostridium perfringens. J. Gen. Microbiol. 87:120-128. Koike, I., and A. Hattori. 1978. Denitrification and ammonia formation in anaerobic coastal sediments. Appl. Environ. Microbiol. 35: 278-282. Illina, T. K., and R. N. Khodakova. 1976. Chemistry of denitrifi- cation in sporeforming soil bacteria. Mikrobiologya. 45:602-606. (English summary) 36 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 37 Inderlied, C. B., and E. A. Delwiche. 1973. Nitrate reduction and the growth of Veillonella alcalescens. J. Bacteriol. 114:1206- 1212. Jones, G. A. 1972. Dissimilatory metabolism of nitrate by the rumen microbiota. Can. J. Microbiol. 18:1783-1787. Keeney, D. R., R. L. Chen, and D. A. Graetz. 1971. Importance of denitrification and nitrate reduction in sediments to the nitrogen budgets in lakes. Nature 233:66. MacRae, D. C., R. R. Ancajas, and S. Salandanan. 1968. The fate of nitrate nitrogen in some tropical soils following submergence. Soil Sci. 105:327-334. Magasanik, B. 1977. Regulation of bacterial nitrogen assimilation by glutamine synthetase. Trends Biochem. Sci. 2:9-12. Middelhoven, W. J., J. Berends, C. Repelius, and A. J. M. vanAert. 1976. Excessive production of ammonium from nitrate by some methanol-assimilating yeast strains. Eur. J. Appl. Microbiol. 2:169-173. Nommik, H. 1956. Investigations on denitrification in soils. Patriquin, D. G., and R. Knowles. 1974. Denitrifying bacteria in some shallow-water marine sediments: enumeration and gas produc- tion. Can. J. Microbiol. 20:1037-1041. Porter, L. K., and W. A. O'Deen. 1977. Apparatus for preparing nitrogen from ammonium chloride for nitrogen-15 determination. Anal. Chem. 49:514-516. Prakash, 0., and J. C. Sadana. 1973. Metabolism of nitrate in Achromobacter fischeri. Can. J. Microbial. 19:15-25. S¢rensen, J. 1978. Capacity for denitrification and reduction of nitrate to ammonia in a coastal marine sediment. Appl. Environ. Microbiol. 35:301—305. Stanford, G., J. O. Legg, and T. E. Stanley. 1975. Fate of 15N- labelled nitrate in soils under anaerobic conditions. In E. R. Klein (eds.) Proceeding of the Second International Conference on Stable Isotopes, Oak Brook, Illinois. Stanford, G., J. 0. Legg, S. Dzienia, and E. C. Simpson, Jr. 1975. Denitrification and associated nitrogen transformations in soils. Soil Sci. 120:147-152. Stanford, G., and S. J. Smith. 1972. Nitrogen mineralization potentials for soils. Soil Sci. Soc. Am. 36:465-472. 27. 28. 29. 30. 31. 38 Wijler, J., and C. C. Delwiche. 1954. Investigations on the denitrifying process in soil. Plant Soil. 5:155-169. Woldendorp, J. W. 1975. Formation l'ammoniaque dans le sol au cours de la reduction des nitrates. Ann. Inst. Pasteur. 109: 316- 327. (English summary) Wolin, M. J., E. A. Wolin, and N. J. Jacobs. 1961. Cytochrome- producing anaerobic vibrio, Vibrio succinogenes, sp. n. J. Bacteriol. 81:911-917. Woods, D. D. 1938. The reduction of nitrate to ammonia by Clostridium welchii. Biochem. J. 32:2000-2012. Zehnder, A. J. B., and K. Wuhrmann. 1976. Titanium (III) citrate as a nontoxic oxidation-reduction buffering system for the culture of obligate anaerobes. Science 194:1165-1166. Chapter III THE DISSIMILATORY REDUCTION OF NITRATE TO AMMONIUM BY A CLOSTRIDIUM SPP. ISOLATED FROM SOIL In anaerobic environments, denitrification is generally considered to be the principal pathway of N03 reduction. Although evidence is sparse, flooded soils (22) and sediments (18,20,29) have shown signifi- cant quantities of N03- reduced to NH4+. While agricultural soils have typically shown no N114+ production from N0 3 when incubated anaerobically + (3,25,34), recent reports correlating increased N03 reduction to NH4 with increased amounts of available carbon (30,31) suggested the poten- tial for N114+ formation exists in such soils. Elsewhere (Chapt. II) evidence was presented that NH4+ was produced through the activity of sporeforming bacteria, principally Clostridium species, via a dissimila- tory pathway. A clearer understanding of the physiology of these NO3 reducing bacteria may reveal approaches which can be used to predict the occurrence of N03_ reduction to NH4+, or possibly to enhance this N- conserving process in agricultural soils. Clostridium perfringens (15), §;_£grtium (l4), and a number of unidentified Clostridium spp. (Chapt. II) have been shown to reduce NO3 to NH4+. All strains capable of this reduction showed increased growth yields. Hasan and Hall (15) have shown the additional ATP production results from a shift in the oxidation state of the fermentation end products in the direction of increased acetate formation which allows increased substrate-level phosphorylation. Cytochromes are involved in anaerobic electron transport to nitrate in Veillonella alcalescens and Selenomonas ruminantium (33), but ferredoxin was reported to mediate 39 40 N03- reduction 1“.§; perfringens (8). The nitrate reductase of C; perfringens has been purified to near homeogeneity (7). It is an inducible, soluble molybdo-iron-sulfur protein. Comparative fermentation balances and measurement of YATP with and without N03- have provided evidence for the dissimilatory function of NO3- reduction in Clostridium spp. However, reliable measurements of the extent of NO3- reduction by these bacteria are lacking. Further- more, little information is available on the regulation of NO3 reduc- tion by Clostridium spp. This study, in addition to providing defin- itive balances of N03— reduction to NH4+, N03--reducing system of Clostridium KDHSZ shares no regulatory features with assimilatory nitrate reductases. presents evidence that the MATERIALS AND METHODS Bacterial strain and culture media. Clostridium KDHS2 was isolated from a Kranzburg silt loam (Chapt. 11) which had been air-dried and stored for several years after collection in South Dakota. Cultures 'were maintained on a basal medium (R.N. Costilow, personal communica- tion) that contained (per liter): vitamin-free casamino acids (Difco), 5.0 g; trypticase (BBL) and yeast extract (Difco), 1.0 g each; and sodium thioglycollate, 0.5 g. Glucose (0.2 % w/v) was supplied as an energy source, and KNO3 was added where indicated at 3.5 mM. Monosodium glutamate (0.2 % w/v) was added to the medium for studies involving glutamine synthetase. 3 ‘medium was determined in batch culture using the basal medium. Cultures The response of Clostridum KDHSZ to the addition of NO to the ‘were grown in Hungate tubes (Bellco Glass, Vineland, N.J.) under 02-free 41 Ar according to the procedure of Bryant and Robinson (6). Growth was monitored at 640 nm and absorbance was converted to dry weight using an experimentally determined conversion factor. Aliquots were analyzed at the beginning and end of exponential growth for glucose, N03-, N02-, and NH4+. Chemical analyses. Glucose was determined colorimetrically using the ortho-toluidine reagent (Sigma Chemical Co., St. Louis, MO) (28). NH4+-N was determined by titration following steam distillation as described by Bremner (2). N03--N was similarly determined following reduction to NH4+ using Devarda's alloy (2). N02--N was measured colorimetrically by the diazo-coupling method (1). Resting cell experiments. Clostridium KDHSZ was cultured in basal medium containing NO3 and harvested in mid-exponential growth (8-10 h) by centrifugation. The cells were washed three times in 0.01 M Tris (hydroxymethyl) aminomethane (Tris)-maleate buffer, pH 7.0, and resus- pended in the same buffer. The suspension was introduced into Hungate tubes containing sufficient pre-reduced, sterile sodium thioglycollate to yield a 0.05 % (w/v) concentration of the reductant. The atmosphere over the suspension was 02-free Ar. The cell suspensions were stored at 4° C until used, the time of storage not exceeding 18 h. Final concen- tration of cells was approximately 2 mg/ml (dry weight). 3 for Clostridium KDHSZ was made by measuring NH4+ production from N03- during a 1 hour incubation at room An estimate of the KS for NO temperature. The reaction mixture contained in 10 ml: 2.0 m1 resting cell suspension, 2.0 mM KN03, 0.2% (w/v) glucose, and 0.05% (w/v) sodium thioglycollate. Aliquots were removed at 10 min intervals and analyzed as described for N03-, N02-, and NH4+. No N02_ was detected in any of 42 these samples. The data were analyzed as a progress curve (9) to deter- 3 I 13 - 13 The production and purification of NO3 and the N detection methods are described in detail elsewhere (12). The 13N03- substrate used usually contained some 13N02_ (5-10%), hence the 13NH4+ produced was from both compounds. The reaction mixture contained in 4.5 ml: 1.0 mine the K8 for N0 m1 resting cell suspension, glucose (0.2% w/v), 1 ug N as KNOB, and sodium thioglycollate (0.05%). Approximately 0.4 mCi 13NO3- was added in 0.5 m1 and the mixture was incubated in Hungate tubes at room temper- ature for 20 min. The reaction was stopped by filtering through a 0.45 um pore size membrane filter. Aliquots of the filtrate were analyzed for 13N03-, 13N02: and 13NH4+ using a high pressure liquid chromatograph (HPLC) equipped with an anion exchange column (Partisil SAX, Whatman, Inc., Clifton, N.J.). The column was eluted at the rate of 6 m1/min with 0.05 M phosphate buffer, pH 3.0. The effluent was monitored by dual 2" x 2" NaI(T1) coincidence detectors, computer-linked for data collection and correction for half-life and background. The effect of a variety of compounds on N03- reduction to NH4+ was measured. NHACl (3.5 mM), glutamate (3.5 mM). and glutamine (1.75 mM) were studied as inhibitors of assimilatory nitrate reduction. L- Methionine-D,L-sulfoximine (Sigma Chemical Co., St. Louis, MO) (0.01 mM and 10 mM) was included as an inhibitor of ammonia assimilation (4,5). Azaserine (1.0 mM) was used as an analog of glutamine (36,37). Also, Na2804 and NaZSO3 (3.5 mM) were studied as possible alternate substrates 3- to NH4+. Similar experiments were done using resting cells grown in basal for the enzyme system reducing NO medium supplemented with glutamate and 0.01 mM and 10 mM methionine 43 sulfoximine. The 13NO3_-reducing activity of these cells was measured in the presence of methionine sulfoximine at the concentration in which they were grown and in the absence of the compound. 35S-sulfate and -sulfite were used to directly determine whether the N03- reducing enzyme system in Clostridium KDHSZ could also reduce these electron acceptors. Resting cells were prepared from cultures grown in basal medium supplemented with KNO3 (3.5 mM) or NaZSO4 (3.5 mM) for these experiments. Sulfite-grown cells could not be used because Clostridium KDHSZ would not grow in media containing 3.5 mM SO3 . The reaction mixture contained in 5.0 ml: 1.0 ml of cell suspension, 0.2% glucose, 0.3 mCi 35803. (or 35$043) plus unlabelled $03= (or 8043) to 3.5 mM, 0.05% sodium thioglycollate, and either 0, 1.0, or 3.5 mM KNOB. The production of S' after 2 hours incubation in Hungate tubes was measured by adding concentrated HZSO4 to the reaction mixture to a final concentration of 1.7 M (D. E. Caldwell, Ph.D. Thesis, Mich. State Univ., 1974) and sparging with air for 10 min into a trap containing 5.0 ml of a saturated solution of lead acetate. One ml of the trapping solution was added to 15 m1 of aqueous scintillation counting solution (ACS, Amersham, Arlington Heights, Ill.) and the amount of 355= measured in a Packard Model 3310 Tri Carb Scintillation Spectrometer. Preparation of cell free extracts. Cultures of Clostridium KDHSZ were allowed to grow into late exponential phase (W 10 hours) in the basal medium containing KNO3. Cells were collected by centrifugation, washed with 0.01 M Tris-HCl buffer, pH 8.0, and resuspended in the same buffer. The organisms were ruptured by passage through a French pressure cell at 16,000 psi. Debris was removed by centrifugation at 9,000 x g for 30 min and the supernatant was used as crude extract. Nitrate 44 reductase was partially purified by (NH4)ZSO4 precipitation. The precipitate at 40-80% saturation (7) was collected by centrifugation at 19,000 x g for 20 min and was immediately dissolved in 0.01 Tris-HCl buffer, pH 8.0, then dialyzed against the same buffer. Clostridium KDHSZ grown on the basal medium supplemented with glutamate and KNO3 and harvested 4-6 hours into stationary phase. The cells were washed with and resuspended in 0.01 M morpholinopropane sulfonic acid (MOPS) buffer, pH 7.0, containing 0.01 M MnCl and 0.001 M 2 mercaptaethanol. Crude extracts were prepared as above and glutamine synthetase was partially purified by (NH4)2804 precipitation. The fraction precipitating at 50-70% saturation (17) was collected by centrifugation at 19,000 x g and immediately dissolved in the MOPS buffer described above, then dialyzed against the same buffer. Protein was determined by the method of Lowry, et a1 (21) using bovine serum albumin as the standard. Enzyme assays. Nitrate reductase activity was measured using a modification of the method described by Chiba and Ishimoto (7). The reaction mixture contained in a final volume of 0.5 ml: 95 umol of Tris-HCl buffer, pH 9.0, 10 umol of KNO3, 1 umol of methyl viologen, 30 umol of Na28204, 20 umol of Na2C03, and approximately 0.7 mg of the partially purified enzyme. The reaction was started by adding Na28204- Na2C03 and stopped after incubation for 15 min at 37° C by vigorous agitation on a vibrator until the violet color of the reduced dye disappeared. One ml of 95% ethanol was added and the mixture was centrifuged at 3000 x g for 5 min. An aliquot of the supernatant was assayed for N02 by the method described above. The amount of N02- formed was proportional to the amount of enzyme preparation used. An 45 estimate of the Km for N03- was made by increasing the volumes of all reagents 10-fold and using progress curves (9). The assay was demon- strated valid for 5 umol and 20 umol KNOB. Glutamine synthetase activity was monitored by measuring the glutamate-dependent liberation of ortho-phosphate (Pi) in the biosynthe- tic reaction (17). The extract would not catalyze the reverse, or y- glutamyl transfer, reaction. The assay mixture contained in 0.4 ml: 0.1 mg protein, 0.0075 M ATP, 0.1 M glutamate, 0.05 M NH Cl, 0.005 M 4 MnCl , and 0.01 M MOPS buffer, pH 7.0. Incubations were carried out in 2 open tubes at 37° C for 15 min. Reactions were initiated by the addition of enzyme. Controls lacking glutamate were included in each incubation, and corrections were made for the phosphate liberated in the absence of substrate. Measured activity of the enzyme was proportional to the amount of enzyme preparation added and was linear with time over 15 min when 0.1 mg of enzyme was used. Activity was expressed as umol Pi formed per min per mg protein. The inhibition of glutamine synthetase activity by methionine sulfoximine and glutamine was measured by including the appropriate amount of each inhibitor in the ATP solution. Methionine sulfoximine was added to the assay mixture at concentrations of 0.01 mM and 10 mM, and glutamine was added at 10 mM. The apparent Km of the enzyme for + NH4 was estimated using initial velocity reactions and the data were analyzed using direct linear plots (11). RESULTS The addition of N03- to the culture medium resulted in more total growth and in a more rapid growth rate for Clostridium KDHSZ (Fig. l). 46 1 0.30 .0 N 0 GROWTH (mg/ml dry wt.) l l l J O 4 8 I? I6 TIME (hrs.l Fig. 1. Growth response of Clostridium_KDHSZ to NO3 in the medium. Growth in presence (0—0) and absence (H) of 3.5 mM NO 3 for inoculum grown without N03-. Growth in the presence of NO3- (H) for inoculum grown in NO 3 -containing medium. 47 Curves labelled P and N represent the growth of the organism in the basal medium containing NO3- after inocula were grown in media with and without N03-, respectively. Curve G depicts the growth of the organism in the absence of NO3-. There was a consistent lag of about 4 hours before initiation of exponential growth. The molar growth yields of the bacterium in the presence of N03 were about 13% higher when compared to those obtained in media without N03- (Table l). The molar growth yields were linearly related to glucose concentration up to 0.3% (w/v) glucose. The extent of NO3- reduction to NH4+ was measured concurrently with the molar growth yields (Table 1). Significant amounts of NH4+ were produced in the medium in the absence of N03", and this value was used as a background to correct the amounts measured in the NO3-containing medium. A similar phenomenon was observed by Hasan and Hall (14,15) and corrections were made using this approach. However, this approach was valid only when applied to N03- reduction during exponential growth. The calculated amounts of NH4+ produced agree quite well with the measured amounts of N03- reduction, these amounts representing approxi- mately 20% of the added N03 . No NOZ- was observed and NH4+ was apparent- ly the only product of NO3 reduction. The kinetics of N03- reduction by Clostridium KDHSZ were studied using intact cells and partially purified N03 reductase. The organism was observed to reduce N03- maximally at a rate of 1.5 ug N/hr mg cells and the concentration of NO3- producing one-half this velocity was estimated as 0.5 mM. For the partially purified NO3 reductase, the Km for N03- was observed to be 0.15 mM. Compounds which are known inhibitors of assimilatory N03 reduction and ammonia assimilation were examined for possible effects on NH4+ 48 Table 1. The reduction of NO ‘ to NH + by Clostridium KDHSZ in axenic culture and its effect on cell yield. Preinduced cells Without N03" With N03' with N03 Nitrogen Balance (ug N/ml)+ N03" reduced - 10.0 9.6 NH4+ in medium Initial 8.2 8.2 8.2 After growth 33.7: 42.7 42.9 From N03" - 9.0 9.2 Glucose used (umol/ml) 6.5 9.8 11.4 Cell yield (dry wt mg/ml) 0.10 0.17 0.20 Molar growth yield 15.4 17.4 (+13.0%) 17.5 (+13.6%) (Dry wt gram/mol glucose) +Media containing NO3 Media with no N0 — contained considerable NH + received 49.9 ug/ml NO -N. 3 This value was used as a background to correct the values determined in the N03 containing media. 49 production by resting cells of Clostridium KDHSZ (Table 2). None of these compounds, including NH4+, glutamate, glutamine, 0.01 mM and 10 mM methionine sulfoximine, and azaserine, had any effect on the reduction 13 13 + of N03 to NH4 . nate substrates for the enzyme system. Sulfate was without effect, but sulfite inhibited 13NO - reduction. Figure 2 illustrates the reduction 3 of 13N03 + 13N02 during the 20 min incubation period. The disappearance of the N02- peak in the samples incubated with no inhibitor indicated N02- is an intermediate in N03- reduction to NH4+, and that the bac- terium is capable of reducing NO Sulfate and sulfite were tested as possible alter- 2-. Note, however, that the N02- peak persisted in the $03 inhibited sample, suggesting the inhibition occurs at the level of nitrite reductase. Although not as apparent because of the large amount of 13NO3- present, 13N03- was reduced to 13NH4+ along with the 13N02-. To provide more information on the apparent $03= inhibition of N03- reduction by Clostridium KDHSZ, the reductibn of $04= and $03= to S= in the presence of two concentrations of NO3 was measured (Table 3). Significant amounts of 8048 were not reduced to S= by Clostridium KDHSZ 3 or SO4 . A low level of $03. reducing activity was observed in cells grown in the absence of grown in basal medium containing either N0 electron acceptors. Slightly higher levels of activity were observed when Clostridium KDHSZ was grown in the 804 -containing medium. A 10- fold increase in activity relative to fermentatively-grown cells was observed for cells grown in the NO3--containing medium, indicating that the enzymes induced by N03- also reduce SO38. There was no inhibition of 803- reducing activity by either concentration of NO3 . 50 Table 2. ngect of Igrious compounds on the reduction of NO to NH by resting cells of Clostridium KDHSE grown in basal medium containing NO3 . Inhibitor 13NH + Produced (log dpm/mg cells)+ Expt. 1 Expt. 2 None 5.39 5.22 NH: 4.99 5.01 Glutamate 5.47 5.31 Glutamine 5.05 5.26 Methionine sulfoximine, 0.01 mM 5.02 4.99 Methionine sulfoximine, 10 mM 5.56 5.30 Azaserine, 1.0 mM 5.53 5.09 303’, 3.5 ml! 4.43“ 4.34“ 304', 3.5 mM 5.14 5.10 Autoclaved cells --None detected-- +Incubated at room temperature for 20 min with 14.3 nM unlabeled N03--N. ** Significantly different at 95% confidence level. 601 so O ”IN (I04 dpm/Z sec interval) 51 PLUS SULFITE | I “\\ «— N I I I I N'Hd’ N92" I 05" _ I I -‘ I _ M I UNA—L- NO INHIBITOR I I I I | - I I Mimi STERILE CONTROL I “hit! kxfidLNflflda. I i i Fig. 2. 2 4 6 RETENTION TIME (min) The reduction of 13N03' and 13N02' to 13NH4+ by resting cells of glostridium KDHSZ, by resting cells in the presence of 3.5 mM 803:, and by autoclaved cells. 52 .muson N mma uoauoa coaumnsocH .A new uov com 28 m.m mo :ofiumuucmocou Hmcfim ca A mow uov cow mm mmm Hue m.o no:HMumOo musuxwa cofiuommm + on 0H OH owes once owmm uqom oq ma Hm ooamm oomcm oommm nmoz om ma Na omom oamm oaqm oaoz umoz nmoz nmoz -moz umoz nmoz sesame suaouw a“ :5 m.m za o.H oz :5 n.m as o.H oz Houamoum nouuowfim AmHHmo we\EQv NoHv qom BOHM UMUSQOHQ m mm Amaamona\eav NOHV mow aouw couscoum m mm umumm m op mom u mm n mm .muounmoum couuomao uamquMH + «om mo coauuavmu on» so a“ a 1 mm . m nufiz suzouw oz mo uuommm .m magma 53 Glutamine synthetase was produced by Clostridium KDHSZ (Table 4). Glutamine at 10 mM inhibited the enzyme by 60%. Methionine sulfoximine caused 32 inhibition when added at 0.01 mM, but resulted in 94% inhibi- tion when the concentration was increased to 10 mM. The Km for N114+ for Clostridium KDHSZ glutamine synthetase was calculated to be 4.0 x 10-4 M (Fig. 3). To ascertain whether glutamine synthetase exerted any regulatory influence on the synthesis or activity of the enzyme system reducing N03- to NH +, Clostridium KDHSZ was grown in the basal medium containing 0, 0.01, and 10 mM methionine sulfoximine. The data in Table 5 depict the ability of these cells to reduce N03 to N114+ in various concentra- tions of methionine sulfoximine. No effect was observed on activity or synthesis of the N03--reducing pathway. DISCUSSION Clostridium KDHSZ grew more rapidly in the presence of N03- and also exhibited a greater yield of cells per mole of glucose utilized. This increase in cell yield suggested that more efficient utilization of 3 present. Hasan and Hall (14) observed the same dual effect on Q; the energy derived from glucose catabolism was permitted when NO was tertium, but with Q; perfringens only an increase in growth yield was observed (15). With §:_tertium the observed increase in YATP in the presence of NO3- was attributable to the increased growth rate, but the increase in cell yield could only be explained if the N03 was being reduced by some type of dissimilatory mechanism. 2 the only detectable intermediate. Because the concentration of N03- in Nitrate was reduced solely to N114+ by Clostridium KDHSZ, with N0 54 .muuoawuonxo oumummom mounu Eouw mamas mum umuuoamu mosam> + He NH.o 25 0H .meaamuaao cm No.0 :8 OH .mcfiaflxomaom oaHGOHnuoz m om.o :8 Ho.o .oaaaHxOMHSm msfi:0finuwz I Hm.o oaoz Auv Aaucaououn we H m Hoanv noufinancH coauaaaneH _ +aua>auo< .NmeM Ebfiuauuwoao mo muomuuxo moumlaaao scum woummoua mmmuonushm oaHBMuaaw mo muw>wuom so muouunfissw mo uommmm .q manna 55 (Faun .pauuo; d sa|omu)/\ N. m. cm mcHEmuaaw HOw + .NmeM Eswvaumoflo mo ammumzucmm E «:2 HOW :ofiumefiumm M «o uoHa ummcfia uomuwa AEEV +¢IZW I 1 : -o: oészzEg :23: .m .mE 56 .Ho>oH monouamsoo Nmm msu um mosam> wan mo mam amo3uwn musouo moamumMMHw unwowmaamfim oz + mm.m I m¢.m OH I No.m Hm.m Ho.o mq.m Ho.m +¢m.m O OH Ho.o o £5 £5 cowmcommsm Hams wcfiummu a“ mawEHwaaam o:w:oanuoz aawwma nuaouw :H m mcfieHx0maaw oswsowsuoz Amaaou w8\anu onV couscoum + mZMH «E m .NmmnM asfivfiuumoao mo mHHoo wawummu mp Ou 02 mo :oauoavou onu so mafiawxomaam mfiwdomMumEI mo ummmmm .m manna 57 the growth medium was selected for minimal N02 accumulation, no N02 was observed in any of the cultures, however. N02- is toxic to many bacteria and in.§g_perfringens the ability of N03 to support enhanced growth yields is limited to 3.5 mM NO or less, because of N02 3 accumulation (15)., A similar pattern was observed for Clostridium KDHSZ. The disappearance of N02- in the 13N03- accumulation of N02- as the product of the partially purified nitrate experiments and the reductase provide evidence for N02- being an intermediate. The nitrogen balances obtained are reasonable and consistent with those observed for both 9; perfringens and §:_tertium. As mentioned earlier, the amount of NH4+ produced from NO3 could only be calculated during exponential growth. The difficulty in balancing the nitrogen species after the organism began to enter stationary phase was most likely the result of a physiological change in the NO3--reducing system. Chiba and Ishimoto (7) observed the nitrate reductase of g; perfringens was produced at high specific activity only during exponential growth. 3 reduction by Clostridium species, and is consistent with a dissimilatory The data presented thus far confirms earlier reports of NO reductive process. Additional evidence supporting the dissimilative function of the N03_ reduction is supplied by the lack of inhibition by NH4+, glutamate, and glutamine (Table 2). NH4+ has been demonstrated to inhibit activity of assimilatory NO - reduction (13,27), whereas dissim- 3 ilatory N03- reduction is unaffected in facultative bacteria (13,24,35). Glutamate and glutamine are readily utilized as nitrogen sources by bacteria and would be expected to inhibit assimilatory NO3 reduction. One proposed mechanism for N114+ production is via decomposition of organic nitrogenous compounds formed by assimilation of NO3 . Brenchley, 58 et a1 (4) observed that 0.01 mM methionine sulfoximine caused a 70% inhibition of activity of glutamine synthetase, but had no effect on the activity of glutamate synthase. Increasing the concentration of methionine sulfoximine to 10 mM completely inhibited both enzymes. Thus, if the N114+ was being produced according to the proposed mechanism, the pres- ence of methionine sulfoximine would enhance the amount of N114+ produced. However, no such effect was observed (Table 2). Additional evidence disproving this assimilation-release theory is provided by the absence of any effect exerted by azaserine on the amount of NH4+ produced (Table 2). Azaserine is a competitive inhibitor of glutamine in reactions involving amino group transfer (37). Therefore, if the proposed hypo— thesis were valid, the presence of azaserine would decrease the amount of NH4+ produced. These data further emphasize the non-assimilatory features of the Clostridium KDHSZ NO --reducing enzymes. 3 Enzyme preparations from Escherichia coli and baker's yeast that possess the capacity for N02 reduction have been shown to be primarily 803: reductases in_vivo (19,26). If this were the case for the enzyme system of Clostridium KDHSZ, SO4 and/or SO3 would be expected to suppress the production of N114+ from NO3-. Sulfate exerted no influence on the production of NH4+, but $0 = did inhibit the reaction (Table 2). 3 The data in Table 3 suggest that 803= reducing activity was induced by growth on N03 . But, surprisingly, the presence of up to equimolar amounts of N03- had no effect on 35$03= reduction to 358:. Since $03= inhibition of N114+ production from N03- occurred at the level of N02- reduction (Fig. 2), this apparent anomaly can be explained. Nitrate has no effect on $03: reduction, unless some critical level of N02 accumulates. 59 It appears the N02--reducing enzyme of Clostridium KDHSZ is inducible and can reduce both N02- and 803:. Two possible explanations exist for the inability of the organism to reduce $04- to 5-. The cell may lack a dissimilatory sulfate reduc- tase. Harrison and Laishley (Abstr. Ann. Meet. Amer. Soc. Microbiol, 3 -reducing system in Q; pasteurianum, which was distinct from the 804- reduction accomplished by 1978) found an inducible, dissimilatory SO this organism, which was restricted to the assimilatory pathway. Alternatively, Clostridium KDHSZ may not readily transport SO4 . The two-fold stimulation of $03'-reducing activity by cells grown in the presence of SO4 suggests, however, that 804 does enter the cell. The Clostridium KDHSZ glutamine synthetase is similar to the glutamine synthetase from 9; pasteurianum with respect to inhibition by glutamine (l7). Glutamine synthetases from Bacillus species have also been reported to be inhibited by glutamine (10,16). The Km of 4.0 x 10.4 M for N114+ is the same as reported for the enzyme from g; subtilis (16). This low Km suggests the existence of a low-NH4+ assimilating pathway similar to that found in Klebsiella (5). The absence of any regulatory effect by glutamine synthetase on the reduction of N03- to N114+ is shown in Table S. Newman and Cole (23) observed no influence of glutamine synthetase on nitrite reductase in E; coli. The product of NO2 reduction by this organism is N114+ and the reaction is similar to the reduction of N03- to NH4+ accomplished by Clostridium KDHSZ. 0n the other hand, the N02 reductase of Klebsiella pneumonia may be regulated by glutamine synthetase (32). Therefore, this lack of regulatory control by glutamine synthetase on N03 60 reduction in Clostridium KDHSZ is consistent with the thesis that NO3 — reducing enzymes catalyze a dissimilatory reaction. Thus, it appears that the N03 reduction to NH4+ carried out by Clostridium KDHSZ is linked to the energy metabolism of the cell and is truly a dissimilatory process. The competitiveness of this pathway with denitrification remains a question. In a previous report (Chapt. II), evidence was presented that this organism, when introduced into soil, could effectively compete with denitrifying bacteria for N03 under anaerobic conditions if an appropriate carbon source is supplied. A thorough investigation of the interaction of denitrifying bacteria and organisms reducing N03- to NH4+ is needed before the natural signifi- cance of this pathway can be understood. Such knowledge may permit exploitation of the bacteria involved to conserve nitrogen in agricul- tural soils. ACKNOWLEDGEMENT The author expresses appreciation to M. R. Betlach for assistance in preparation of Figure 2. 10. ll. 12. LITERATURE CITED Bremner, J. M. 1965. Inorganic forms of nitrogen, pp. 1179-1237. In C. A. Black, ed., Methods of soil analysis, Part 2. Chemical and Microbiological properties. American Society of Agronomy, Madison, Wisconsin. Bremner, J. M., and D. R. Keeney. 1966. Determination and isotope- ratio analysis of different forms of nitrogen in soils: 3. Exchangeable ammonium, nitrate, and nitrite by extraction-distilla- tion methods. Soil Sci. Soc. Amer. Proc. 30:577-582. Bremner, J. M., and K. Shaw. 1958. Denitrification in soil. I. Methods of investigation. J. Agric. Sci. 51:22—39. Brenchley, J. E. 1973. Effects of methionine sulfoximine and methionine sulfone on glutamate synthesis in Klebsiella aerogenes. J. Bacteriol. 114:666-673. Brenchley, J. E., M. J. Prival, and B. Magasanik. 1973. Regula- tion of the synthesis of enzymes responsible for glutamate formation in Klebsiella aerogenes. J. Biol. Chem. 248:6122-6128. Bryant, M. P., and I. M. Robinson. 1961. An improved nonselective culture medium for ruminal bacteria and its use in determining diurnal variation in numbers of bacteria in the rumen. J. Dairy Sci. 44:1446-1456. Chiba, S., and M. Ishimoto. 1977. Studies on nitrate reductase of Clostridium perfringens. I. Purification, some properties, and effect of tungstate on its formation. J. Biochem. 82:1663-1671. Chiba, S., and M. Ishimoto. 1973. Ferredoxin-linked nitrate reductase from Clostridium perfringens. J. Biochem. 73:1315-1318. Cornish-Bowden, A. 1976. Principles of enzyme kinetics. Butterworths, London. 206 pp. Deuel, T. F., and E. R. Stadtman. 1970. Some kinetic properties of Bacillus subtilis glutamine synthetase. J. Biol. Chem. 245: 5206-5213. Eisenthal, R., and A. Cornish-Bowden. 1974. The direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters. Biochem. J. 139:715-720. Firestone, R. B., M. K. Firestone, M. S. Smith, M.l§. Betlach, and J. M. Tiedje. 1977. Production and detection of N-labelled NH , NO , N0, N O, and N for denitrification studies. Annual Report, Cyclotron Laboratory, Mich. State Univ., East Lansing, pp. 82-83. 61 l3. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 62 Hadjepetrou, L. P., and A. H. Stouthamer. 1965. Energy production during nitrate respiration by Aerobacter aerogenes. J. Gen. Microbiol. 38:29-34. Hasan, M., and J. B. Hall. 1977. Dissimilatory nitrate reduction in Clostridium tertium. Z. Allg. Mikrobiol. 17:501-506. Hasan, S. M., and J. B. Hall. 1975. The physiological function of nitrate reduction in Clostridium perfringens. J. Gen. Microbiol. 87:120-128. Hubbard, J. S., and E. R. Stadtman. 1967. Regulation of glutamine synthetase. VI. Interactions of inhibitors for Bacillus licheni- formis glutamine synthetase. J. Bacteriol. 94:1016-1024. Hubbard, J. S., and E. R. Stadtman. 1967. Regulation of glutamine synthetase. II. Patterns of feedback inhibition in microorganisms. J. Bacteriol. 93:1045-1055. Keeney, D. R., R. L. Chen, and D. A. Graetz. 1971. Importance of denitrification and nitrate reduction in sediments to the nitrogen budgets in lakes. Nature 233:66. Kemp, J. D., D. E. Atkinson, A. Ehret, and R. A. Lazzarini. 1963. Evidence for the identity of the nicotinamide adenine dinucleotide phosphate-specific sulfite and nitrite reductases of Escherichia coli. J. Biol. Chem. 238:3466-3471. Koike, I., and A. Hattori. 1978. Denitrification and ammonia formation in anaerobic coastal sediments. Appl. Environ. Microbiol. 35:278-282. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265—275. MacRae, D. C., R. R. Ancajas, and S. Salandanan. 1968. The fate of nitrate nitrogen in some tropical soils following submergence. Soil Sci. 105:327-334. Newman, B. M., and J. A. Cole. 1977. Lack of a regulatory function for glutamine synthetase protein in the synthesis of glutamate dehydrogenase and nitrite reductase in Escherichia coli K12. J. Gen. Microbiol. 98:369-377. Nicholas, D. J. D., W. J. Redmond, and M. A. Wright. 1964. Effects of cultural conditions on nitrate reductase in Photobacterium sepia. J. Gen. Microbiol. 35:401-410. Nommik, H. 1956. Investigations on denitrification in soils. Acta Agri. Scand. 6:197-228. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 63 Prabhakararao, K., and D. J. D. Nicholas. 1970. The reduction of sulphite, nitrite, and hydroxylamine by an enzyme from baker's yeast. Biochem. Biophys. Acta 216:122-129. Riet, J. van't, A. H. Stouthamer, and R. J. Planta. 1968. Regula- tion of nitrate assimilation and nitrate respiration in Aerobacter aerogenes. J. Bacteriol. 96:1455-1464. Sigma Chemical Company. 1974. The colorimetric determination of glucose in whole blood, plasma, or serum. Sigma Tech. Bull. No. 635, 17 pp. Sdrensen, J. 1978. Capacity for denitrification and reduction of nitrate to ammonia in a coastal marine sediment. Appl. Environ. Micrbiol. 35:301-305. Stanford, G., J. O. Legg, S. Dzienia, and E. C. Simpson, Jr. 1975. Denitrification and associated nitrogen transformations in soils. Soil Sci. 120:147-152. Stanford, G., J. O. Legg, and T. E. Stanley. 1975. Fate of ISN- labelled nitrate in soils under anaerobic conditions, pp. 667-673. In E. R. Klein and P. D. Klein (eds), Proceeding of the Second International Conference on Stable Isotopes, Oak Brook, Ill. Tubb, R. S. 1974. Glutamine synthetase and ammonium regulation of nitrogenase synthesis in Klebsiella. Nature 251:481-484. deVries, W., W. M. C. van Wijck-Kapteyn, and S. K. H. Oosterhuis. 1974. The presence and function of cytochromes in Selenomonas ruminantium, Anaerovibrio lipolytica, and Veillonella alcalescens. J. Gen. Microbiol. 81:69-78. Wijler, J., and C. C. Delwiche. 1954. Investigations on the denitrifying process in soil. Plant and Soil 5:155-169. Wimpenny, J. W. T., and J. W. Cole. 1967. The regulation of metabolism in facultative bacteria. III. The effect of nitrate. Biochem. Biophys. Acta 148:233—242. Wolk, C. P., J. Thomas, P. W. Shaffer, S. M. Austin, and A. Eglonsky. 1976. The pathway of nitrogen metabolism after fixation of N- labelled nitrogen gas by the cyanobacterium Anabaena cylindrica. J. Biol. Chem. 251:5027-5034. Zalkin, H. 1973. Anthranilate synthetase. Adv. Enzymol. 38: l— 39. APPENDICES APPENDIX A THE ABSENCE OF A REGULATORY FUNCTION FOR GLUTAMINE SYNTHETASE IN THE SYNTHESIS OF ENZYMES INVOLVED IN DENITRIFICATION Glutamine synthetase (GS) has been shown to possess, in addition to its biosynthetic role, a regulatory function controlling the synthesis of several proteins involved in nitrogen metabolism. In the enteric bacteria, GS regulates the synthesis of enzymes subject to nitrogen catabolite repression (10,15), in addition to autogenously regulating its own synthesis. In organisms other than the enteric bacteria, the regulation of gene expression by G8 has not been extensively studied. Nitrogen fixation in Rhizobium (9) may be regulated by CS, and, in _ Bacillus subtilis, the synthesis of CS is apparently autogenously regulated (2). Since GS regulates the expression of the enzymes involved in assimilation of nitrogen from a number of nitrogenous compounds, the question arises whether the enzymes catalyzing assimilatory nitrate reduction are subject to similar control. This question has not been addressed experimentally but synthesis of nitrite reductase in Klebsiella aerogenes is repressed by NH4+ (13), suggesting GS may play a regulatory role. Regulation by GS of the assimilatory enzymes, but not of the enzymes of the dissimilatory NO3--reducing system, is easily postulated. However, the differences between the assimilatory and dissimilatory nitrate reductases have not been well-delineated. In fact, van't Riet, et al (13) have suggested that the catalytic subunits of the enzymes are 64 65 identical, the distinction lying in assembly with different regulatory subunits. The dissimilarity became less clear with the observation that nitrate reductase B may serve either assimilatory or dissimilatory functions, depending upon the bacterium from which it is obtained (12). Studies with a series of five nitrate reductase mutants of Paeudomonas aeruginosa led van Hartingsveldt and Stouthamer (6) to suggest the existence of a molybdenumrcontaining cofactor common to both the assim- ilatory and dissimilatory systems. The purpose of this study was to ascertain the presence of GS in Pseudomonas fluorescens, to partially characterize the enzyme, and to examine the effect of CS on the syn— thesis and activity of the denitrifying enzymes in this organism. Pseudomonas fluorescens, strain 72, isolated by Gamble, et a1 (5), was cultured in a basal mineral salts medium (M. R. Betlach, personal communication) that contained (per liter): glucose, 9.0 g; NaH2P04- H20, 2.7 g; NaZHPO4-7H20, 8.2 g; vitamin-free casamino acids (Difco) and yeast extract (Difco), 0.1 g each; KNO 2.0 g; Na Moo ~2H O, 0.15 g; 2 4 2 0, 1.0 mg; FeSO 3’ CaCl ~2H O, 0.05 g; MgSO 0, 0.20 g; MnCl 2 2 4 2 2 2 4 2 0.55 mg, and citric acid, 0.52 mg. Monosodium glutamate was added at ~7H '4H -7H 0, 0.2% (w/v) to the medium as a nitrogen source. .2; fluorescens cells were harvested by centrifugation 4-6 h after entry into stationary phase during anaerobic incubation. and cell-free extracts were prepared by passage through a French pressure cell. Glutamine synthetase was partially purified by collecting the precipitate at 50-70% saturation with (NH4)ZSO4 (7). The precipitate was dissolved in 0.01 M morpholino- propane sulfonic acid (MOPS) buffer, pH 7.0, containing 0.01 MMnC12 and 0.001 M mercaptoethanol. Glutamine synthetase activity was monitored using the biosynthetic reaction described by Hubbard and Stadtman (7), 66 except the MOPS buffer above was substituted for imidazole buffer. Controls lacking glutamate were included in each incubation, and cor- rections were made for the ATP hydrolysis that occurred in the absence of substrate. Measured activity of the enzyme was proportional to the amount of enzyme preparation used and was linear with time up to 15 min. The activity of glutamine synthetase in the partially-purified enzyme preparation from E; fluorescens and by glutamine and methionine sulfoximine are shown in Table l. The apparent Km for NH4+ was 4.5 x 10.4 M (Fig. l). The data in Table 2 indicate GS exerted no positive control on the activity or the synthesis of denitrifying enzymes in P; fluorescens. Previous reports indicate that the biochemical and immunological properties of CS from Gram negative bacteria are similar (14). §;_ fluorescens was inhibited 60% by 10 mM glutamine, whereas the enzyme of Salmonella typhimurium was reported to be inhibited only 20% (7). Hubbard and Stadtman (7) were unable to measure glutamine inhibition by the E; fluorescens enzyme because of an interfering enzyme activity. Similar problems were not encountered in the extracts used in this study. The §;_fluorescens GS also appeared to be less sensitive to 0.01 mM methionine sulfoximine than other Gram negative bacteria. The enteric GS is inhibited 70% by this concentration (1), but only a 30% inhibition was observed for P; fluorescens. Also, the KIn for NH4+ for the.§; fluorescens enzyme is an order of magnitude lower than the 1.8 x 10-3 M value reported for Escherichia coli (16). But, 3; fluorescens, a soil bacterium, is likely to encounter lower concentrations of NH4+ in its natural habitat. Indeed, this Km is similar to the value 4.0 x 10-4 M reported for the GS of two bacteria found in soil, Bacillus subtilis (3) and a Clostridium spp. (Chapt. III). 67 .vumvamum mnu mm awasnam Bonus mafi>on spa? va Hm um .hHBoA mo monuma oau magma wousmmoe was samuoum .mucmawummxm umunu mo mamas mum mmsam> .swa ma you 0 ohm ad was cowumnsosH .o.n as .pmmmsn mac: 2 Ho.o was .Naosz z moo.o .Huemz z no.o .musssusam z H.o .mH< z mnoo.o .sfimuona we H.o ”H8 «.0 ca vocfimusoo unauxwa hmmm< + H0 Ha.o as ea .msasmusflo ooH o as ea .mswsfixomfism msfisofieumz mm o~.o 25 Ho.o .mafiafixomasm mGH:0finuw2 I mm.o I oaoz _ANV AHI :Hmuoun ma HIcwE m Hoe:v HouanwnaH sosuanassH +sus>fiuo< msmommHOSHm mucoEovsmmm Eoum ommumnuaau mafiamusaw no muH>Huom so muouwnwncfi mo uommmm .H manna 68 pauuo; d $9|OLU um ('_quI- CII'PIII'TI. ' .ANN cfimuumv sow +¢mz How cowumawumo EM mo Aqv uoHa ummcwa uumufio .H .wwm Ev. . o t V AEEV+ Izm ’ q _. Nu m- s- 2.10: neurazzesv. =28: mun- 69 .Hm>mH mucmvfimaOU Nmm mnu um mmumu macaw usooo mmosmumMMfiw uamofimwawfim oz .maoamuoum Bum H.o mo moammmua :H sowuuswoua o a 2 mm vmuammma mum» cowumowwfiuuwcma + m.“ 0H HH m 28 0H o.m OH I m o 28 0H m.n ma HH n 25 H.o N.n ma I o o 28 H.o H.w ma NH 5 SE OH m.w 0H NH m SE Ho.o m.m ma HH n o o Aflln HIwHHoo we H1v z o.N a m.H s o.H dogmamamsm asfivms cowuosvopa Omz AHImHHmo me any coauusvouq oNz wwmwfiMMWmMMmmcfismwmmww a mo 3% mo cowumuuamoaoo .mcmomouosam mucoEocsmmm mo mHHoo wafiumou an +c0fiumofimwuuacmw mo mums mnu so mafiafixomasm maH:OHnumE mo uummmm .N manna 70 The absence of any regulatory effect by glutamine synthetase on denitrifying enzymes in E; fluorescens is not surprising. Newman and Cole (11) found no influence of GS on nitrite reductase in §;.E2l£' An earlier report (Chapt. III) presented evidence that the dissimilatory NO3--reducing enzymes in a Clostridium species isolated from soil were not subject to regulation by CS. Thus, it appears that the regulatory control exerted by CS on enzymes involved in assimilatory nitrogen metabolism does not extend to the dissimilatory NO --reducing enzymes. 3 10. 11. 12. LITERATURE CITED Brenchley, J. E. 1973. Effects of methionine sulfoximine and methionine sulfone on glutamate synthesis in Klebsiella aerogenes. J. Bacteriol. 114:666-673. Dean, D. R., J. A. Hoch, and A. I. Aronson. 1977. Alteration of the Bacillus subtilis glutamine synthetase results in overproduc- tion of the enzyme. J. Bacteriol. 131:981-985. Deuel, T. F., and E. R. Stadtman. 1970. Some kinetic properties of Bacillus subtilis glutamine synthetase. J. Biol. Chem. 245: 5206-5213. Eisenthal, R., and A. Cornish-Bowden. 1974. The direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters. Biochem. J. 139:715-720. Gamble, T. N., M. R. Betlach, and J. M. Tiedje. 1977. Numerically dominant denitrifying bacteria from world soils. Appl. Environ. Microbiol. 33:926-939. van Hartingsveldt, J., and A. H. Stouthamer. 1973. Mapping and characterization of mutants of Pseudomonas aeruginosa affected in nitrate respiration in aerobic or anaerobic growth. J. Gen. Microbiol. 74:97—106. Hubbard, J. S., and E. R. Stadtman. 1967. Regulation of glutamine synthetase. II. Patterns of feedback inhibition in microorganisms. J. Bacteriol. 93:1045-1055. Lowry, D. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265—275. Ludwig, R. A., and E. R. Signer. 1977. Glutamine synthetase and control of nitrogen fixation in Rhizobium. Nature 267:245-248. Magasanik, B. 1977. Regulation of bacterial nitrogen assimilation by glutamine synthetase. Trends Biochem. Sci. 2:9-12. Newman, B. M., and J. A. Cole. 1977. Lack of a regulatory func- tion for glutamine synthetase protein in the synthesis of glutamate dehydrogenase and nitrite reductase in Escherichia coli K12. J. Gen. Microbiol. 98:369-377. Payne, W. J. 1973. Reduction of nitrogenous oxides by micro- organisms. Bacteriol. Rev. 37:409-452. 71 13. 14. 15. 16. 72 Riet, J. van't, A. H. Stouthamer, and R. J. Planta. 1968. Regula- tion of nitrate assimilation and nitrate respiration in Aerobacter aerogenes. J. Bacteriol. 96:1455-1464. Tronick, S. R., J. E. Ciardi, and E. R. Stadtman. 1973. Comparative biochemical and immunological studies of bacterial glutamine synthetases. J. Bacteriol. 145:858-868. Tyler, B. 1978. Regulation of the assimilation of nitrogen com- pounds. Ann. Rev. Biochem. 47:1127-1162. Woolfolk, C. A., B. Shapiro, and E. R. Stadtman. 1966. Regulation of glutamine synthetase. I. Purification and properties of glutamine synthetase from Escherichia coli. Arch. Biochem. Biophys. 116: 177-192. APPENDIX B NITRATE-STIMULATED MINERALIZATION OF AMMONIUM IN ANAEROBIC SOILS The phenomenon referred to as "priming" is defined by Jenkinson (1971) as a change in the decomposition rate of native soil organic matter, either stimulation or retardation. It usually results from the addition of fresh organic matter, but the same events may be triggered by the addition of inorganic nitrogen or fertilizer salts (Broadbent and Nakashima, 1971; Westerman and Tucker, 1974). As a result of priming action, either mineralization or immobilization of soil N occurs (Smith and Douglas, 1971). The quantitative significance of this effect has not been established, and the mechanism remains controversial. Laura (1974) proposed that the decomposition of organic matter is entirely chemical, the result of the protolytic action of water, while Westerman and Tucker (1974) implicated enhanced activity of soil microorganisms. Neither theory adequately explains the phenomenon, although the latter seems more reasonable. In the course of studying dissimilatory reduction of N03- to NH4+ in anaerobic soils (Chapter II), N037-stimulated minerali- zation of organic matter was observed. This note points out the magnitude of this effect and its significance to ecological studies on the nitrogen cycle. Soil (5 g) was placed in Hungate tubes and amended with 10 ml water containing carbon and/or nitrate as indicated. Tubes were sealed and incubated anaerobically under 02-free Ar for 5 days at 28° C. After + 4 + NH4 -N was measured as described elsewhere (Chapter incubation the soils were extracted with 1N KCl and analyzed for NH (Bremner, 1965). 15 73 74 II) using the ratio mass spectrometer in the laboratory of J. 0. Legg, USDArARS, Beltsville, Md. Four tubes per treatment were prepared; the extracts of two were pooled and the mean of the duplicate determinations reported. The accumulation of NH4+-N and 15NH4+-N in three soil samples is shown in Table l. The increase in NH4+ accumulation following NO3 addition was evidenced by the larger amount of NH4+ in the soils to which NO 7 was added. The origin of the NH4+ was not N03-, since 3 15 - 15 + - reduction of N03 to NH4 was minor. Thus, NO3 -stimulated min- eralization was responsible for the NH4+ formation. In the fresh, 3 the amount of mineralization. For glucose-amended soils, the apparent unamended soils, addition of NO resulted in an 22-25-fold increase in stimulation was only a factor of 14-18, probably because of enhanced immobilization. Parnas (1976) suggested the rate of mineralization was a function of the C/N ratio of the substrate, with mineralization favored when this ratio was low with respect to the optimum C/N ratio for the growth of the carbon-decomposing bacteria present. Immobiliza— tion (or assimilation) would be enhanced if the change in C/N ratio resulted in a ratio higher than this optimum. Simultaneous amendment with carbon and N03- does not lower the C/N ratio as much as addition of N03- alone; thus, the observed effect is consistent with this hypothesis. The higher levels of mineralization in the dry Kranzburg soil is signif- icant. Drying of soils, a common practice for many soil studies, apparently alters the organic N fraction of the soil rendering it more susceptible to microbial attack. The addition of carbon to the soils increased the amount of 15NO - 3 15 converted to N—organic N reflecting the increased demand for nitrogen 75 .N.0 muo3 maoaumcfiahoumv q q no muouuw um um . w m was . Sous . m c . m m o + mZmH was + :2 m 0 0 um 0 00 ZmH m mm 00 u OZmHM macaufivc oz0H u m + H.H m.H 0.5m ~.H 0.0 0.0 0.m H.0 0.0 00 0 .mudumo< H.0H H.0 n.0H m.HN 0.0 0.0 0.0a 0.0 m.m 00 0 .mmousao 0.0 H.0 0.0m 0.H 0.0 N.HH 0.0 ~.0 0.0 00 0 I I 0.0 I I 0.0 I I ~.0 0 0 w e a w s e ass a a m ms mm 2%... is: is 3...“... is: is z ”220 is: is :2 V :s 0H 0H 0H . kquMMdnnamux nmwnmlwusnusmhx Smouwlum>oaoo + mozma donumo AHHom m\ZI+qumw 0:0 vmuawoun adfiaoaa< muamswama< M .mHHOm NNHSU Ou msowuauvm sonumo was 02 ou uncommon aw soaumnsoaw oanoummsm whoa m umumm mOZmH menu +¢mZmH was +¢mz Hmuou mo cowuosvoum .H mflan 76 for growth of bacteria. Note that the addition of glucose tremendously stimulated N03- assimilation, while the addition of acetate, a carbon source utilized by only a limited portion of the bacterial flora, resulted in a considerably lower amount of N03 assimilation. One mechanism that could account for N03--stimulated priming in anaerobic soils is that N03- would serve as a terminal electron acceptor, allowing more oxidative metabolism to occur (by denitrifying bacteria, for example). Acetate is not readily metabolized by fermentative bacteria, but is used by virtually all denitrifiers. If this mechanism were operable, acetate should be more rapidly used by denitrifying 3 the priming effect. Acetate caused reduced priming in only the fresh bacteria than soil organic carbon, thereby removing the N0 and reducing Kranzburg soil, suggesting that oxidative respiration is not the only mechanism responsible for N03--induced priming. From the data presented, it is apparent that ecological studies in soil involving nitrogen require use of a tracer such as 15N if quantita- tive measurement of N cycle processes are to be made. Since carbon and nitrogen are closely related in priming action, carbon tracer studies are also mandated. Priming action raises a potential problem with use of isotope tracers. If a method assumes steady-state rates, for min- eralization or immobilization in the case of N, clearly then data measured would be subject to large errors, compounded because priming may result in either a positive or negative effect. Experiments based on isotope dilution require such assumptions. The soils used in this study were incubated anaerobically which may have enhanced NH4+ accumulation relative to that which occurs in the 77 partially anaerobic soils more commonly encountered in nature. Never- theless, because of the considerable extent of N0 --induced priming, the 3 phenomenon can not be dismissed as being trivial. 78 References Bremner, J. M.: Exchangeable ammonium, nitrate, and nitrite by steam- distillation methods. In: Methods of soil analysis. 2. Chemical and microbiological pr0perties (C.A. Black, ed.), pp. 1191-1206. Madison, Wisconsin: Amer. Soc. Agron. 1965 Broadbent, F. E., Nakashima, T.: Effect of added salts on nitrogen mineralization in three California soils. Soil Sci. Soc. Amer. Proc. 35, 457-460 (1971) Jenkinson, D. 8.: Studies on the decomposition of C14 labelled organic matter in soil. Soil Sci. 111, 64-70 (1971) Laura, R. D.: Effects of neutral salts on carbon and nitrogen min eraltion of organic matter in soil. Plant Soil 41, 113-127 (1974) Parnas, H.: A theoretical explanation of the priming effect based on microbial growth with two limiting substrates. Soil Biol. Biochem. 8, 139-144 (1976). Smith, J. H., Douglas, C. L.: Wheat straw decomposition in the field. Soil Sci. Soc. Amer. Proc. 35, 269-272 (1971) Westerman, R. L., Tucker, T. C.: Effects of salts and salts plus nitrogen-lS-labeled ammonium chloride on mineralization of soil nitrogen, nitrification, and immobilization. Soil Sci. Soc. Amer. Proc. 38, 602-605 (1974) IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIHII IIIIIIIIIIIIIIIIII 1293 696