Transport and Deamination of Amino Acids by a Gram-Positive, Monensin-Sensitive Ruminal Bacterium

Transcription

1 APPLID AND NVIRONMNTAL MICROBIOLOGY, JUIY 199, p /9/ $2./ Copyright 199, Amerian Soiety for Mirobiology Vol. 56, No. 7 Transport and Deamination of Amino Aids by a Gram-Positive, Monensin-Sensitive Ruminal Baterium GUANGJIONG CHN1 AND JAMS B. RUSSLL' 2* Department of Animal Siene, Cornell University,1 and Agriultural Researh Servie, U.S. Department of Agriulture,2 Ithaa, New York Reeived 26 February 199/Aepted 4 May 199 Strain F, a reently isolated ruminal baterium, grew rapidly with glutamate or glutamine as an energy soure in the presene but not the absene of Na. Monensin, a Na+/H+ antiporter, ompletely inhibited baterial growth and signifiantly redued ammonia prodution (85%), but 3,3',4',5-tetrahlorosaliylanide (a protonophore) and valinomyin had little effet on growth or ammonia prodution. Diylohexylarbodiimide, a H+-ATPase, inhibitor had no effet. The kinetis of glutamate and glutamine transport were biphasi, showing unusually high rates at high substrate onentrations. On the basis of low substrate onentrations (<1,uM), the Km values for glutamate and glutamine were 4 and 11,uM, respetively. Strain F had separate arriers for glutamate and glutamine whih ould be driven by a hemial gradient of Na. An artifiial A* was unable to drive transport even when Na was present. The glutamate arrier had a single binding site for Na with a Km of 21 mm; the glutamine arrier appeared to have more than one binding site, and the Km was 2.8 mm. Neither arrier ould use Li instead of Na. Histidine and serine were also rapidly transported by Na-dependent systems, but serine alone did not allow growth even when Na was present. Beause exponentially growing ells at ph 6.9 had little A* (-3 mv) and a slightly reversed ZApH (+17 mv), it appeared that the membrane bioenergetis of strain F were solely dependent on Na irulation. Ruminal amino aid deamination is a nutritionally wasteful proess that often reates an exess of ammonia. Previously isolated ruminal bateria produed little if any ammonia (1) and ould not aount for speifi ativity of mixed ultures (25). Reently isolated ruminal bateria grew rapidly with amino aids as a sole energy soure and produed at least 2 times more ammonia than other ruminal bateria (5, 6, 23). Strain F, a gram-positive, irregularly shaped rod, was found at highest numbers in vivo, and it fermented glutamate, glutamine, histidine, and serine at a rapid rate (6). Other amino aids were not deaminated (6). arly work with sherihia oli indiated that baterial transport ould be oupled to protons (15, 16), but subsequent experiments showed that glutamate transport was stimulated by Na (11). It has sine beome apparent that Na gradients an serve as a driving fore for transport in a variety of bateria (1, 18, 27, 3). The rumen is a Na-rih environment (approximately 9 mm), and reent work indiated that the ruminal bateria Streptoous bovis (24) and Peptostreptoous strain C (7) used Na symport mehanisms to take up neutral amino aids and branhed-hain amino aids, respetively. The results presented here indiate that the amino aidfermenting ruminal baterium strain F (i) had separate transport systems for glutamate, glutamine, histidine, and serine whih ould be driven by a hemial gradient of Na; (ii) was unable to grow or produed little ammonia in the presene of monensin, an ionophore that exhanges Na+ for H+; and (iii) had little Ai\ and a slightly reversed ZApH. On the basis of these results, it appeared that strain F was solely dependent on a Na gradient for transport. MATRIALS AND MTHODS Cells and growth. Strain F has been desribed previously (6). Cells were grown anaerobially in a basal medium * Corresponding author ontaining (per liter) 292 mg of K2HPO4; 292 mg of KH2PO4; 48 mg of Na2SO4; 48 mg of NaCI; 1 mg of MgSO4. 7H2; 64 mg of CaCI2 H2; 6 mg of ysteine hydrohloride, vitamins, and mirominerals (8). Casamino Aids (15 g/liter; Difo Laboratories, Detroit, Mih.) were usually used as an energy and arbon soure for baterial growth. When ells were grown with glutamate, glutamine, or histidine,.5 g of Casamino Aids was added per liter as a arbon soure. The amino aid mixture (.5 g/liter) alone supported little baterial growth (<.2 optial density [OD] units) and gave little ammonia prodution (<2 mm). When the ells were grown in a sodium-defiient medium, Na salts were replaed by K and Casamino Aids were replaed by a purified amino aid mixture with a similar omposition. Growth rates were estimated from inreases in OD at 6 nm in 18-mm tubes. When the rates of ammonia prodution were estimated, washed ells (1. OD unit) were inubated in the basal medium ontaining only glutamate, glutamine, histidine, or serine. The inubation temperature was 39 C, and the medium ph was 6.7. The ph was sometimes dereased by the addition of onentrated HCl. Monensin, valinomyin, 3,3',4',5-tetrahlorosaliylanide (TCS), and diylohexylarbodiimide (DCCD) were prepared in ethanol, and the final onentration of ethanol was less than 1%. Ammonia was assayed by the olorimetri method of Chaney and Marbah (4). Transport assay. Cells (8 ml, 14,ug of protein per ml) were harvested by entrifugation (11, x g, 5 min, 5 C) and washed twie with potassium phosphate (1 mm K, ph 6.5). Washed ells were suspended in potassium phosphate (1.5 ml) and treated with valinomyin (5,uM) for 3 min on ie. K-loaded ells (4,ul, 3 ug of ell protein) were then diluted 5-fold into holine phosphate (1 mm, ph 6.5, 2 RI) to reate an artifiial A&i in the absene of Na. An artifiial A4 in the presene of Na was produed by diluting K- and Na-loaded ells into sodium phosphate (1 mm Na, ph 6.5). A hemial gradient of Na (AuNa) was formed by

2 VOL. 56, 199 AMINO ACID-FRMNTING RUMINAL BACTRIUM STRAIN F 2187 diluting K-loaded ells into potassium and sodium phosphate. All transport rates were orreted for nonspeifi binding (K-loaded ells into K or K- and Na-loaded ells into K and Na [1 mm eah]). The uptake of 14C-labeled glutamate, glutamine, histidine, or serine (1.5,uM) was measured from to 12 s, and initial rates were measured over a period of 5 s. Transport was terminated by adding 2 ml of ie-old 1 mm LiCl and filtering through ellulose nitrate membrane filters (pore size,.45,um). The filters were washed one with 2 ml of 1 mm LiCl and dried for 2 min at 15 C. The filters were then ounted by liquid sintillation. Competition experiments were onduted with a 1-fold exess of unlabeled amino aids. Cell protein was hydrolyzed in.2 N NaOH at 1 C for 15 min and measured by the method of Lowry et al. (17). Proton-motive fore. Cells were grown with 1 mm glutamate and.5 g of Casamino Aids per liter at ph 6.7 and 5.7, respetively. Samples (2 ml) of an exponentially growing ulture (213,ug of protein per ml) were anaerobially inubated with [14C]tetraphenylphosphonium bromide ([14C] TPP+) (1.,uCi, 3 uci/,umol), [7-14C]benzoate (1.,uCi, 21.8,uCi/umol), [U-14C] taurine (1.,uCi, 115 jici/umol), or 3H2 (4. jici, 3.6,iCi/,mol) for 15 min. Cultures were entrifuged through silion oil (a mixture of equal parts of Dexter Hysol 55 and 56; Hysol Co., Olean, N.Y.) in miroentrifuge tubes (13, x g, 5 min, 22 C), and 2-pdl samples of supernatant were removed for sintillation ounting; the tubes were then frozen (-15 C). Bottoms of tubes ontaining ell pellets were removed with dog nail lippers and dissolved in sintillation fluid for ounting. A*i was alulated from the uptake of [14C]TPP+ aording to the Nernst relationship, and nonspeifi TPP+ binding was estimated from ells whih had been treated with valinomyin (5 or 5,uM) or toluene (1%). Intraellular ph was estimated from the distribution of benzoate (21), and TCS-treated ells were used as a ontrol. Intraellular volume was alulated from the differene between 3H2 and [U-14C] taurine. A/l and ZApH were orreted for extraellular ontamination. The ability of K diffusion to generate an artifiial A4j was estimated from the uptake of [14C]TPP'. Valinomyintreated ells (see above) were loaded with 1 mm K and diluted into 1 mm Na phosphate whih ontained.1 jici of TPP+ per 2 ul. Uptake was terminated by adding 2 ml of ie-old buffer (5 mm MOPS [3-N-morpholinopropanesulfoni aid], 5 mm MS [2-N-morpholinoethanesulfoni aid], 1 mm MgCl2, ph 7.) and filtering the ells through ellulose aetate membrane fiters (pore size,.45,um). The filters were washed one with another 2 ml of buffer and prepared for sintillation ounting as desribed above. Less than 2% of the TPP+ was bound to the filters. Materials. 14C-labeled ompounds were obtained from Amersham Corp., Arlington Heights, Ill. All hemials and reagents were reagent grade. RSULTS Cell growth. When strain F was inoulated into medium (ph 6.7) ontaining glutamate (6 mm) as an energy soure and small amounts of other amino aids (.5 g/liter) as a arbon soure, the growth rate was.51 h-1 (Fig. la). No growth was observed in a Na-defiient medium, and monensin, an ionophore whih exhanges Na+ for H+, inhibited growth ompletely. Valinomyin, a K ionophore, aused a 5-h lag in growth, but the ulture eventually grew at a rate of.43 h-'. The protonophore TCS aused only a short lag (2 h) in growth, and DCCD, an inhibitor of H+-ATPases, had C z -j C-) u lo TIM (h) FIG. 1. Growth of strain F in medium ontaining (a) 6 mm glutamate or (b) 1 mm glutamine. Cultures were also treated with 5,uM DCCD, 1 um TCS, 5,uM valinomyin, Na defiieny, or 5,uM monensin as indiated. no effet on baterial growth. In all ases, the final OD was proportional to ammonia prodution (data not shown). When washed ells (1. OD unit) were reinubated with medium ontaining glutamate (from.5 to 1 mm) for 2 h to estimate initial rates of ammonia prodution, the apparent Km and Vmax values were 17.8 mm and 91 nmol/mg of protein per min, respetively (data not shown). When the initial ph was dereased to 5.7, there was a longer lag time, but the growth rate was not signifiantly affeted (data not shown). Monensin, valinomyin, and DCCD had similar effets at ph 5.7, but TCS aused a omplete inhibition of growth at this lower ph value (data not shown). Strain F grew less rapidly on glutamine than on glutamate (Fig. lb), but monensin, valinomyin, DCCD, and TCS had similar effets on growth. When Na was omitted from the medium, some growth was observed, but the rate was very slow (.9 h-1) and the final OD was less than.4. Initial rates of glutamine utilization (ammonia prodution) by washed ells (2 h in.5 to 1 mm glutamine) yielded apparent Km and Vmax values of 16.2 mm and 777 nmol/mg of protein per min, respetively. Transport. Preliminary experiments indiated that the rates of glutamate and glutamine transport were diretly proportional to ell protein so long as the assay ontained less than.3 mg of protein per ml and the transport time was less than 1 s (first-order kinetis). Preliminary experiments also indiated that amino aid transport was not signifiantly affeted by the presene of oxygen (data not shown). When

3 2188 CHN AND RUSSLL APPL. NVIRON. MICROBIOL..' 4. A + au Na Oz \ ^a~~~~&u Na 3 - ( \ * a~ (+Na) 2 A No fore or g2 C._ L.. L- m _ 6 + L I CD TIM (Oe) FIG. 2. Transport of glutamate by strain F ells with an artifiial Aifi plus AuNa, AuNa, A4i in the presene of Na, and no driving fore, or Aip in the absene of Na. The ph was 6.5. valinomyin-treated, K-loaded ells were diluted into holine phosphate (ph 6.5) to reate an artifiial &i in the absene of Na, no uptake of glutamate (1.5,uM) was deteted (Fig. 2). However, rapid transport was observed when NaCl was added to the holine phosphate (Al\ + AuNa). Sine ells whih were loaded with K and diluted into potassium and sodium phosphate took up glutamate and glutamine rapidly, it appeared that a AuNa alone was able to drive transport. No uptake was observed when Na was replaed with Li (data not shown). Little transport was observed when ells were loaded with K and Na and diluted into sodium phosphate buffer (Ai4 in the presene of Na but no AuNa, Fig. 2). When ells were loaded with K or K plus Na and diluted into K or K plus Na, respetively (no driving fore), no uptake was deteted. Similar results were obtained with glutamine (data not shown). The inability of an artifiial A*i to drive glutamate and glutamine transport ould not be explained by a resistane of the ells to valinomyin. When valinomyin-treated ells were loaded with K and diluted 5-fold into sodium phosphate, there was a rapid uptake of the hydrophobi ation [14C]TPP+ and this result indiated that an artifiial Aq was indeed reated (Fig. 3). On the basis of an internal volume of 3.,ul/mg of protein, the Af at 1 s was 129 mv, and this value was in reasonable agreement with that for the theoretial dilution (5-fold), or 15 mv. When K-loaded ells were diluted 5-fold into sodium phosphate (Aij + AuNa) at phs ranging from 7.5 to 4.5, there was a linear derease in the rate of glutamate uptake, but there was little effet on glutamine transport (Fig. 4). A 1-fold exess of nonradiolabeled glutamine, histidine, serine, asparate, alanine, or aspargine had little effet on [14C]glutamate transport, and nonradiolabeled glutamate, histidine, serine, asparate, alanine, or aspargine had little effet on [14C]glutamine transport (data not shown). When K-loaded ells were diluted into sodium phosphate (ph 6.5) ontaining 1.5 to 1,uM [14C]glutamate or [14C]glutamine, the apparent Km and Vmax values for glutamate or glutamine were 4,uM and 82 nmol/mg of protein per min and 11,uM and 5 nmol/mg of protein per min, respetively. However, at higher substrate onentrations (1 to 2,M), the adie- Hofstee plot (vls versus v) was biphasi, and the transport rate was abnormally high at high substrate onentrations (Fig. 5). Ammonia prodution by washed ells was diretly TIM (se) FIG. 3. Uptake of TPP+ by valinomyin-treated ells whih were loaded with 1 mm K and diluted into 1 mm K (O [no A*i]) or loaded with 1 mm K and diluted into 1 mm Na ( [A* plus AuNa]). In eah ase, the ph was 7.. proportional to the glutamate or glutamine onentration (Fig. 5, insets), and this result suggested that a failitated diffusion was also involved in the uptake proess. There was a dramati inrease in glutamate transport as Na onentration was inreased from to 5 mm (Fig. 6a), and an adie-hofstee plot (Fig. 6a, inset) indiated that the Km for Na was 21 mm. A Hill plot of the data indiated that the glutamate arrier had only one binding site for Na (napp = 1, Fig. 6b). The glutamine arrier had a muh higher affinity for Na (Km = 2.8 mm, Fig. 7a) and more than one binding site (napp = 1.41, Fig. 7b). Sine the Koshland ooperativity fator (the ratio of substrate yielding a veloity of.9 Vmax versus the amount needed to ahieve.1 Vmax) was 26.7 (a value less than 81), Na binding to the glutamine arrier showed positive ooperativity. Beause the affinities for Na were very low, it was impossible to determine the exat stoihiometry of Na symport with 22Na. Proton-motive fore. When exponentially growing ultures were inubated with [14C]TPP+ and orreted for nonspeifi binding (ells treated with 5 or 5 um valinomyin, 1 um TCS, or 1% toluene), Aqi was only -3 mv. Very little ['4C]benzoate was taken up, and the intraellular ph (6.67) w o _1.JU) L- L-C (._ <2 ob C <: o 5 a Ji * Glutamate * Glutamine I* UI vi I ph FIG. 4. ffet of ph on the initial rate (5 s) of glutamate and glutamine transport by strain F. Cells were loaded with 1 mm K and diluted into buffers ontaining 1 mm Na. U

4 VOL. 56, 199 AMINO ACID-FRMNTING RUMINAL BACTRIUM STRAIN F 2189 *I- C - *_ a.9 2 L N. C/ v/s (nmol /mg protei n/mi n/um) FIG. 5. adie-hofstee plots of the initial rate (5 s) of (a) glutamate and (b) glutamine transport by strain F ells whih were loaded with K and diluted 5-fold into sodium phosphate (ph 6.5) (U). The insets show ammonia prodution from glutamate or glutamine by washed ells (1. OD unit) (). was lower inside than outside (ph 6.9). This reversed ZApH (+ 17 mv) was eliminated by TCS. If the ulture ph was dereased to 6.3, there was no detetable A* or ZApH. Histidine and serine. K-loaded ells whih were diluted into sodium phosphate (ph 6.5) transported histidine and serine (1.5 um) at a rapid rate (4.3 and 5.6 nmol/mg of protein per min, respetively), and no uptake was observed in the absene of Na (data not shown). Strain F grew rapidly on histidine (.17 h-1, 423 nmol of ammonia/mg of protein per min), but little growth and ammonia prodution were observed when Na was deleted from the medium (data not shown). Serine was fermented at a rate of 256 nmol/mg of protein per min, and serine alone ould not support baterial growth (Fig. 8). However, when the ultures were provided with glutamate and serine, the OD was greater than with glutamate alone (Fig. 8). Little serine was deaminated when the Na salts in the medium were replaed by K. DISCUSSION Most living ells maintain a lower onentration of Na inside than outside, but the importane of Na irulation in baterial bioenergetis has only reently been reognized. As Skulahev (26) noted, "some anaerobi or alkali-tolerant speies extrude Na by Na-motive enzymes utilizing a orresponding energy soure with no AuH' involved, and the * I- L - %-O F: -i CD x % i 5 1 NaCl 15 (mm) Log S FIG. 6. (a) ffet of added NaCl on the initial rate (5 s) of glutamate transport (1.5,uM) by strain F ells loaded with K and diluted 5-fold into holine phosphate (ph 6.5). An adie-hofstee (vls versus v) plot is shown in the inset. (b) Hill plot of the data shown in panel a where napp is the slope of the plot. AuNa generated by these enzymes is utilized to perform all the main types of work inherent in baterial membranes." Aidaminoousfermentans, Peptostreptoous asaharolytius (originally designated Peptoous aerogenes [14]), and Clostridium symbiosium utilized glutamate as a sole soure of energy, had an absolute requirement for Na, and used a glutaonyl oenzyme A (CoA) dearboxylase to generate a Na gradient aross the ell membrane, but the mehanism of glutamate transport was not studied (2, 3, 31). Sine glutamate and glutamine were only taken up by strain F in the presene of Na, and sine transport ould be driven by a Na gradient, it appeared that these amino aids were transloated aross the ell membrane in symport with Na. Some Na-dependent transport systems are able to utilize Li as well as Na (7, 12, 28, 29), but those of strain F only used Na. The Na-oupled serine-threonine transport system of. oli had a Km for Na of 21,uM (13), but the glutamate and glutamine arriers of strain F had muh lower affinities for Na (21 and 2.8 mm, respetively). The glutamate arrier had a single binding site for Na, but the glutamine arrier appeared to have more than one site. Some Na symporters an be driven by either a AuNa or a A* (24, 27), but the glutamate and glutamine arriers of strain F ould not be driven by a A4*, even when Na was present, and little

5 219 CHN AND RUSSLL 1 U- APPL. NVIRON. MICROBIOL. 2 ) 12 C).5 1- C w z H -J (I 8 4' ' NaCI (mm) A~ Serine o Glutamate ai.2 * Glutamate + Serine - C-) TIM (h) FIG. 8. The growth of strain F in medium ontaining 15 mm serine, 1 mm glutamate, or 15 mm serine plus 1 mm glutamate. A small amount of Casamino Aids (.5 g/liter) was provided as a arbon soure. I x _ --J Log S FIG. 7. (a) ffet of added NaCl on the initial rate (5 s) of glutamine transport (1.5 FtM) by strain F ells loaded with K and diluted 5-fold into holine phosphate (ph 6.5). (b) Hill plot of the data shown in panel a where napp is the slope of the plot. inrease in transport rate or aumulation was noted when a A4+ was added to a AuNa (Fig. 2). The inability of Al to drive transport in the presene of Na may be related to the harge of the transported speies. If glutamate and glutamine arried a negative harge (-1) and were taken up in symport with one Na atom, the overall transport proess would be eletroneutral and AiJ would not ontribute to the driving fore. Sine the pls of glutamate and glutamine are 3.22 and 5.65, respetively, speies arrying a net negative harge would predominate at physiologial ph. Poolman et al. indiated that the glutamate/glutamine arrier of Streptoous remoris was only able to transport the protonated form (2). The failure of Ak, to drive transport of glutamate and glutamine might also be related to intraellular Na. In order to reate an artifiial Ak, in the presene of Na without AuNa, it was neessary to load the ells with Na. If the rate-limiting step in transport involved the release of Na from the arrier, uptake ould be inhibited by intraellular Na. Yamato and Anraku (33) reently indiated that the Na+/proline arrier of. oli was inhibited by Na aumulation. Valinomyin-treated ells whih were loaded with K and diluted into Na phosphate took up TPP+ rapidly (Fig. 3), but exponentially growing ells took up little, if any, TPP+. On the basis of these results, it appeared that strain F did not have a membrane At and was solely dependent on a AuNa. Sine an artifiial A1f was unable to drive transport (see above), the absene of a 1\+ would not neessarily be a problem. The mehanism of A* dissipation has not been determined, but K uptake might be responsible. The inhibition of ammonia prodution by monensin, an Na+/H+ antiporter whih would not dissipate A+, and the resistane of strain F to valinomyin and TCS were onsistent with the idea that transport was driven by a hemial gradient of Na rather than by AlJi. Beause aetate and butyrate were produed in the same ratio (2:1) as the glutamate fermentation of P. asaharolytius (31), no transport was observed in the absene of Na, and the ATP yield from substrate level phosphorylation is very low (1.5 mol of ATP per mol of glutamate or glutamine), it is likely that strain F used a biotin-linked glutaonyl CoA dearboxylase to expel Na (Fig. 9). Strain F was unable to grow on serine alone, even though ammonia prodution was greater than 25 nmol/mg of protein per min, and the absene of growth may be related to Na expulsion. The pathway of serine fermentation involves pyruvate dearboxylation, but suh reations usually are linked to thiamine rather than biotin. In the absene of a biotin-linked dearboxylase, ATP arising from substrate-level phosphorylation would be needed to expel Na. When serine was added to a glutamatefermenting ulture, there was a nearly twofold inrease in ellular OD (Fig. 8), and this synergism may be explained by the observation that glutaonyl CoA dearboxylases an theoretially expel more than one Na atom (9). If glutamate is taken up in symport with one Na and the glutaonyl CoA dearboxylase expelled more than one Na, the ATP from serine fermentation ould be used for growth rather than Na export (Fig. 9). Glutamate and glutamine transport was driven by a AuNa, but the kinetis were biphasi (Fig. 5). When strain F was inubated with low onentrations of glutamate or glutamine (<1,uM), the arriers had a high affinity for substrate (Kms of 4 and 11,uM, respetively), but the Vmax values were 1-fold lower than those of ammonia prodution by whole ells. At higher substrate onentrations, there was a marked inrease in the transport rate. These results indi-

6 VOL. 56, 199 AMINO ACID-FRMNTING RUMINAL BACTRIUM STRAIN F 2191 o2 ser 3ser -Q ptyruvate - <; aet 1 CoA ATP nna--,nna? NH3,j ATP ADP +Pi malate + aetate n Na latate o gin gl n aetate + +;4--- rotonyl CoA NH3 1.5 ATP n Na? gi U g--l ooguat. glutaonyli CoA n Na gl u ;~~~F glu JN NH3 n Nat NH3 FIG. 9. A hypothetial sheme showing the transport and fermentation of amino aids by strain F. ated that the hemial gradient of glutamate and glutamine ould also ontribute to the driving fore. Sine amino aid onentrations are often low in the rumen (32), AuNa would usually be the primary driving fore for transport. ven at high substrate onentrations, Na was still required, but the rumen always ontains an abundane of Na. Protein is usually the most expensive ingredient in ruminant rations, and the "5N studies of Nolan (19) indiated that more than 25% of the dietary protein was lost as ruminal ammonia. The ionophore, monensin, has been used a a feed additive in beef attle rations for more than 1 years, and more than 9% of the attle in feedlots are urrently being fed ionophores. Ionophores have usually been desribed as ruminal methane inhibitors, but some of the benefit an be asribed to a derease in ruminal ammonia (22). The observation that strain F transports amino aids in symport with Na via mehanisms whih are sensitive to monensin and not dependent on a A* provides an explanation for the proteinsparing effets of ruminal ionophores. ACKNOWLDGMNT This work was supported by the U.S. Dairy Forage Researh Center, Madison, Wis. LITRATUR CITD 1. Bladen, H. A., M. P. Bryant, and R. N. Doetsh A study of baterial speies from the rumen whih produe ammonia from protein hydrolysate. Appl. Mirobiol. 9: Bukel, W., and R. Semmier A biotin-dependent sodium pump: CoA dearboxylase from Aidaminoousfermentants. FBS Lett. 148: Bukel, W., and R. Senmnler Purifiation, haraterisation and reonstitution of glutaolylcoa dearboxylase, a biotin-dependent sodium pump from anaerobi bateria. ur. J. Biohem. 136: Chaney, A. L., and. P. Marbah Modified reagents for determination of urea and ammonia. Clin. Chem. 8: Chen, G., and J. B. Russell Fermentation of peptides and amino aids by a monensin-sensitive ruminal peptostreptoous. Appl. nviron. Mirobiol. 54: Chen, G., and J. B. Russell More monensin-sensitive, ammonia-produing bateria from the rumen. Appl. nviron. Mirobiol. 55: Chen, G., and J. B. Russell Sodium-dependent transport of branhed-hain amino aids by a monensin-sensitive ruminal peptostreptoous. Appl. nviron. Mirobiol. 55: Cotta, M. A., and J. B. Russell ffet of peptides and amino aids on effiieny of rumen baterial protein synthesis in ontinuous ulture. J. Dairy Si. 65: Dimroth, P Sodium ion transport dearboxylase and other aspets of sodium ion yling in bateria. Mirobiol. Rev. 51: Ferhihi, M., D. Hemme, and M. Nardi Na+-stimulated transport of L-methionine in Brevibaterium linens CNRZ 918. Appl. nviron. Mirobiol. 53: Frank, L., and I. Hopkins Sodium-stimulated transport of glutamate in sherihia oli. J. Bateriol. 1: Fujimura, T., I. Yamato, and Y. Anraku Mehanism of glutamate transport in sherihia oli B. 2. Kinetis of glutamate transport driven by artifiially imposed proton and sodium ion gradient aross the ytoplasmi membrane. Biohemistry 22: Hama, H., T. Shimamoto, M. Tsuda, and T. Tsuhiya Properties of a Na+-oupled serine-threonine transport system in sherihia oli. Biohim. Biophys. Ata 95: Hino, T., and J. B. Russell The effet of reduing equivalent disposal and NADH/NAD on the deamination of amino aids by intat and ell-free extrats of rumen miroorganisms. Appl. nviron. Mirobiol. 5: Horeker, B. L., J. Thomas, and J. Monod Galatose transport in sherihia oli. J. Biol. Chem. 235: Kabak, H. R Transport studies in membrane vesiles. Siene 186: Lowry,. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: MaDonald, R.., R. V. Greene, and J. K. Lanyi Light-ativated amino aid transport systems in Halobaterium halobium envelope vesiles: role of hemial and eletrial gradients. Biohemistry 16: Nolan, J. V Quantitative models of nitrogen metabolism in sheep, p In I. W. MDonald and A. C. I. Warner (ed.), Digestion and metabolism in the ruminant. University of New ngland Publishing Unit, Armidale, Australia. 2. Poolman, B.,. J. Smid, and W. N. Koning Kineti properties of a phosphate-bond-driven glutamate-glutamine transport system in Streptoous latis and Streptoous remoris. J. Bateriol. 169: Reibeling, V., R. K. Thauer, and K. Jungermann The internal-alkaline ph gradient, sensitive to unoupler at ATPase inhibitor, in growing Clostridium pasteurianum. ur. J. Biohem. 55: Russell, J. B., and H. J. Strobel ffet of ionophores on ruminal fermentation. Appl. nviron. Mirobiol. 55: Russell, J. B., H. J. Strobel, and G. Chen nrihment and isolation of a ruminal baterium with a very high speifi ativity of ammonia prodution. Appl. nviron. Mirobiol. 54: Russell, J. B., H. J. Strobel, A. J. M. Driessen, and W. N. Konings Sodium-dependent transport of neutral amino aids by whole ells and membrane vesiles of Streptoous bovis, a ruminal baterium. J. Bateriol. 17: Skerman, V. B. D., V. MGowan, and P. H. A. Sneath Approved lists of baterial names. Int. J. Syst. Bateriol. 3: Skulahev, V. P Baterial sodium transport: bioenergeti funtions of sodium ions, p In B. P. Rosen and S. Silver (ed.), Ion transport in prokaryotes. Aademi Press, In., New York. 27. Tsuhiya, T., S. M. Hasan, and J. Raven Glutamate transport driven by an eletrohemial gradient of sodium ions in sherihia oli. J. Bateriol. 131: Tsuhiya, T., M. Oho, and S. Shiota-Niiya Lithium ion-sugar otransport via the melibiose transport system in sherihia oli. J. Biol. Chem. 258: Tsuhiya, T., Y. Yamane, S. Shiota, and T. Kawasaki Cotransport of proline and Li' in sherihia oli. FBS Lett.

7 2192 CHN AND RUSSLL 168: Vri, W., R. A. Buithuis, P. R. Iwaarden, and W. N. Konings Mehanism of L-glutamate transport in membrane vesiles from Baillus stearothermophilus. J. Bateriol. 171: Wohlfarth, G., and W. Bukel A sodium ion gradient as energy soure for Peptostreptoous asaharolytius. Arh. APPL. NVIRON. MICROBIOL. Mirobiol. 142: Wright, D.., and R.. Hungate Amino aid onentration in rumen fluid. Appl. Mirobiol. 15: Yamato, I., and Y. Anraku Mehanism of Na+/proline symport in sherihia oli: reappraisal of the effet of ation binding to the Na+/proline symport arrier. J. Membr. Biol. 114: