MSU. LIBRARIES “ RETURNING MATERIALS: P1ace in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. ASPECTS OF METABOLIC REGULATION IN RUMEN BACTERIA BY Douglas Bertram Bates A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science 1985 ABS‘IRACI' Aspects of Metabolic Regulation In Runen Bacteria BY Douglas B. Bates Aspects of metabolic regulation in runen bacteria were studied using in vitro culture techniques. Growth rate of runinal bacteria was varied by changing energy source and substrate limitation (glucose and nitrogen). Ribonucleic acid:protein ratios of runen bacteria were highly correlated with specific growth rate, H. As H increased, RNA/protein and RNA/INA values increased. The regression of RNA/protein on #1 (six microorganism) was Y = .6211 + .23. Glucose and nitrogen limitation affected (P<.Ol) RNA:protein ratios of seven predaninant runinal strains. Nitrogen limited cultures had the highest RNAzprotein ratio (.52) , followed by nutritional sufficiency (.28) and glucose limitation (.12). 'me mmprotein ratio of runinal bacteria was not affected by variation in growth rate. Macrmolecular composition of runen bacteria may be used as an indicator of microbial activity in the runen. Proteolytic activity was assayed using azocasein as substrate. An interaction (P<.001) was observed between growth substrate and stage of growth as factors that affect empotease activity of Bacteroides tuninicola GA33. A similar interaction (P<.01) was observed between nutritional status and stage of growth. It is conclucbd that energy limitation interferes with the mechanism that normally depresses (P<.Ol) emprotease activity of stationary phase cultures of B. ruminicola GA33. Several other factors including rate of growth (P<.01) and nutritional status (P<.Ol) have been ichntified that influence the proteolytic activity cf this organisn. A plot of emprotease activity vs dilution rate for a glucose limited continuous culture indicates that a complex, interacting set of controls may be involved in regulating the proteolytic activity of B. runinicola GA33. ACKNOWLEDGMENTS The author wishes to express his appreciation and gratitude to Dr. W. G. Bergen for his guidance and support throughout the duration of the graduate program. His willingness to interact and challenge made an important contribution to my professional growth. Sincere gratitude is also extended to Dr. M. T. Yokoyama for his friendship and technical expertise. His door was always open. Appreciation is also expressed to Dr. J. C. Waller for his understanding and valuable counsel. In addition, the author wishes to acknowledge Dr. J. T. Huber for his advice and review of this manuscript. I wish to express special thanks to Mr. R. Van Meter and Dr. M. Villereal for their assistance and loyalty during the course of my graduate program. Appreciation is also extended to the many fine students with whom I interacted during my graduate training includ- ing, among others, Dr. C. Isichei, Dr. W. Rumpler, Dr. G. Weber, Mr. S. Barao, Ms. K. Johnson and Ms. J. Gillett. I would like to thank my parents, Dr. and Mrs. J. B. Bates, for their love and devotion. I would not have attained this goal were it not for their confidence in me. I especially would like to thank my wife, Gilda, for all the sacrifices she has made in order for me to pursue this goal. She has endured all trials put before us and has been the beacon in my life. ii 'mBLE OFCIN'IENIS Lm 0’ m.OOOOOOOOOOOOOOOO00.00.0000...0.0.0....OOOOOOOIOOOOOOOOV m OF HGWOOCOOOOOO...00......OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOVii WGMOOOOOOOOOOOOOOOOOOOOOI00.0.00...0.00000000000000000000000I m was EMooooooooooo0.00900000000000000000000000001 m Wmm-OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOZ mam w AWS mm.00....0.0.0.0.000000000000000004 m 096m mmIG m THE m0.COO...OOOOOOOOOOOOOOOOOOOG mm m m WIC 1316me mm.0.0.0.0000000000000009 m 1.0.0.0....CCOOOOOOOOOOOO0.0.0.0....00......0.0.0.0000000000012 W mLWOOOOOOOOOOOOOOOOOOOO00......00.0000000000000012 mmMDm®00000.0000000000000000000.0.00.000000000000016 or as....0...00.0.0...O.OOOOOOOOOOOOOO.0.0.0.00000000000016 muv.um00.0.0.00.0.......0.0.0...COO...0.0.00.00000000000016 ma.0.00.0.0.0...0....OOOOOOOOOOOOOO..00000000000000000000017 Wiaum in erth mteOOOOO0.0....O0.0.0.0.000000000000000017 Determination of Growth Limiting Conditions..................19 Experiment Protocol - Experiment 1...........................20 mm HWI - WIM 2...0.0.0.000000000000000022 suumm mwOQOOOOOOOOOO.OOOOOOOOOOOOOOOOOOOOOOO.0000.24 mm...OO...OOOOOOOOOOOOOOOO.O.OO0..OOO.0.0.0.000000000000000024 mm 1...OOOOOOOOOOOOOOOOO.0...O0......0.0.0.000000000024 ”rim ZOOOCOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0.00000029 mm.0.00000....0...O...0.0.0.0...O..0OOOOOOOOOOOOOOOOOOOOOB‘ mammal MW at m ”Charla In V1WOOOOOOOOOOOOOOO3‘ mtabolic Characteristics of Stationary Phase Cells..........43 Regulation of Protein Weaning fistem in Prokayotes......45 mad” In Standary m wtmeBOOOOOOOOOOOOOOO0.0.0.50 INA/Protein In Nutrient Limited Oontinmus wtme V8 Batd‘ mltmeOOOOOOOOOOOOOOOOOOO.OOOOOOOOOOOO.0.0.51 INA/Protein During Static Growth (based by “trim meumOOOOOOOOOOOOOOOO.OOOOOOOOOOOOOOOO0.0.00.0052 MM“ M m Balm“. mWOOOOOOOOOOOOOOOOO0.0.57 music Acid -}V'rotal-N as a Marker of Microbial Protein.....60 iii m II.IOOOOOOOOOOOOOOOOOO.0......00.0.0.0...00.0.00000000000000062 m m LMREOOOOOOOOOOOO0.0.000000000000000000000000.0.0.62 Manual 0f “aerial wtamlim0.00...00.0.00000000000000062 Regulation of mgotease in Non Runinal Bacteria............64 mad“! Of Prwedytic AdiViw in me RmmOOOOOOOOOOOOOOGS 'me Effect of mnensin on Nitrogen Metabolisn mRWBOOOOOOOOOOOOOOOOOOOIOOOOOOOOOOOOOOOOOO0.0.0.0....69 M MD mm.0..OOO.CO...O.COO...O0.0.0.000000000000000070 mgmBOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO00......0.0.0.00000000070 “MQOOOOOOOOOOOOOO0..OOOOOOOOOOOOOOOOOOO...0.0.0.0000000071 ma.’OOOOOOOOOOOOOOOOOOOOOOOO000......00.00.000.0000000000071 mmmm Mt‘re d mamrmm..0.00.000.00.0000000000071 Rama maadms.O...OOOOOOOOOOOOOOOOOOOOO0.0.00.0...74 M.OOOOOOOOOOOOOOOOOOOOOOOO00.......0.00.000.000.00000000075 ma]- mrm HMI.O.OOOOOOOOOOOOOOOOO0.0.0000000076 WIN BOOOOOOOOOOOOOOOO0.0..0.0.0.00.0.0000000000000000077 ”rm ‘0.0.0.0.000...OOOOOOOOOOOOOOOOOOOOO0.00.00.00.00077 ”rim SOOOOOOOCOOOOOOOOO000......0.00.00...0.0.0.000000078 mrm 6.0.0.0...O.IO.I...OOOOOOOOOOOOOOOOO0.0.00.00.00.078 mtm 7.00.00.00.0000000000.0...OOOOOOOOOOOOOOOOOOO0.00.79 mm.0......OOOOOOOOOOOOOOOOOO0.0.0.0....0.0.0.00000000000000079 mt 3..0.0...00.......0.000......0.0.0.00000000000000079 ”rim 44.0.0000000000000OOOOOOOOOOOOOOOOOOO0.00.00.00.085 ”rim 7.0.0.0....OOOOOOOOOOOOOOO0.0.0.0....0.0.0.000000095 MWOOOOOOOOOOOOOOOOOOOCOOOOOO00.00.000.00...00.0.00000000098 Factors Affecting Empotease of B. Runinioola GA33..........98 Effect of Diet on Proteolytic Activity in the Rm.........103 my is'lhereaDecreaseinRLmiml Protease During mu Mm.0.0...OOOOOOOOOOOOOOOOO0.00.00.00.0000000000104 WWOOOOOOOOOOOOOOOOOOOCO...OOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.00.108 WOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.00.00.00.00...0.0.0.00000000110 8mm.OOOOOOOOOOOOOOOOOOOOO...OOOOOOOOOOOOOOOOOOO0.0.0.0000000117 iv LIST OF TABLES TPBIE PAGE Sta-nary cf Experiments Reporting Microbial Protein Yielda.........13 Experimental mdiun for Pure Culture of Rtminal Bacteria .........18 Adjmunents to Experimental Median to impose Nutritional mud“ Mir'g the Statiomry W.O0.0.00.0...0.0.0.0000000023 (beerved and Predicted RNA/Protein Values for Zero Growth 1nmBaaeriaOOOOOOOOOOOOO00......OO.COO...0.0.0.000000000000028 Effect of Ntxtrient Limitation on the RNA:Protein Ratio of Several Prerhninant Runen Bacteria in the Stationary Phase of Growth......32 Nitrient Limitation as a Main Effect on the RNAzProtein Ratio a W magi-a.0.0.0....OOOOOOOOOOOOOOOOOOOOOOOOO000.00.000.033 Species Variation as a Main Effect on the RNAzprotein Ratio d W maeriaOOOOOOOOOOOOOOOOOOOOOOOCOO0.00.00.00.000000000033 INAxProtein Ratios oleree Runinal Bacteria Fran Sheep Fed ma mfferem mas .0.0.00000000000000000.0.000...0.0.00.00000036 RazProtein Ratios of Inherent Runinal Bacteria Frat Sheep M m” mfferetit mas O0.00.0000...OOOOOOOOOOOOOOO0.0.0000000037 Interaction of Physical State and Diet on the RNAzProtein Ratios i W meria Frm M O0......O0.0...0.0.000000000000000038 Overall mans Showing the Effect of Pl'lysicnl State, Diet and Time AfterlFeeding on the RNA:Protein Ratio of Runinal Bacteria Rm M OOOOOOOIOOOOOOOOOOI00.0.0.0...00.0.0.0...0.0.0.0000000038 12 Basal mdiun Used in the Protease Experiments.....................72 13 the Effect of Substrate, Monensin and Stage of Growth on me much” M1v1ty i B. mmla @30000000000000000000086 14 Interaction of Substgate and Stage of Growth on ”gm M1V1ty i B. Minimla “3.0.0.0000...00.0.0.0...88 15 Interaction of Nutritiogal Status and Stage of Growth on Emprotease Activity of B. Runinicola GA33.......a............88 16 Effect of Stage of Growth on the Emg’otease Activity x 8. Minimla @300...OOOOOOOOOOOOOOOOOOOOOOOOOO00.0.00000000093 17 Cellular Protein as a Percent of mm Cell lass During Nutrient Sufficiency, Glucose Limitation and Nitrogen Limitatign...........93 18 Inflmnoe of Growth Substrate on Emrrotease Activity ‘ B. mmla @300000000000000000000000.0.0.0....0.0.0.00000094 19 Inflmnce of Mmensin on the Emrrotease Activity of B. mmla “3.0.0.0...OOOOOOOOOOOOOOO000.00....OOOOOOOOOOOOOO” 20 Interaction of Mmensin and Growth Substrate on Emprotease wv1w i B. male 633.000....0..O0......0.000000000000000” @ O N) m Ul b “NH (35 Appendix Tahle Page emprison of Protease Activity in Several Runinal Bagteria...u.ll4 Effect of Cell Cbncentration on Azocasein Degradation tar meta-as Rmmla W3000OOOOOOO0.00....0.0.0000000000900000115 Effect of (bnoentration of Azomsein Used in the Assay m Hm m1v1q i B. Minimla GmBOOOOOOOOOO... 0.00.00.0115 Decay (i Different (bncentrations of Hydrolyzed Azo Dye .........115 (Inprison of Dry Matter Yield (mg/ml) When Cells a Were Harvested With or Without Fonnaldehyde—NaCl Pretreatment ...116 (imprison of Absorption of Moisture During Gravitimetric Determinatign of (211 Dry Weight Using Filters, Metal Pans or 61m ms 00.00.000.000...OOOOOOOOOOOOO...00.0.0000000000000000116 38.283?- vi LIST OF FIGURES FIGURE PAGE 1 01mm x m maion.0.0.000...00......0.0.0.00000000000000011 2 Flow Chart of the Methods Used to Determine the Relationdiip Between Specific Growth Rate and RNA:Protein Ratio of Runen maria.OOOOOOOOOOOOOOOOO000......O0.0.0....0.0.0.0000000000000021 mAxProtein Ratios at Specific Growth Rates Fan .1 to 1.2 mum ”I am for Six Rm Baaeria.0.00.00.00.000000000026 lemma Ratios at Specific Growth Rates Fran .1 to 1.4 Doublings per Hour for Five Runinal Bacteria.....................27 MsProtein Ratios at Specific Growth Rates Fran .1 to 1.4 Domlings per Hour for Five Runinal Bacteria.....................30 mAzProtein Ratio at Specific Growth Rates Fran .4 to 2.0 m‘mm pr HM for Streuomm M18.OOOOOOOOOOOOOOOOOOOOOOBI 7A Growth of Bacterial (blony Attached to a Solid Surface GUI.“ Narims.00.00....OOOOOOOOOOOOOO00.00.00.000...0..00.40 Containing B Sharp Diffmion Gradient Exists Fan the Exterior of the ml” mar$ the InteriorOOOO.OOOOOOOOOOOOOOOO0.0.000000000000040 8 Relationship Between Growth and Emgotease Production by Bacteroi¢s runinicola G183 Grown on Glucose.....................80 9 Relationship Between Growth and Emprotease Production by Bacterolcbs runinicola G183 Grown on Cellobiose..................81 10 Relationship Between Growth and Empretease Production by Bacteroicbs runinicola GA33 Grown on Maltose.....................82 11 Relationship Between Growth and Exorrotease Production by Bacteroicbs runinicola G183 Grown on Lactose.....................83 12 Relationship Between Growth and Exorrotease Production by Bacteroiés runinimla GA33 Grown on Soluble Starch..............84 13 Effect of Addition of 2-deoxyglucose on Empotease of meta-*8 rmmla GMBOOOOOOOOOOOOOOOO0.0.0.0.0000000000000089 14 Effect of Addition of cyclic AMP on Exorrotease of mad¢£ rmmla GBBOOOOOOIOOOOOOOOOOOO0.00.00.00.0000000090 15 Effect of Addition of Levallorrhan on Empotease of BC¢9I01&8 [mmla WBOOOOOOOOOOOOOOOO0.0.0.00.000000000000091 16 Emprotease of Bacteroichs runinioola G183 Expmssed as a Function of Dilution Rate of Glucose Limited Continuous ontmeOOO000......OOOOOOOOOOOOOOOO0.0.0.0000...0.00.00.00.00000097 17 Four Patterns of Enzyme Promotion in Bacteria Grown in mm unme(W1um et al.’ lye)OOOOOOOOOOOOOOOOOOO0.00.102 vii Appendix Pignes Page A1 Stanchrd Curve of Protease (Signa # P5380) Activity Possessing 12 Units of Activity per Milligram......°.............111 A2 Decay cf Bydrolyzed Azo We (Storage in 5% T0. at O C)...........112 A3 ample Showing the Determination of Ks Value (Glucose) for 8. WWOOOOOOOOOOOOOOOOOOOOO.00...0.0.00.0000000000000113 viii IN'IROIIICI‘ICN A truly synbiotic relationship exists between the host runinant and its acoonpanying microbes. Structural polysaccharicbs of plants cannot be @graéd by animal digestive processes and the herbivore relies on the microbes in the runen to convert these to products which it can utilize for energy and growth. A salient feature of the runen is the rather constant set of conditions which support a conplex microbial ecosystan (mmgate, 1966). Runen bacteria are anaerobic and require rather exacting conditions to flourish. 'Ihe presence of extrenely dense microbial populations in the runen (Gall et a1., 1947: Bryant and Burkey, 1953; Bryant and Robinson, 1961; Caldwell and Bryant, 1966) indicate that these conditions are being met. here are several factors that influence microbial growth in the runen. lumen tenperature is maintained at approximately 39 kgrees centigracb (mitigate, 1966) and an influx of feed and water insures that the bacterial population in the rmen is a dynamic one. 'Ihe pH of the runen is normally buffered between pH 6.0 and 7.0 (Bryant, 1977) by bicarbonate and phosphate {resent in saliva (McDougall, 1948). Runinal absorption of volatile fatty acich (Pemington, 1952; Pennington, 1954) and We (Bmgate, 1966) also inflmnoes rune: IE. Canotic pressure is generally lypotonic with respect to blood (Clarke and Bauchop, 1977) . 'Ihe 2 gs [base above runen contents is highly anaerobic and contains gases which arise fron the active fermentation in the runen. 'Ihe Eh of men contents ranges from a -250 to -450 mV. 'Ihus, the runen consists of a highly refined environnent capable of supporting the growth of anaerobic microorganism uprising the runen ecosysten (Bryant, 1977) . 'Ihe onplex interactions between runen microorganism foster a striking diversity within the microbial population. Apgoximately 200 species of bacteria and 20 species of protozoa have been isolated fron the rmen (Russell and Hespell, 1981) . Several emlarations have been proposed to account for the abundance of niches which exist within the rminal ecosystem. ‘Ihefeedof nminantsisveryomplexandcontainsavarietyof carbohydrates (both cunplex and simple), uoteins, fats and numerous other organic ampom& such as alkaloicb and minerals (ngate, 1966). 'ihose orgnians carnhle of vigorous growth on the available nutrients will survive. Bacterial species can conpete for a given niche by optimizing one or more of several factors (Russell and Bespell, 1981) . ‘Ihese incluch maximum growth rate, substrate affinity, tolerance to low 13, growth efficiency, the ability to metabolize a variety of substrates, the ability to metabolize recalcitrant substrates or resistance to pedation. An important (hterminant of relative species success is the maximal growth rate which a given species can achieve. Streptococcus bovis can achieve a chubling time of around 20 minutes (Russell et a1., 1979), almst triple that of other runinal strains. When substrate is plentiful, S. bovis can hirsute the runinal ecosystem (Bartley et a1., 1975). his condition ocasionally exists when cattle enter a feedlot or are mifted 3 from a primarily roughage diet to one high in concentrates (Bartley et a1., 1975) and the pH of the runen Mines dramatically. mring this period the uechninate bacteria are Megaslhaera elschnii, Streptococcus bovis, and eventually a variety of lactobacilli. Under these rather specific conditions tolerance to pH appears to be an important factor regulating the population mix within the runen. Ruminal soluble substrate concentrations are usually very low (Takahashi and Nakamura, 1969) . At low substrate concentrations the relationship between substrate concentration and growth rate in a batch culture can be &so:ibed by the Monod egmtion: u=u maxx (S’IKSi'Sl) where: S = concentration of the growth limiting nutrient, u = specific growthrateandu max=maximunspecificgrcwth rateobtainableinthe absence of any substrate limitation. 'Ihe term KS is a constant and is Mined as that growth limiting substrate concentration which allows the organism to grow at one half the maximum specific growth rate. It is a measure of the affinity of an organism for the growth limiting nutrient and as sad: is also a major ecologial cbterminant in the runen. In the early 1960's Bauch0p and Elscbn (1960) preposed that yield (YAI‘P) of bacterial cells (g/mole ATP) was a constant at aromd 10.5. In the late 1960's and early 1970's many contradictions to this constant appeared in the literature (Hobsen and Smners, 1967; Bobsen and Simers, 1972; Bowlett et a1., 1976; Jerkinson and Woodbine, 1979). 'meoreticnl calculations by Stouthallner (1973) indicated that YATP could be as high as 32.5 for growthof anorganisnina conplexmediun. Stouthanner and Bettethamen (1973) extrapolated froa experimental results and suggested that much of the discrepancy could be explained by the utilization of ‘0! ti l"... ‘4 l.) "l [u' .I’I ._I V q 1‘.’ £31 4 increasingly greater proportions of energy for maintenance as the bacterial culture grows at slower growth rates. 'Ihis relationship has been (hfined tar Pirt (1965) and is expressed as: u/yd‘ml + [(u/m) + (mfmmn where m = minterance coefficient (mole/hr/g dry wt); u = specific growth rate (hr-1) and YMATP extrapolated yield (g/mole) at u = °° . Mainterance for individual species can vary greatly (Russell and Baldwin, 1979) , even though the maintenance energy of the total runen p0pulation is low unrated to other bacteria (Issacson, 1973) . The relative amount of energy required for maintenance can also be a determinant which affects the final population characteristics of the rumen ecosysten. Predation by Eratozca may be involved in the selection process as well. he rate of bacterial engulfment in the runen can be quite substantial and under certain conditions approaches 20,000 bacteria/protozoan/hr (Coleman, 1975) . Coleman and Sandford (1979) have elegntly Wrated that Entodiniun and other runinal protozoal species show a selective response in their engulfinent of mixed bacterial papilations. For instance, Entodina species preferentially engilfed cellulytic bacteria as opposed to other runinal strains. Hungate (1966) proposed that the mechanics of the anaerobic fermentation in the runen are comparable to those of a vat with a constant supply of fred: nutrient in which the culture constituents (bacterial cells, fluid and fermentation end prodicts) are renoved at a rate eqmllingthe supplyoffreshmediun. Suchasystenhasbeendefinedasa continmus feed systal akin to the continuous oilture commonly utilized uncbr laboratory conditions. Bmgate (1966) has outlined the mathematical &sigx used to (hfine the dilltion of a pulsed marker adcbd to a continmus fermentation systen with a given turnover rate. 'Ihe average time that runen contents remain in the ruaen must equal the time required for an equivalent amount of food to enter the runen. Otherwise, the volune of the runen would change. ibny researchers have used these mechanics to study runen function through the use cf app-opiate markers that flow with the various runinal fractions (Faidmey, 1975) . his information has been used to calculate digestion of different caponents of the diet (Schneikr and Flatt, 1975) , microbial protein flowing to the lower gastrointestinal tract (Snith, 1975) and total non-amonia nitrogen contribution to the nitrogen status of the host animal (Orskov, 1982) . 'Ihree different types of markers have been used to correspond to the three Lhases ollnonly associated with the runen: 1) particulate markers - markers of the flow of undigested feed particles which remain insoluble within the runen; 2) liquid mariners - markers of the flow of liquid from the runen; 3) microbial markers - markers which specifically measure the flow of microorganism irrespective of their {basic distribution. Faichney (1975) defined the ideal marker in the following manner: 1) It must be non-absorbable 2) It mmt not effect or be affected by the gastrointestinal tract or its microbial population 3) It must be nysially similar to or intimately associated with the material it is to mark; and 4) Iilhe analytical grocedure used to estimate it must be specific and sensitive and must not interfere with other analysis. Soluble markers oomnonly used are me and Cr-EIIl'A while 0:203, rutheniun and a variety of rare earths (i.e., Lanthaniun and Yutterbiun) have frequently been used as particulate markers (Faidmey, 1975). Microbial markers that have been used inclucb RNA (Snith, 1975), mm (Wolstrup and Jenson, 1979), Dianinoptimelic acid (Theurer, 1982), and D-alanine (Garrett et a1., 1982) . 'Ihe latter two are conponents of the bacterial cell wall. WM 'Ihe analogy of the runen to a continuous fermenter is acceptable when used to (hfine runen turnover. (1) the other hand, direct application of the continuous oilture model to define microbial growth in the runen has sane serious limitations. 'Ihe major assunption ixixerent in continmus culture @319: is that a defined growth median is used which contains a single growth limiting nutrient (Pirt, 1975). Because a direct relationship exists between the dilution rate and the conoexxtration of growth limiting nutrient reaching the cells, an equivalence bebween dilution rate and growth rate of the bacterial culture exists at steady state. 'ihis relationship is defined by the following eqmtion: D = ([11 um I: Sl/Rsl) where: D = dilution rate and S = steady state concentration of the growth limiting nutrient in the culture vessel. At steady state u= D such that an increase in Dwill result in a conparable increase in n. 11:13 condition c‘bes not exist universally in the runen. Intake is sporadic, especially when animals are fed only one or two times daily: runen values is not constant; rate of mlivary flow is not constant (mngate, 1966): and the rate at which material leaves the rtnen is not constant, regardless if attributable to digestion (VanSoest, 1982) 7 fermentation (ngate, 1966) , absorption (Annison et a1., 1957) or Spillover to the lower gastrointestinal tract (Faidiney, 1975). Transient feed sunny would result in a burst of microbial growth followed by periog during which growth is stationary (KOdl, 1971). Gas and VFA prodiction (mitigate, 1966), p33 labeling (Bucholtz and Bergen, 1973) and simulated cell {reduction with time after feeding (Baldwin and Dexhan, 1979) all indicate periog of growth transition in the runen. A bacterial culture mgrgoing growth transients spends much time in periods of unbalanced growth, undergoing both diifts up and chm during which it 'gears up" for growth then gstroys or alters much of its metabolic machinery in an effort to econonize its maintenance expenditures (Koch, 1971). ‘Ihe continuous culture, on the other hand, exists in a state of balanced growth where every conponent, both structural and metabolic, gubles at emctly the same rate (Mandelstam and Mcnuillen, 1973) . El Shazley and ngate (1965) extrapolated runinal bacterial growth rate frm increase in maximal rates of gas production between initial runen sanples and samples that had been incubated for one hour. 'Ihese authors assune that fermentative activity is coupled to growth of microorgnisls. Because uncoupled growth has been reported in runen microorganims (Jenkinson and Woodbine, 1979), this procedure may overestimate microbial growth. Using this techniqm, ngate (1966) proposed that the average growth constant of runen microbes in vivo was .O6-.08 per hour. He states “The runen resenbles a continmus fermentation system with a growth rate similar to a late stag of a batdx culture . . . where specific growth rate is .066 populations per hour' and further points out that substrate is limiting and prodicts inhibitory airing much of the feeding cycle (Bmgate, 1966). Chrford (1964) takes 8 this one step further when he suggsts “The fact that runen bacteria seen to be acting so well within their theoretical potentialities, apparently dividing on the averag only once or twice a day instea of several times per hour as many bacteria are capable of doing at 39 C, surely meam that as biochemical agnts they are more often acting in the resting state or eventhe stag of declinethaninthelogritl'micphaseofgrowth . . . it seens likely that their logorithmic phase lasts for only very brief periods immediately after fresh fodgr reaches the runen. But their enzymic makeup will be very different according to stag of growth.“ The attachment of bacteria to feed particles during the digstion process is another confounding factor. Hungte (1966) noticed a larg gcline in gs prediction when samples containing little or no particulate matter were used as innocula. Direct microscopic examination shows that much cf the bacterial population is bond to feed particles (Akin, 1979: Chexg and Costerton, 1980) . Cheng and Hungte (1976) found higher counts of meteria in media containing solid preparations fron alfalfa fiber than in media containing dissolved fiber oligosaccharigs. Minato and Suto (1978) used Tween80 to detach bacteria frcn digsta particles and showed that approximately equal preportions of bacteria were associated with feed particles as with rmen fluid. Viable counts of acmerent and free bacteria separated by hmognization also indicate that approximately one half cf the bacteria present in the runen are bound (Leedle and Hespell, 1980: Gillett, 1982). Furthermore, Forsberg and Len (1977) found that 75% of the MP measured in runen smples was associated with feed particles. 'Ihe resignce time of largr fibrom feedstuffs in the runen is longr than the liquid residence times (on the orgr of 25 vs 10 hours) (Ellis, 1982) . If a direct correlation exists between dilution rate of mean contents and growth rate of runen bacteria, then a two pool mochl must be invoked to gscribe microbial growth on feed particles which turn over at one rate and microbial growth in the runen fluid which would tend to turn over with the liquid fraction. 9 If one asslnes that the feed particles passing through the runen are maximally colonized, or at least colonized to the .e ggree, than an exponential rate constant would by applicable. However, time required for glonization is greater than that required for turnover of the initial particulate fraction after its ingstion (Akin, 1979: Van Soest, 1982) . A mochl describing linear growth of bacterial colonies on agr has been proposed (Pirt, 1975) . Application of this model to microbial growth on feed particles introduces a variable that renders unacceptable direct application of a continuous culture model to runen microbial growth. 'ihe nutrition of the runinant depends heavily on the canplex microbial interactions and metabolien that constitute runen function. Up to 85% of the digstible dry matter of the normal production diet is digsted by the runen microbes (Bryant, 1977) with ensuing accunulation of volalite fatty acids, (1)2, methane, m3 and ultimately, microbial cells. 'Ihe volatile fatty acids serve to meet the energy requirenent of the host animal while microbial glls (whidx flow <1an the digstive tract) contribute sigifigntly to the potein mmriture of the animal (Bmgte, 1966) . Because many of the runen microorgnisms can synthesize essential B vitamins, the rminant an be maintained on diets devoid of otherwise essential vitamins (mmgte, 1966). lbs major carbohydrate constituents of the rminant diet freqmntly inclug fibrous materials such as cellulose and henicellulose whidx are not susceptible to direct enzymatic attack by malian emymes produced by either nonrminants or ruminants. 'Ihere are no vertebrates that synthesize the enzymes gpable of l'ydrolyzing the B linkags associated 10 with the chemical structure of these materials (Prins, 1977). The runen ecoqrstm, however, possesses the ability to cleave B glycosidic linkags and, in the pogss, provide a substantial contribution to the nutritional status of the host animal. A diagren indicating the points of runen function that might best be manipulated to the host's advantag is shown in Figure 1. The diet of a ruminant contains both carbohydrates and proteins which are subject to hydrolysis and utilization by the runen microbes. Proteins are subjected to enzymatic processes which first degrade then to amino acids and peptigs and finally to ammonia and carbon skeletons. Energy is conserved as ATP during fermentation of sugrs arizing fran the digstion of mplex polyaccharigs. Anabolic reactions which utilize ATP as an energy currency the: construct cellular materials fran the end products of these digstive gogsses. Manipulation of these events to maximize the runen's contribution to host fixysiology can take the following forms: 1) Change in the fermentation to either imwe dry matter digstibility or alter the stoichionetry of the end products (Chalupa, 1980) . 2) Decreases in proteolysis of dietary protein to increase the amount which 'bylnsses' runen ggradation and reaches the lower gstrointestinal tract (GIT) intact (Chaluupa, 1975). 3) Increases in both efficiency and yield in microbial growth (Bergn and Yokoyana, 1982) . The following chapters address various aspects of manipulation and/or assesaent of mean function. ll RUMEN FUNCTION FEED Dietary protein \ ”P” @ PROTEOLYSIS cn o NH3 Polysacchorlde k3u90r8 ADP'PI'J‘T p @FERMENW’ PROTEIN ENERGY FIGURE 1. DIAGRAM OF RLMEN FUNCTION. (IiAPI‘ERI PUG-BIC ACID - mam RMIOS OF PURE QIL'IURES AND MIXED RUMINAL BACI'ERIA W Effective nitrogn utilization by the ruminant requires a supply of gotein of suufficiexxt quantity and quuality to satisfy the requirenents of the animal for both maintenance and produuction. An adequate description of nitrogn reaching the enall intestine requires measurenent of the contributions mag by both runinally undergraded dietary protein and microbial protein synthesized during the fermentation of the feedstuff. (he cannon approach has been to gug the microbial contribution by using an internal marker of microbial protein such as ribonucleic acid (RNA) . This apgroach assumes that most RNA reaching the lower gut is of microbial origin (Snith and McAllan, 1970) . The poportion of microbial nitrogn (N) in abomasal or duognal N can be calculated by multiplying the ratio [(total N/bacterial dry matter) (RNA-N/bacterial dry matter)] by {(RNA-N/abonasal dry matter) (NonAunonia Nitrogen (MM/abomasal dry matter)}. Microbial flow is then gtermined by multiplying this figure by total NAN passag to the lower GIT. wviously, a prerequisite of this marker technique is the maintenaxm of a constant ratio of WA-Nmotal-N in the bacterial population leaving the runen. Ellis and Pfander (1965) and Smith (1969) suggested that this relationship was constant in runen bacteria under a variety of dietary 12 13 conditions and that it might be useful as a marker of microbial protein synthesis. Analyses of runen microorgnisns taken fran meep fed diets gvoid of nucleic acids indicated that a relatively constant (10.4 - 14.8%) of the total microbial nitrogn could be attributed to ribonucleic acid N. 'Jhese estimates were calculated assuming a constant Nitrogn: Phosgiucrus ratio of 15:3.8. Snith (1969) reported that approximately 19% of the total microbial N was nucleic acid. Smith (1969) also conduucted an analysis of five species of rumen bacteria and concluded that RNA-N:total—N ratios were less variable than DNA-N/total-N. Since these early studies, many workers have reported digsta passag studies where RNA-N/total—N has been utilized as a marker for microbial protein synthesis (Theuurer, 1980) . Unfortunately, a consigrable rang exists in the estimates of microbial yield using this technique (Table 1). This variation may be due to species variation, a physiological response of the runen microogniens or to difficulties with sample analysis. We 1. Gunnery of Entperiments Reporting Microbial Protein Yield a W now MICKBIAL MARKER MARKER MEAN RPME (hrcmiun RNA 12 6-20 Lignin RNA 19 12-23 1'vizeurer, 1979 bGrails Microbial N/Kg non Several gable-s have been igntified with the use of this technique. 'Ihese inclurh contribution of dietary nuucleic acid to the pool of nucleic acid measuured postruninally (McAllan and Snith, 1973) and fluctuations mc QL Jr.~ 14 over time in the RNA-Nztotal-N ratio of mixed runen bacteria (Smith, 1975). Recently, a correction factor of .85 has been prode to account for the contribution of exogenous dietary RNA to nucleic acid measurements mach postruninally (Mdkllan and Smith, 1973) . Mind runen bacteria ampled just before feeding showed significantly lower RNA—N/total-N then bacteria taken 4-6 hrs after feeding (Snith and McAllan, 1974). (herakawski (1976) reported that the relative composition of microbial preparations varied more with diet than with time after feeding. mk-N/total-N varied fran .09 for a hay diet to .18 for diets containing high levels of grain. Consichrable variation in the RNA-Nztotal-N ratio between individual animals and different diets was observed by Ling and Buttery (1978) . Workers at Kansas State (Arambel et al. , 1982) found extensive variation in RNA-Wtotal—N and mn-N/total-N of 17 pure runinal strains harvested in the stationary phase. Exact growth conditions were not reported. Most in vivo RNA-Wtotal-N values vary fran .16 - .20 for samples taken at several times after feeding and pooled prior to analysis (Poos et a1., 1979 and Isidiei, 1980) . mA-N/total-N values in the literature vary frcn .03 (Ellis and Pfanchr, 1965) to .07 (Snith, 1969). By using appopriate conversion factors (6.25 x N for protein, and 6.76 x N for RNA) (Ling and Buttery, 1978) the following calculation an be mach: (6.76 x .18) [(1 - .18 - .05) x 6.25] = .25 11118 value regesents a conversion fro: m-N/total-N to RNA/protein and is representative of values reported by Gillett (1982) and Barao (1983). Work with enteric bacteria has indicated that microbial INA content is notcnnstantper mitofcellmassandhememaybemsuitableasa 15 microbial bianass marker (Maaloe and Kjeldgard, 1966). RNA content varies with the [hysiological state or specific growth rate of bacteria (Haaloe and Kjeldgaard, 1966; Rosset et a1., 1966; Neirlida, 1978; and Ingram et a1., 1983). INA/protein values of .l-.2 are usually obtained for enteric orgnisns growing near stationary phase (Koch, 1971) . Bacteria in the runen can be broadly divicbd into free floating orgnisns in the small particle-liquid phase and attached (aduerent) or bound organisns in the large particule phase (Cheng and Costerton, 1980) . Hungate (1966) and Oxford (1964) have indicated that runen bacteria are 10 to 1011 present at 10 per g runen contents and grow at an average rate of .06 to .07 doublings/hr. If all runinal bacteria were growing at this rate, the RNAzprotein ratios would be expected to be low and constant (Nierlidl, 1978; Bergen et a1., 1982). Variations in overall INA/protein ratios of runen bacteria would only occur if a sizeable fraction of the population were growing more rapidly. 11118 is likely for free bacteria after feeding, especially when large quantities of readily fermetable carbohydrate are {resent in the diet. 'lhe objectives of the work reported in this chapter were 1) 'no determine BEA/protein and RNA/protein values of several predcninant runiml bacteria when cultured at different specific growth rates and harvested at mid log and stationary mass 2) 'no apply this information to interpretation of existing data on the variation of INA/protein that has beenotservedinthe runenwithtimeafter feedingand3) Todetermine the effect of nutrient limitation on the RNA/protein of predaninant ruminal bacteria. l6 mtgials and Methods W Butyrvibrio fibrisolvens D1, Selenanonas ruminantiun GA192, Ruminococcus albus 7, and Bacteroides succinogenes S85 were obtained from the culture collection of the Department of Dairy Science, University of Illinois. Butyrivibrio fibrisolvens H106 and Ruminococcus flavefaciens (94 were a kind gift fran Dr. B. A. Dehority, Department of Animal Science, (1110 Agricultural Research and Developnent Center. Bacteroides runinicola subsp brevis (ATCC, 19188) and subsp ruminicola (ATCC, 19199) and Selananonus runinantiun subsp lactilytica (ATCC 19205) were purchased from the American Type Culture Collection. Streptomccus bovis was isolated frcm the rumen of a steer fed alfalfa hay. A brigit orange colony was isolated fran a 108 dilution into a roll tube containing medium 98—5 (Bryant and Robinson, 1961) . 'Ihe colony was one of the first to appear during a 24 hour incubation. 'Ihe isolated cells are gram positive cocci that exist as singlets or pairs. 'lhis species fernents glucose, sucorse, naltose, mamose, cellobiose, lactose, fructose, galactose and soluble starch: but not xylose or arabinose. It is a halofermentative lactate [roducer and grows with a specific growth rate over 2 .0 am most substrates. Routine transfers of stock cultures were performed at monthly intervals. Gram stains and wet mounts of the respective microorganisms were examined bimonthly as a check for culture purity. W 'lhe Hungate technique was used in the preparation of media and cultivation of microorganisms. Inoculation and sampling were performed while continmusly gassing with 02 free (I) 2whiduhadbeenpnssed 17 through a heated quartz column containing elenental cOpper. Cultures were grown at a constant 39°C. indie Bacteria were maintained on slants of Medium 10 (Caldwell and Bryant, 1966) suppleIuented with 10% (v/v) clarified rumen fluid and modified to mntain .18 (v/v) of the volatile fatty acid mix. 'lhe éfined experimental medium is shown in Table 2. W Specific growth rate of a bacteria species is a function of the rate at which a given substrate is metabolized and the genetic potential of that strain to synthesize cellular material and diviub. Growth rate of runinal bacteria an be varied by changing energy source (Russell et a1., 1979) or limiting sugar concentration in the growth medium (Russell and Baldwin, 1979) . Both of these techniques were used in this work to grow runinal strains at a diversity of growth rates. S. ruminantium was grown in media containing as the primary energy substrate .5% (w/v) fructose, glucose, sucorse, galactose, nualtose and cellobiose, respectively (listed in the orkr reflecting ability to support rapid growth); B. ruminicola in media containing glucose, galactose, sucrose, fructose, lactose, maltose and cellobiose; B. fibrisolvens in media containing sucrose, fructose, cellobiose, galactose, glucose, xylose, maltose; B. succinogenes in media containing glucose and cellobiose; R. albus in media containing cellobiose and glucnse: and R. flavefaciaus in media containing cellobiose and glucose. In addition, each of these strains was grown in a medium containing .18 (w/v) glucose and the celluloylytics (B. succinogenes, R. albus and R. flavefaciens) were grown in a cellobiose medium frcm which the amino acid solution was deleted. Five separate growth trials and 18 Table 2. Experimental Indium for Hire Culture of Ruminal Bacteriaa mm Perm carbohydrate sourceb 1 to .5 (w/v) Yeast extract c .01 (“v/v) Vitamin solution 62 1.0 (v/v) Amino acid soluti? 1.0 (v/v) Mineral solution 7.5 (v/v) Mineral solutign 11f 7.5 (v/v) Rain soluti .5 (WW VFA solution .33 (v/v) (1) solution (8%) f 5.0 (v/v) examine-BC]. Nazs 9820 solution 2.0 (v/v) Deionized water 76.0 (v/v) :Prernred um&r (D 3 final :8 6.8. Carbohydrate sourges varied to achieve varying growth rates; glucose at various concentrations, maltose, cellobiose, lactose, galactose, c xylose, fructose and sucrose used at .5%. Contained in mg/liter: thiauuine HCL, 20; Calcium Inntothenate, 20; nicotiani&, 20; riboflavin, 20; pyridaxine—HCL, 20; para-aminobenzoic acid, 1; biotin and , .2. dContained .5% of aspar c acid, glutamic acid, threonine, serine, proline, glycine, alanine, valine, nethinonine, isoleucine, leucine, tyrosine, phenylalanine, lysine, tyrptorhan, histidine, arginine, e asparagine, glutamine and cystine. .l% 'l‘rypticase was substituted for mino acid solution during cultivation of B. ruminicola. (bran et at. (1977) . ntained in ml/liter: acetic acid, 548.4; propionic acid, 193.5; n-butyric acid, 129.0; n-valeric acid, 32.33 isovaleric acid, 32.3; isobutyric acid 32.3; 11. 2-methylbutyric acid, 32.3. l9 sapling periw were anployed. 'lhe growth rate observed with a given bacterium on a particular substrate was not always constant. Russell et a1. (1979) reported a similar observation in their study of maximum growth rates of rumen bacteria and suggested that genetic variation in the inoculum could affect mandmum growth rate observed umder a given set of conditions. 'Ihe Ks value of an organisn for a given substrate is defined as the concentration of growth limiting substrate that will support one half maximal growth rate (Lynch and Poole, 1979). An approzdmate KS that could be used to limit growth of the bacteria used in emeriment one was chtermired for glucose, m3, amino aci$ and peptiés. Varying concentrations of each of these nutrients were utilized to achieve a stepwise increase in both growth rate and yield up to a maximum value for both praneters. 'Ihe approrriate modifications of the experimental medium inclu¢d the following: 1. Use of .01, .05, .1, .2, .3, .4, .5 and 1.0% (w/v) glucose in the experimental medium to test for limitation by glucose concentration. 2. Use of .005, .01, .025, .05, .1, .15, .25, .5 and 1.0% (v/v) of an acidified (pH 4.0) amino acid mix (.5% (w/v) glycine, alanine, valine, leucine, isoleucire, serine, threonine, methionine, aspartic acid, asparagire, glutamic acid, glutamire, arginine, lysine, histidine, phenylalinine, tyrosine, tryptopuan and proline) to test for amino acid limitation. 3. Use of .01, .05, .l, .2, .3, .4, .5, 1.0, 2.0, 3.0 and 7.5% (v/v) Mineral 2 solution in the experimental medium modified by raoval 20 of the amino acid mix. 11:18 set of media were used to test for N33 limitation. Other minerals renaired constant through appropiate mireral supplenentation. 4. Substitution of .005, .01, .025, .05, .1 and .2% Trypticasem (BBL Microbiology systems) for the amino acid mix to test for limitation by peptide. Bacteria were grown in 500 ml roumd botton flasks containing 300 ml of the experimental medium. Medium conposition was desigued to contain only trace Tricloracetic acid (TOD precipitable protein or nucleotide souurces. Cultures were taken through several serial transfers in a medium icentiunl to the experimental medium before they were used to inoculate experimental flasks. mly cultuures in mid-log phase were used a inocula. Inocula represented 3% of the final volume of the emerimental cultures. Growth of bacteria was monitored by measurenents of optical cenisty (600 mu) of S-ml mples taken fr- experimental flasks at variouus time intervals. The specific growth constant (:1) was conputed by taking the slope of the 1n optical chnsity vs time curve. Preliminary growth curves for the various bacteria were prepared and final cell dry matter yield and the start of the retardation [base determired. 'Ihirty five ml samples for RNA, IRA and protein analysis were initially taken at apgoximately mid—log phase of grcwflu, which was defined as batch growth to an optical chmity cf .5;I-_ .1. Bob culturewas then grownfor atotal of48handa final sanple representing stationary phase bacteria was taken. All culture growth had plateaued at final sampling. Fifty percent 'I'CA was added to the sample to yield a final concentration of 5% and the sample was placed on ice for at lease 30 min. 21 .Smmfiam 55m ”5 22m zanmflSlE 92 Eé Exam Balsam 5: $5222.31 at MEEEQ 8 am: 895: me: lo E3 30.: .m was: zopdzgmwhwo <20 20.22.5qu 23:2: zofiazififio 42m 352.2 m ....om..:o z 2“ . a E: 2. 9.550%»: 5.1:: ._zSmtmzm0 .._o- mmmmm l 1 I p (hr'1; Specific Growth Rate) FIGURE 4. RNAuDNA RATIOS AT SPECIFIC GROWTH RATES FROM .1 TO 1.4 DOUBLINGS PER HOUR FOR FIVE RUMINAL BACTERIA. 28 Table 4. Cheerved and Predicted RNA/Protein Values for Zero Growth in Rumen Bacteria RNA-protein at RNA-protein Correlation between stationary [base Iredicted when u and RNA/protein Bacteria u :0 B. fibrisolvens .24 i .07“1 .27 .93 R. albus .18 i .01ID .08 .70 B. succinogenes .18 1 .05b .29 .85 B. runinioula .23 i .04a .26 .90 R. flavefaciens .23 3; .03a .17 .97 s. runinantium .25 1 .05a .14 .97 s. bovis .46 1 .13° .26 .94 Arithnetric mean .25 .21 sau .06 a'b' C, p<001 29 The INA/protein (Figure 5) of ruminal bacteria growing at u fron .l to 1.2 ranged frouu a low of .65 to a high of 1.5, with most values falling between .85 and 1.15. 'lhus, specific growth rate did not systematically affect bacterial INA/Protein values. INA/protein values of S. bovis are plotted against specific growth rate (It) in Figure 6. The values represented are the conposite mean of 3 separate observations for growth on each of the substrates listed. The regression for RNA/protein vs I1 for this organisn was YE-.28u+ .26, r=.94. The linear relationships appears to strictly adhere up to u of appoximately 1.5 after which a plateau is observed. 'Jhe sIOpe of this regression line is .24 as confered to .65 for the composite of the other six predmirant ruminal strains tested. This indicates that at any given growth rate, less RNA is required by S. bovis than is required by other ruminal bacteria. Protein synthesis in this strain would appear more efficient than with any of the other six ruminal strains tested. Streptococcus bovis also appears to retain RNA as it enters stationary Lease proviubd a high RNA/protein ratio was established in the exponential Lease of growth. This is eviuenced by the relatively large standard deviation associated with the S. bovis mean (low INA/protein values associated with low growth rates renained low, whereas high RNA/protein values associated with bigu growth rates renained high). The bacteria in the other two groups tend to assume a constant macrcuuolecular ccmposition in stationary Lhasa regardless of growth rate during exponertial growth. W The effect of nutrient limitation on the RNA/protein ratio of several runen moteria is presented in Table 5. No siguificant species x treatment interaction was observed, although S. bovis tended to respond 30 isms/em .225“ “at eon. m8: 3.. 82358 :4 E H. 5E $31 565 Salaam 2 mofié 255mm"; .m 58$ .22. £265 6:325 ”15 1 Q; m; «.9 w.p O; 0. 0. h. 0. n. v. n. N. w. -l a J a a q u q u q q q q a .fi + I + mm? 0 + + N x 4 8 x .. a. x u £02553 .0 x 0 W 53:355.: .m + m. mocooofioosm .m ‘ I m u mcozofltbz .m 0 520396: .E I 31 .m.>om msoooooeaweem so. ”.8: am... 82358 9N E 3. 5E 33. 558 2.5% E SE. 298...? .A. $52. :23 use .3305 creases 0." V.“ O.N 0.. N.. o. v O S 9 3 9 9 1 MON... 3 m v v 831% H D 1 n wwlo a o w o 0505 l S l S SSS 0 3 0 3 I 3 a: S S I N. 3 n 3 w. m n u a i m. m I s N # l C 3 m a u w I . d u I o m I‘ B W N I I 6.. '0. 0- ON.¢DN. n> . _ _ c . _ .4... 32 ho. NN. mu. om. mm. mm. em. be. .5380 no cause 05033“... 05 5 3.883 5.5m gum Hancomm no ofism 503% 05 co 5333c 535:2 no ”Bonus .m_0dnt 33 Table 6. mtrient Limitation as a Main Effect on the RNA:Protein Ratio of Runinal Bacteria. maggot Wu Nutrient Suffidency .23“I Glucose Limitation .12b Nitrogen Limitation .52c $94 .08 a,b,c P<.01 Me 7. Species Variation as a Main Effect on the RNAzProtein Ratio of Rmiral Bacteria. m. W. S. bovis .47 B. nninioola .34 S. rmimntim .23 B. fibrisolvens .32 B. succinogenes .30 R. flavefaciens .25 R. albus .22 SEN .07 P<.01 34 differently to nutritional status than did the other six species. 'lhe RNA/motein valm of stationary phase S. bovis was similar between nutritionally sufficient and nitrogen limited cultures whereas this ratio was much lower in nutritionally sufficient vs nitrogen limited cultures of the other microbial strains tested. 'Ihe RNA/protein ratio of stationary {base cultures of seven runen bacteria was affected by glucose (P<.01) and nitrogen limitation (P<.01) (Table 6). Nitrogen limited cultures had the highest ratio (0.52 across all species), followed by cultures grown with aéqmte energy and nitrogen (0.28) and glucose limited cultures (0.12). Significant species variation (P<.01) was also observed (Table 7). S. bovis had the highest overall mean stationary phase RNA/protein ratio. A group including B. runinicola, B. succinogenes and B. fibrisolvens had intermediate value whereas S. runinantitn, R. flavefaciens and R. albus exhibited the lowest RNA/protein ratios across the three nutritional regime. 121mm Bact i o Schaechter et a1. (1958) first reported that macrcnolecular cmposition of bacteria is determined by rate of growth. Since then, nany other researchers have verified the validity of this concept (Neidlard; and nagasanik, 1960; Kjeldgaard and Kurland, 1963; Rosset et a1., 1966: Dennis and Braer, 1974: Quann et a1., 1980). For a given microbial species, RIB/protein and MIA/INA have been shown to be a fraction of growth rate. INA/protein, however, is indepemhnt of specific growth rate (neick, 1968). 'Jhe macroolecular cmposition of runinal bacteria reported in this study (as reflected in RNA/protien, RNA/INA, and INA/protein) is similar to other reported values for runinal (Smith, 1969; 35 Mink et a1., 1982: Russell, 1983) and nonruninal bacteria (Sdiaedlter et a1., 1958; Dennis and Brener, 1974). As expected, MIA/protein and RNA/INA inaeased with growth rate whereas growth rate had little effect on BIA/protein. The mA:RNA synthesis ratio has been suggested as a measure of munity growth rate in cunplex bacterial ecosystens (Karl et a1., 1981 a,b: Kirchnan et a1., 1982) . John (1984)‘concluded that RNA/DNA in runen bacteria followed the .e general pattern as fermentation activity and microbial growth rate within the runen. In a similar vein, RNA/protein is higily correlated with growth rate and physiological status of runen bacteria in vitro. In this study, the relationship was not as strong over growth rates representative of in vivo conditions. However, it was still higlly significant (P<.05) over a range of u fran .1-.5. Sane general observations can be made by extrapolating fran the plot of INA/protein vs u generated in vitro. First, RNA/protein app'oaching .2 clearly indicate that a bacterial population is growing very slowly if at all (Table 4). Second, INA/protein less than .5 indicate that the bacterial population in the runen is growing at a rate less than the maximun permitted la! the genetic potential of the bacteria mder optimal conditions. Fluctuations of INA/protein due to experimental treatment may reflect variation in microbial growth rate, marked changes in population mix within the runen or analytical error. line results of the RNA/protein of runen bacteria in vivo in a sheep study (Bates et a1., 1985) are dam in Tables 8—11. A simificmt (P<.001) interaction of Lhysical state (free vs bound) with diet was observed (Table 10). The EVA/protein of free floating bacteria isolated from corn fed sheep was higaer (P<.05) than the ratio in free bacteria 36 Table 8. mAzProtein Ratios oleree Runinal Bacteria Fran Sheep Fed Three different Diets Diet am. after feeding 12 113 III4 0 .25 i .01: .25 i .01: .38 i .02: 2 .29 .1 .01‘31 .31 i '02a .36 3: .03a 4 .32 i .0151 .34 3; .04a .40 .t .03ID 6 .31 .t me .27 .t .0451 . 2 1 .1431 o 8 .31 3: .01a .27 5; .0151 .47 :2 .09a' 10 .29 3: .01a .27 1 .01a .40 1 .03a 12 .26 i .01 .27 + .02 .34 i .03 Mean .29¢_.006C .29 + .011c .41 + .025d gates et a1. , 1985 3Com 4Corn-(Dom silage a rn silage ' alues in colmms (0—12h) not sharing cmnon superscripts different c a: (P<.05) . ’ alues (in rows) not maring cannon superscripts different at (P<.05) . Differenms at various sampling intervals across diets not tested. 37 Table 9. INA:Protein Ratios oflAdlerent Runinal Bacteria Fran Sheep Fed lnurse Different Diets . Diet10n$§r Hr. after feeding 12 II3 III4 0 .24: .13: .21: 2 .22a .15a .18a 4 .22a .18a .24a 6 .22a .13a .22a 8 '22a .15a '22a 10 .21a .16a .23a 12 .22 .12 .22 Mean .23C .15‘il .21“ SEM .007 .005 .005 éBates et a1., 1985 3Corn 4Corn-Corn silage a rn silage ' alues in colunns (0—12h) not sharing cannon superscripts differnet t (P<.05) . c'aValues (in row) not sharing cmmon superscripts different at (P<.05) . Differences at various sampling intervals across diets. 38 Table 10. Interaction of Physical State and Diet on the RNA:Protein Ratios of Runinal Bacteria Fran Sheep . Diet State 1 11 111 Eknxui .227 .147 .216 Free .291 .288 .409 .001. tes et a1., 1985 Table 11. Overall Means Showing the Effect of Physical State, Diet and Time After Feeding on the RNA:Protein Ratio of Runinal Bacteria Fran Sheepl. 133:1 ENAAErgtein N’ Probabilitz___ State Free .332 77 Bomd .194 77 sen .004 <.0001 .Diet 1 .259 42 11 .217 56 111 .313 56 sen .004 <.001 lunatafter_feedinga o .243 22 2 .252 22 4 .286 22 6 .279 22 8 .280 22 10 .263 22 12 .242 22 saw .004 <.14 iShows a significant quadratic effect (p<.001) . Bates et a1., 1985 39 fran the corn silage or corn-corn silage diet (.4 vs .29). Less marked variation was observed in the attached bacteria, but the RNA/protein of attached bacteria when corn-corn silage was fed was sigiificnntly laver (P<.05) than with either of the other diets (.15 vs .22) . Substrate limitation is generally accepted as the factor which most freqmntly limits microbial growth within the runen (ngate, 1966; Russell, 1984). Feeding a grain diet increases the concentration of soluble carbohydrate in runen fluid (Takakashi and Nakamura, 1969) . Increased RNA/protein of free floating bacteria when corn is fed may reflect greater substrate availability. If this is the case, two issues must be addressed: 1. The RNA/protein fran corn fed sheep acherent bacteria dues not differ fran the RNA/protein of corn silage — attached bacteria 2. The INA/protein fran corn-corn silage fed sheep attached bacteria is (huessed Gravth of acherent bacteria which colonize the surface of feed particles is supported 1y emymatic attack of canplex carbohydrates contained in plant material. Sugars that are released during the digestion process support a &nse popflation of cells which form microcolcnies (Oosterton and Cheng, 1980) . Growth in the center of a mlony is limited by the sharp diffusion gradient which exists fr:- outsié to insich (Pirt, 1975) . Soluble sutstrate mncentration an increase (as in the case with high grain diets), but substrate availability within the microcolcnies of attached bacteria may still be limited by restricted substrate diffusion into the center of the colony (Rigire 7) . 4O .6523 ME malice >238 ME .6 855 m5 SE 8.55 Emaéo Santa n55 .meszSz 2.2258 85mm 2.3 < E Bxuat< >238 dambé no :53 hszhzz makings»: mo uZON ¢ “US$33“ Dunk 822.558 mks-g» bio—ll, uu¢m§m ONUL :b’gmw anti... what 8kg: Zh’OCG 353...“! ‘ ..¢¢u:e.¢me >238 455.55 mega—mam cum“. Ob DMIOSLR £0.50 A<_mmhom zogaoma mmfieomaoxm 92 :Esé 235mm Ezmzezdz :2. we: on S 3 «N 3 2 ~. 5 e a o _ _ _ _ a _ .I! _ - a u \1 \uu \ \ \ I \ \ \ \ T R .3323 .33 O x seashore o I u u \ as n 1 v .. - O T 1 - o u u h b _ b _ P _ _ 7 O. N.— T. w.- a. 3.” NN 1N QN 0N EECLWCQ .x use; 81 9 RE 582;”. mua§m5m zotbaoomm mméomcoxm Qz< Itasca zdtfimm EImZOCsz .OH ”#50: 33mm: “WE/0W AllMLDV 3SV3iOHd .mmobé 20 7208 2:: us: on Na 2.. ea 8 2 ~. a O — - _ _ d _ _ ”a O \ I \ \\ \o \ . - a n 5.85 .33 0 ll. Q\ umeupoce 0...... x - \ ~ - \ - \ c a .. - \ e . - a - - w l l h — _ _ _ _ b _ b o N. .. e. a. 3 an 0.. n n H ~.. 3 on. n . a e. W m I. m: A uw a. m C /l\ o.~ ~.~ . ed as m.~ 83 . @855 20 23ch RE 30255”. Seaweed a 5:25er $55.85 92 SEE Eczema $520:sz .2 mad; 3:: mg... on. am am cu o~ c. m. a c c 0.. _ _ a _ _ _ _ Q\\w\.u\\-\\ o . \ _ \ - \ \ - w _ x 1. \\ u v. \ record jug all. \ - mmeueczc ouuu Eu -. a. \\ u I Q. n V 35’43108d N I A a I I m. e (009v) Ailmeani aani‘mo ‘dfliWflD SUNS/0W Ail/Hi. V I Q N 3 {—71 Om. 84 36 I I I u I r a 9 3.4 —- — 32 ‘— l/lo "" 8 3.0 - Ix” - 2.8L- ,x’ - 7 "o 2.6.— x0 _____ .0/ .. ” 8 24 _ $7 - 6 E 4 22 ’l 2 v e '- I -u D 3 so ’I E g I: " 5 E _. _ > :3: LB C u... U u l.6 - .. 4 < s . a: ._ I .. 5 '4 I a D I F- 0 I2 b u - 3 8 I a ' 0 ' $5 --- c PROTEASE° ‘ .8 — 0 CELL GROWTH _ 2 .6 _ .4 ._ I .2 ._ o 1 J 1 u 1 J o O 4 8 l2 IS 20 24 28 32 TIME (hrs) FIGURE 12. RELATIONSHIP BETWEEN GRowm AND EXOPROTEASE PRODUCTION BY BACTEROIDES Rummucouu GAB GROWN ON SOLUBLE STARCH. 85 This indimtes a decline in the specific activity of emgotease as the cultuures enter the declining phase of their growth cycle. A decrease in activity was seen in four of the stationary phase cultures. The sugars that supported fastest growth (glucose and cellobiose) resulted in the lowest absolute activity. 'IWO sugars that supported intermediate growth rates (maltose and lactose) had activity curves intermediate in their final valuues. Soluble starch, with which the activity curve never crossed the growth curve, auows the highest protease activity. This activity increased sonewhat during the stationary phase. Growth on soluble starch ceased at a conparatively low cptiml density of 1.8. W The results of Experiment 4 are shown in Table 13. A siguifimnt interaction (P<.001) again was observed between growth substrate and stage of growth (Table 14). With all substrates, except soluble starch, protease activity substantially declined during stationary phase. When B. ruminicola GA’33 was grown on soluble starch, protease activity renained relatively stable (decline frcm 4.78 to 3.99 nil/composite mg protein- mg dry matter) during shift frcn exponential to stationary phase. Growth of B. ruminicola GA33 on soluble starch is initially very rapid, but ceases at a culture turbidity below that on other substrates (final optiml density of 1.6 - 1.8). This may be an indimtion that B ruminicola GP33 undergoes substrate accelerated death when grown on soluble starch. Suubstrate accelerated death has been attributed to a cell's inability to adequately regulate transport processes during transition fron nutrient mficient to repleted conditions. This mfect ca-II-nn veniué I\.flI~I-- .- u... r, Exponential Stationary 86 Table 13. The Effect of Substrate, Monensin and Stage of Growth on Emprotease Activity of B. Ruminioola GA33 Glucose Maltose Lactose Cellobiose Soluble Starch +14a -14 +14 -M +14 -14 +14 -14 +14 -14 C '74 23 8.6 7.9 8.6 11.2 15.8 11.3 5.0 8.6 10.9 5.8 as B 94 0‘ $5 3.8 2.9 3.5 3.4 3.3 3.9 3.0 3.6 3.0 2.6 3 ‘3 era {'3 5.7 4.8 3.0 6.3 " 4.1 5.7 4.5 7.2 11.9 3 E 2‘ $5 1.1 107 .4 2.1 - 2.0 1.1 1.1 1.6 5.4 a2 .5 ug momnsin/m1 87 may ultimately result in a depletion of critiml metabolic intermediates, thereby simulating an emrgy starved state (Koch, 1971) . An interaction also was observed between nutritional status of the growth culture and its stage of growth (Table 15). Glucose limited cultuures did not experience the decline in stationary {base proteolytic activity observed with the other two nutritional treatments (Experiment 5). It appears that when culture growth ceases due to a limitation of emrgy, exoprotease activity stabilizes at a valuue comparable to that observed during exponential growth. Further evidence for this conclusion is derived frcn the response of B. ruminimla GA33 to addition of inhibitors of glucose transport (Figure 13) . Addition of a methylglucosicb and 2 deoxyglucose to an exponentially growing culture mused an inmediate cessation of growth. Proteolytic activity stabilized and remained constant over a six houur period. Derepression of exoprotease activity due to increased cAMP (mtabolite repression) appears to be ruled out bemuse no net increase in activity was recorded. Indeed, little response to exogenous cAMP was observed (Figure 14). Rather, it can be concluded that, during emrgy starvation, something interferes with the mechanism that normally muesses emp'otease activity of stationary phase cultures (Table 16) . A clue may be fouurud in the response of B. ruminicola GA33 to levallorphan (Figure 15) . This compound, which stimulates ppGpp production in enteric bacteria, severely perturbed the emgotease activity of this microorganisms. Although the activity vs growth curve (following levallorghan addition) showed a cycliml pattern (first stimulation and then repression of activity), final proteolytic activity appeared to be mpressed. Nitrogen starvation, whidu should stimulate 88 Table 14. Interaction of Substgate and Stage of Growth on Emprotease Activity of B. Runinicola GA’33 Stage 0mm 511m Wial Stationary Glucose 5 .26 1 .79 Cellobiose 5.72 1 .96 Maltose 6 .92 1 .97 lactose 7 .78 2 .12 Soluble Starch 4.78 3 .99 .001 311an protein - dry matter (least square means) Table 15. Interaction of Nutrisional Status and Stage of Growth on Exngotease Activity of B. Ruminicola G183 Stage of Growth W Emanuel Stationarv Adequite 5.4 2.1 Glucose Limitation 6.6 5.3 Nitrogen Limitation 3.4 .7 .001 mg protein - dry matter (least square means) - 89 amass: Ewfififi .3 “2: “+558 8:..de .m m2: has». 328 .N we: Eczema 3858 A mzsc .93 5022.22 mmeommbé to $9885 20 mmo§c>xomo-m do 20:58 to Barn .2 was: an... v w::. h 0 0 V n N m 3 o a a a a a a o 0951 33209—309 N m '1 .1 I. n a 3 I. a m ”01 mm 3 0 w. 1 3 0A a ’ V 9 OT 0 O ( 9] fl. - D h P .P b N.- 9O Acmfi .5 20:52 to Sat .2 cello: 2352:. a. c o e n a _ o o a a . a a _ c .2.:2 >w._ N 1 N. m e u v n n a 3 a m o r a m. 3 m V l n u o ) V o - a. m m. 0. 7 0.. u. p p p B — — N.- 92 ppGpp production as well, also resulted in a depression of protease activity in stationary phase cultuures (Table 15) . A stimulation of ppGpp production should result in an increase in intracellular proteolysis (St. John and Golduerg, 1978) . Guanosim polthOsphates accumulate during stationary phase of stringent E. coli (Kraner et a1., 1981) and may be associated with an increase in intracellular proteolysis and decreased emgotease normally observed in stationary phase cultures of B. ruminicola (Mandelstam and Mcnuillen, 1973). In nonruminal bacteria, intracellular proteolysis has been shown to be an emrgy mpendent process (St. John and Goldberg, 1978). Thus, starvation for energy could conceivably result in an accumulation of ppGpp (Cashel, 1975) without a corresponding increase in intracellular proteolysis. Cellular Irotein as a percent of total cell mass during nutrient sufficient, glucose limited and nitrogen limited growth of B. ruminicola 6183 is shown in Table 17. This valuue increased fron 34% to 44% as glucose limited cultures entered stationary uhase. Gluucose limitation has been shown to decrease the RNA:protein ratio of B. ruminicola (Bates and Bergen, 1984) . Nongotein cell conponents must turn over faster than protein in this bacterium when it is in the glucose limited state, suggesting a degession of intracellular proteolysis. 93 Table 16. Effect of Stage of Growth on the Emprotease Activitya of B. Runinicola.GA33. ___S£ssifl£_Act iv ity WI! mU/mc 9W Exponential 9.18 3.42 6.22 Stationary 3 .49 2.55 2.93 SE24 .42 (.001 east square means Table 17. Cellular Protein as a Percent of Total Cell Mass Duuring Nutrient Sufficiency, Glucose Limitation and Nitrogen Limitation SW bummed: 3W Nutrient Sufficiency 37% 34% Nitrogen Limitation 29% 29% Glucose Limitation 34% 44% 94 Table 18. Influence of Growth Substrate on Emmotease Activitya of B. Ruminicola G183. Substrate Specific activityb Glucose 3 .48 .49 Cellobiose 3 .74 .43 Maltose 4.45 .13 Lactose 5.74 .17 Soluble Starch 4.45 .43 SEM .78 .0 st square means Emu/mg protein - dry matter Specific growth rate 95 Growth substrate as a main effect is seen in Table 18. In gemral, as growth rate increased, euaogrotease activity declined. A relationship between bacterial growth rate and proteolytic activity has been previously reported (Wiersma et a1., 1978) . Momnsin was not siguifimnt as a main effect although exoprotease activity tended to decline with momnsin when expressed on a dry matter basis (Table 19) . In addition, an interaction between substrate and monensin was observed (Table 20). Monensin decreased emprotease activity of B. ruminicola with all substrates except glucose (with which activity increased). The decline for maltose monensin and soluble starch monensin was proportionately snaller when specific activity was expressed on a protein as Opposed to a dry matter basis. W TWO peaks were observed in a plot of dilution rate (D) vs exoprotease activity (Figure 16). Cm peak occurred at D = .18, the other at a dilution rate approaching the maximum growth rate of the organism. 96 Table 19. Influence of Monensin on the Ebaoprotease Activity of B. Runinicola GA‘33 SpecifioAcbivitv W mU/mq protein mU/mq dry matter Control 7.6 2.9 Monensina 7.8 2.3 a2.5 g/ml Table 20. Interaction of Monensin and Growth Substrate on Exogotease Activity of B. Ruminicola GA33 Treating]; mt ate Control Monensin Glucose 4.3 4.8 Maltose 5 .8 3 .9 Lactose S .3 Cellobiose 4 .5 3 .7 Soluble Starch 6 .4 5.7 (.01 3110/ mg protein - dry matter 97 wk; 20.5.10 n. h. w. n. '. .mczehau m:o::__z:u cae_z—n mmocauo do as... 22555 no 2252:“. a 2 Swansea Ea 59:55.. m5....,___:0 5 ‘U D E \ :3 Id 9' U 52 B m in I g... 2 6 m C 2 >.. N Z U u. 0 ti < (I D DILUTION RATE (1.") FIGURE 17. FOUR PATTERNS OF ENZYME PRODUCTION IN BACTERIA GROWN IN CONTINUOUS CULTURE (WIERSMA ET AL.. 1978). 103 repression by the growth limiting nutrient is shown in curve C. The rate of emyme production declines with increased dilution rate bemuse the rate of substrate utilization increases at the higher dilution rate. Curve D has been attributed to a canbination of regulatory processes including induction and repression (Clarke and Lilly, 1969) . The activity profile of the glucose limited continuous culture of Bacteroides ruminicola was very similar to that for maltose limited continuuous cultures of Bacteroides amyloghilus (Henderson et a1., 1969) . One peak in activity is seen at a dilution rate of .18 and another at .5. This later value is approaching the maximum growth rate of this organism. This profile indimtes the involvenent of sane type of induction and repression. Induction by anino acim and peptims could not be denonstrated in batch cultures of this organism. The nature of the inducer renains undefined. Regulation by mtabolite repression maybe responsible for the descending slope of the curve after D = .18. eat at o ote ti Activi in e Rumen The rate of hydrolysis of protein in the rumen is influuenced by diet. Typimlly, proteolysis is elevated when high roughage diets are fed (Ganev et a1., 1979). Nugent and Mangen (1981) recently reported that the rate of hydrolysis of alfalfa fraction 1 protein was nine times higher when aueep were fed alfalfa than when fed a hay-concentrate mix. Substrate limitation is gemrally accepted as an important factor that limits microbial growth in the rumen (Hungate, 1966). Ruminal sugar concentrations should be elevated when grain is fed (Takakashki and Nakanura, 1969). Thus, the results of these in vivo studies are consistant with the observation that proteolytic activity of B. ruminicola is higher during glucose limited growth than when glucose is not limiting. is There a Decrea - A protein sparing effect has recently been attributed to mauensin (Van Nevel and Deneyer, 1W7; Poos et a1., 1979). Decline in rumen aunonia is routimly observed when ionoghores are used as feed additives (Van Nevel and Deneyer, 1977: (halupa, 1980). Short (1978) reported inhibition of proteolysis in the presence of momnsin using mixed rumen cultures in vitro. Barao (1983) observed a depressiau in both protease and deaninase activity in rumen bacteria in vivo when momnsin was fed. An increase in the Ixoportion of dietary protein esmping ruminal degrachtion has frequently been observed with diets containing monensin (Isichei, 1980: Poos et a1., 1979). Thus, momnsin may increase both the quantity and/or quality of protein reaching the lower gastrointestinal tract for digestion and ataorption. Several researchers (Cheu and Wolin, 1979; Wallace et a1., 1981) have established that a shift in the microbial ecology of the rumen resuults when ionoghores are fed. Gran positive bacteria (The [redminant acetate, tydrogen and formate poducers in rumen) are inhibited by momnsin (Chen and Wolin, 1979) . Gran mmtive strains, many of which produce suuccinate, are not inhibited to the sanue mgree (Chen and Wolin, 1979: Barbrson et a1., 1981) . Dickerson (personal cannunimtion, 1983) observed a similar response to lasalocid using pure cultures of rumen bacteria in vitro. It has been suggested that ionoghores muse a diversion of reducing equdvalents fran methane production to proximate production as a consequence of the selection process which allows gran mgative organism to proliferate and dcminate the rumen fermentation (Chen and Wolin, 1979) . the explanation for the observed shift lies in the physical structure of gran mmtive bacteria. The outer membrane of gran mgative species 105 serves as a penetration barrier which protects these cells fran antibiotics (Kadaer and Bassford, 1978) . The growth energetics of gram negative strains may also be involved (Bergen and Bates, 1984) . Given the inefficiency of amercbic fermentative processes (Bmgate, 1966), the availability of ATP is often consi&red the limiting factor controlling rate and extent of bacterial growth in the runen (Hungate, 1966). (bllapse of the proton gradient catalyzed ty monensin would impinge on available ATP supplies to varying degrees (depending upon the mechanisn by which protons are expelled frcn the cell). Those bacteria which couple this process to electron transport would protect their valmble ATP stores and have a selective advantage over strains which depend heavily on direct utilization of intracellular ATP. Anaerobic respiration in general and the funarate reductase system in particular, is more prevalent in gram negative microorganism. Thus, many of the runen bacteria selected for by ionomores possess the capacity to generate a groton motive force by electron transport thereby minimizing the contribution required by the intracellular ATP supply. Research conducted in the early 1960's indicated that the primary proteolytic organisns in the runen are gram negative (Blackburn and Hobsen, 1962). Sane gram positive strains have been implicated (Blackburn and Bobsen, 1960: Russell et a1., 1981), but these should be inhibited by monensin (Chen and Wolin, 1979) . It would seem that if a simple bacterial shift explains all monensin effects on the runinal fermentation, runinal proteolysis should actually increase airing monensin feeding. The observation that total proteolytic activity in the runen did not change with different dietary protein levels lead Allison (1970) to speculate that many runen bacteria possess the constitutive paperty for 106 at least a low level or protease prediction. This observation, coupled with research that indicated that protein, pepticbs or amino acids did not indice protease in BacteroiE< “.o mbz: NH gmmmwmoe E254 Emma a $.28 $55”: no 58 5355 .2 new: 3.... 338.... we on on ea 2 N. o o . q . u . 4 on. e. Noo.oanO. er 112 8 co 2 <8. nm 2. $5.88 96 8.4 8&6; no Ema .N< $22... m>ck and W.A. Hanilton (Ed.). Microbial Energetics. pp 241-284. unabridge University Press, (zmbridg. Koch, A.L. 1971. The adaptive responses of Escherichia coli to a feast and fanine existence. Adi. Microbial Plysiol. 6:147. Koch, A.L. and C.S. Deppe. 1971. In vivo assay & protein synthesizing capacity of Escherichia coli fran slowly growing chenostat ailtures. J. Molec. Biol. 55:549. Kepeaiy, J. and R.J. Wallace. 1982. Cellular location and sane p‘eperties of gateelytic enzymes of mnen bacteria. Appl. Erwiern. Microbial. 43:1026. 126 Kornberg, ILL. and M.C. James. 1977. The prosthetransferase system as a site of allular control. In: B.A. Hadtbck and W.A. Hanilton (Ed.) Microbial Energetics. pp 217-240. Cambridge University Press, Cambridge. Kramer, 14., E. Keeshes and I. Horvath. 1981. Guanosine polyphosghate prediction of Escherichia coli stringent and relaxed strains in the stationary [base of growth. (heal. Acad. Sci. Proc. 28:165. Langheinridi, W. and K. Ring. 1976. 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Leick, V. 1968. mtios between contents of INA, INA and protein in different microorganims as a fmction of maximal growth rate. Nature. 217:1153. Lenenager, B.P., P.N. Ownes, B.J. Shockey, R.S. Lusby and R. Tbtmek. 1978. mnensin effects on turnover rates, 24 how VFA patterns, nitrogen cmponents and cellulose disappearance. J. Anim. Sci. 47:255. Ling, J. and P.J. Buttery, 1978. The sinmltaneous use of ribonucleic aci , 2,6 dianinopimelic acid and 2 anineethylphcsrhoric acid as markers of microbial nitrogen at the duocbnun of sheep. Brit. J. Nitr. 39:165. Liditfield, C.D. and J.N. Prescott. 1970. Regulation of proteolytic enzyme prediction 1y Aeranonas proteolytica I. Extracellular encbpeptidase. Can. J. Microbial. 16:17. Lynch, J.N. and N.J. Poole. 1979. Microbial Ecology. A conceptml approach. pp 22-63. Blaclewell Scientific Publications, Iondm. 127 Maalee, O. 1979. Regulation of the protein synthesizing machinery- ribosanes, tRNA, factors, and so on. In: R.F. Geldaerger (Ed.). 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A canparison of the chenicnl canpo- sitien of mined bacteria harvested fran the liquid and solid fractions of reuse digests. Brit. J. Nutr. 50:701. Miller, C.J., C. Heiman and C. Yen. 1976. mtants of Salmnella tyrhimuriun chficient in endoprotease. J. Bacteriol 127:490. Minate, H. and T. Sutto. 1978. Technique for fractionation of bacteria in the runen microbial ecosysten. II. Attadment of bacteria isolated fran bovine runen to cellulose in vitro and elation of bacteria attached there fran. J. Gen. Appl. Microbiol. 24:1. Mink, R.W. and R.B. Hespell. 1981a. Survival of mgasrhaera elsdinii diring starvation. Curr. Microbial. 5:51. Mink, R.W. and R.B. Hespell. 1981b. Long term nutrient starvation of continmusly cultured (glucose limited) Selenanonas raninantiun. J. Bacteriol. 148:541. Mink, R.W., J.A. Patterson and R.B. Hemll. 1982. Charges in viwnity, cell composition and enzyme levels during starvation of continuome cultured (annnnia limited) Selenanonas runinantiun. Appl. Environ. 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