Vol. 42, No. 1APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1981, p. 89-960099-2240/81/070089-08$02.00/0

Regulation of Urease and Ammonia Assimilatory Enzymes inSelenomonas ruminantium

C. JEFF SMITH,lt ROBERT B. HESPELL,' AND MARVIN P. BRYANT".*Microbiology Division, Department of Dairy Science,1 and Department ofMicrobiology,2 University of

Illinois, Urbana, Illinois 61801

Received 5 February 1981/Accepted 30 April 1981

Urease and glutamine synthetase activities in Selenomonas ruminantium strainD were highest in cells grown in ammonia-limited, linear-growth cultures or whencertain compounds other than ammonia served as the nitrogen source and limitedthe growth rate in batch cultures. Glutamate dehydrogenase activity was highestduring glucose (energy)-limited growth or when ammonia was not growth limiting.A positive correlation (R = 0.96) between glutamine synthetase and ureaseactivities was observed for a variety of growth conditions, and both enzymeactivities were simultaneously repressed when excess ammonia was added toammonia-limited, linear-growth cultures. The glutamate analog methionine sul-foximine (MSX), inhibited glutamine synthetase activity in vitro, but glutamatedehydrogenase, glutamate synthase, and urease activities were not affected. Theaddition of MSX (0.1 to 100 mM) to cultures growing with 20 mM ammoniaresulted in growth rate inhibition that was dependent upon the concentration ofMSX and was overcome by glutamine addition. Urease activity in MSX-inhibitedcultures was increased significantly, suggesting that ammonia was not the directrepressor of urease activity. In ammonia-limited, linear-growth cultures, MSXaddition resulted in growth inhibition, a decrease in GS activity, and an increasein urease activity. These results are discussed with respect to the importance ofglutamine synthetase and glutamate dehydrogenase for ammonia assimilationunder different growth conditions and the relationship of these enzymes to urease.

The production of ammonia from urea is animportant aspect of nitrogen metabolism inmammals, including humans (21, 27). Largeamounts of urea enter the rumen and bowelfrom the blood stream, saliva, or dietary sourcesand are hydrolyzed by bacterial ureases to CO2plus ammonia (14, 16). Obligately anaerobic bac-teria are the most frequently isolated ureolyticorganisms in the gastrointestinal tract (13, 26,28). However, it is difficult to evaluate the con-fribution of these organisms to the total ureaseactivity in the gastrointestinal tract withoutknowledge of the factors in the environmentwhich regulate their urease activity.Selenomonas ruminantium strain D has been

isolated from rumen contents and is a typicalrepresentative of the ureolytic gastrointestinaltract anaerobes (13, 26, 28). Urease activity inmost of these organisms is very low or undetect-able during growth with excess ammonia or com-plex nitrogen sources present in the medium orboth (13, 26, 28). Control of urease activity bythe alterations in the availability of nitrogen alsoappears to be the most common form of regula-tion in the aerobic and facultatively anaerobicbacteria (15). In these bacteria, high levels of

urease activity are produced only during nitro-gen-limited growth, and the levels are repressedin the presence of excess ammonia. Detailedstudies of this control of urease by nitrogenlimitation in Klebsiella aerogenes (11) suggestthat there is a relationship between the regula-tion of urease and the regulation of an ammoniaassimilatory enzyme, glutamine synthetase(GS).Although ammonia is the major and often a

required source of nitrogen for growth of thepredominant gastrointestinal tract bacteria (5,6), there generally is a paucity of informationconcerning the pathways and regulation of am-monia assimilation in these organisms (1, 25).However, our previous work demonstrated twopotential routes of ammonia assimilation in S.ruminantium strain D (25). The glutamate de-hydrogenase (GDH) pathway possesses enzy-matic properties best suited for growth in thepresence of excess ammonia, whereas the GS-glutamate synthase pathway appears to be bet-ter suited for growth when ammonia concentra-tions are low. The present studies were designedto determine what parameters influence the ac-tivities of urease, GS, and GDH and whether

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90 SMITH, HESPELL, AND BRYANT

any interactions between the regulation of am-monia assimilation and urease activity exist.

(Preliminary accounts of this work appearedpreviously [C. J. Smith, M. P. Bryant, and R. B.Hespell, Abstr. Annu. Meet. Am. Soc. Microbiol.1978, K14, p. 129] [24].)

MATERIALS AND METHODSBacterial growth conditions. S. ruminantium

strain D (13) was obtained from our culture collectionand was maintained by monthly transfer on carbohy-drate agar slants (7). The anaerobic methods for me-dium preparation and culturing of bacteria were aspreviously described (4, 12). A chemically defined,nitrogen-free, mineral salts medium was used forgrowth of strain D and has been described previously(25). Nitrogen sources were added to the medium, asdescribed below, and glucose was deleted in experi-ments in which glucose (energy)-limited growth wasrequired. Glutamine, urea, histidine, and methioninesulfoximine (MSX) were filter-sterilized, but all othernitrogen sources were autoclaved in the medium, un-less otherwise indicated.The inocula for all experiments were mid-log-phase

cultures grown in nitrogen-free mineral salts mediumcontaining excess NH4Cl (8 mM). For studies on thelevels of enzyme activity, cultures were routinelygrown in 300-ml side-arm flasks or in modified 500-mlreagent bottles, and growth was measured in 13-mm-diameter tubes (25). Experiments which tested theeffect of inhibitors or nitrogen sources on growth weregenerally done with 10 ml of medium in rubber-stop-pered culture tubes (18 by 150 mm) and were alwaysdone in triplicate. Growth was measured by absor-bance (600 nm) with a Bausch & Lomb Spectronic 70.An absorbance of 1.0 ± 0.1 in a 13-mm-diameter tubecorresponded to between 3 x 108 and 5 x 108 cells perml.The anaerobic linear-growth culture system used

has been described previously (25). The linear growthrate was maintained between 0.1 and 0.15 absorbanceunit per h. Experiments with glucose (energy)-limitedcultures containing a constant amount of nitrogensource in the culture vessel were performed by addingsufficient nitrogen source to the glucose reservoir toreplace that which was utilized during growth (ca. 0.3,umol of nitrogen added per ml of culture per h).

Preparation of cell suspensions and cell ex-tracts. The cultures were harvested by centrifugationat an absorbance of 1.0 ± 0.2 (mid-log phase), unlessotherwise indicated. The cells for GS or GDH assayswere washed in 1 volume of TKD buffer [50 mMtris(hydroxymethyl)aminomethane-hydrochloride, 1%KCl, 1 mM dithiothreitol; pH 7.4] and then resus-pended at 1/50 to 1/100 the original volume in TKDbuffer (25). These suspensions were stored in stop-pered test tubes with an argon atmosphere on iceovernight; separate experiments showed that GS orGDH activity of cell suspensions stored in this mannerdid not change overnight. For determination of ureaseactivity, the cells were washed with 2 volumes of abuffer (pH 7.2) containing 50 mM potassium phos-phate, glycerol (13%, vol/vol), and 1 mM dithiothrei-tol. The cell pellet was resuspended in 1/5 the original

APPL. ENVIRON. MICROBIOL.

culture volume and stored in tubes with an argonatmosphere at -20°C. Cell extracts were preparedwith a French pressure cell (20,000 lb/in2), followed bycentrifugation for 20 min at 15,000 x g at 4°C, asdescribed previously (25). Protein was measured bythe method of Lowry et al. (17), with bovine serumalbumin fraction 5 as the standard. The protein ofwhole-cell suspensions was solubilized before measure-ment by steaming the cell suspension for 15 min in 0.2N NaOH.Enzymatic activities. Urease activity was deter-

mined by measuring the amount ofammonia producedfrom urea at 30°C. The reaction mixture contained12.5 ,umol of urea, 12.5 ,umol of ethylenediaminetetra-acetic acid, and 0.1 ml of cell suspension in a finalvolume of 0.5 ml of 10mM potassium phosphate buffer(pH 7.2). The urea solution was prepared fresh daily.The reaction was initiated by the addition of pre-warmed cell suspension (30°C) to the reaction mixture,and the reaction was terminated by removing 0.1- to0.2-ml samples to the phenol solution used for theindophenol measurement of ammonia (9). Controlswithout enzyme or without urea were always includedto correct for background ammonia. One unit of ureaseactivity was defined as the amount of enzyme requiredto produce 1 ,tmol of ammonia per min at 30°C. Theurease specific activities of whole-cell suspensionswere equivalent to those found in cell extracts madefrom the respective cell suspension. Ammonia produc-tion in urease assays and ammonia concentration inculture fluids were measured by the indophenolmethod described by Chaney and Marbach (9). Cul-ture fluids containing amino acids or MSX interferedwith the indophenol method, and ammonia concentra-tions in these fluids were measured with an ammoniaelectrode (Orion Research, Inc.).GDH activity was estimated in cell extracts by

measuring the rate of reduced nicotinamide adeninedinucleotide phosphate oxidation (19, 25). GS activitywas measured by the forward reaction assay of Benderet al. (2) as modified by Smith et al. (25). One unit ofGDH or GS activity was defined as the amount ofenzyme required to form 1 nmol of product per min.

RESULTS

Effect of growth conditions on enzymeactivities. Preliminary experiments and pre-vious work (13, 25) showed that urease and GSactivities in S. ruminantium were low when cellswere grown in batch cultures containing initialammonia concentrations varying from 3 to 20mM. These enzyme activities increased several-fold in cells grown with low concentrations ofurea (13; Table 1), a condition in which ammoniadid not accumulate in the media. In contrast,GDH activity did not change significantly.To determine which environmental parame-

ters could influence GS, GDH, and urease, weexamined the effects of a variety of alternativenitrogen sources and growth conditions on theseenzyme activities. Growth conditions in whichlittle ammonia accumulated in the media (Table

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UREASE REGULATION IN S. RUMINANTIUM

TABLE 1. Effect of the nitrogen source on urease, GS, and GDH activities in S. ruminantium strain D

Enzyme activity' NH4+ in medium (mM)

Nitrogen source'Urease GS GDH Preiocul After growth

Glutamine (2.6).3.27 431 67 0.48 0.02Serine (2.9).1.69 183 91 0.27 0.03Threonine (2.0) ...... .. 1.30 220 110 0.05 0.01Histidine (2.1) ... .. 1.10 230 94 0.32 0.06

Aspartate (1.6).0.50 97 101 0.3 0.02Adenine (2.2).. 0.37 104 130 0.03 0.03Casamino Acids (1.5) .0.17 60 53 0.61 0.02

1.5 mM urea (1.05) . 0.38 158 177 0.1 0.095.0 mM urea (1.05) ... 0.08 35 175 0.05 0.4510.0 mM urea (1.05) .<0.02 18 178 0.11 2.20

NH4+ limited ... 1.32 182 142 0.05 0.05NH4Cl (1.05)... <0.02 22 181 9.82 7.96

a Cells were grown in batch cultures containing the 10 mM nitrogen source indicated unless otherwise noted.Casamino Acids was added at 0.4% (wt/vol), and the NH4+-limited culture was a linear-growth culture. Cultureswere harvested at an absorbance of 1.0 ± 0.2. Numbers in parentheses indicate mean generation times.

b Urease and GS were measured by using the whole-cell assays described in the text. GDH was measured incell extracts. Enzyme activities are given in units per milligram of protein (see above) and represent the averageof triplicate assays from two separate experiments.

1) were presumed to be nitrogen limited, sinceaddition of ammonia to these cultures resultedin an increase in growth rate (data not shown).Under these nitrogen-limited conditions, the ac-

tivities of both urease and GS increased relativeto the low basal activities observed in culturescontaining excess ammonia (Table 1). However,the different growth conditions caused variouseffects on urease and GS activities.GDH activity was generally lower in cells

grown with nitrogen sources other than ammo-nia or urea (Table 1). The GDH activity variedfrom very low levels seen during growth withglutamine or Casamino Acids to the moderatelevels observed with adenine.

Overall, when batch cultures were used,urease and GS activities appeared to increaseduring nitrogen limitation, whereas GDH activ-ity decreased or was not significantly affected.To more accurately define the physiologicalroles of GDH, GS, and urease, these enzymeactivities were measured by using glucose (en-ergy)-limited linear-growth cultures. With thesecultures, the urease and GS activities remainedlow whenever ammonia was the nitrogen source,even when concentrations were less than 0.5mM(Table 2). When urea was substituted for am-

monia, both urease and GS activities increasedslightly, but were less than those observed inbatch cultures grown with similar concentra-tions of urea (Tables 1 and 2). On the otherhand, GDH activity was very high and did notvary significantly when the nitrogen source or

concentration was altered (Table 2).Relationship between urease and GS ac-

tivities. Thus far, our results suggested a closeassociation between the levels of urease activityand GS activity. Further evidence for a relation-ship between these two enzymes was observedwhen an excess of ammonia (20 mM final con-centration) was rapidly added to ammonia-lim-ited, linear-growth cultures. The results showedthat shortly after the addition of ammonia, total

TABLE 2. Effect of nitrogen source on urease, GS,and GDH activities in S. ruminantium strain Dgrown in glucose-limited, linear-growth cultures

NH4' in medium **vb(mM) Enzyme activityNitrogen source'

(mM) Preinoc- EndPremoc- of Urease GS GDHulation expt

NH4Cl, 3.0 3.1 2.5 <0.02 22 225NH4C1, 1.5 1.5 1.3 0.07 25 190NH4Cl, 0.5 0.5 0.3 0.05 22 227Urea, 1.0 0.15 1.4 0.17 61 215Urea, 2.5 0.1 3.1 0.15 57 235a S. ruminantium was grown in glucose-limited, lin-

ear-growth cultures with the nitrogen source indi-cated. In addition, sufficient nitrogen source was addedto the substrate reservoir to be pumped into the cul-ture vessel to replace the nitrogen source utilizedduring growth, so that a constant level of nitrogen inthe culture vessel could be maintained.bEnzyme assays were measured as described in

footnote b of Table 1, and activities are given in unitsper milligram of protein.

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92 SMITH, HESPELL, AND BRYANT

activitiemuch sliout amwithin oactivitiethat litt]after thwhen esidly addammonitions inand thealso levifinal co.monia-limic, noand bot]at ratescontrolsments clurease uin growtof ammtures.

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Xs of urease and GS increased at a rate The relationship between GS and urease ac-Lower than that seen in the control with- tivities appeared to be invariable for all growthumonia additions (Fig. 1). Moreover, conditions tested. Pooled data which comparedne culture doubling, both urease and GS these enzyme activities in cells grown under a-s simultaneously leveled off, suggesting variety of nitrogen-limited, glucose (energy)-lim-le or no new enzyme was being produced ited, and nonlimiting conditions showed a verylis time. Similar results were observed high correlation (R = 0.96) between the specificKcess urea instead of ammonia was rap- activities of urease and GS from very low to veryled to ammonia-limited cultures. In the high levels of enzymatic activity.ia-limited cultures, ammonia concentra- Inhibitor studies with methionine sulfox-creased soon after the addition of urea, imine. MSX, a glutamate analog, is a potent!production of urease and GS activities inhibitor of growth, GS, and glutamate synthaseels off. When serine or histidine (10 mM in K. aerogenes (3). S. ruminantium was highlyncentration) was rapidly added to am- sensitive to this analog. When the strain wasimited cultures, growth became logarith- grown in medium containing 20 mM ammonia,ammonia accumulated in the medium, detectable growth inhibition occurred in theh GS and urease total activities increased presence of 0.1 mM MSX. At higher MSX con-similar to those of the unsupplemented centrations, long lag periods ensued and growth(data not shown). These latter experi- rates were further reduced; however, complete

learly showed that the effects on GS and inhibition of growth of S. ruminantium was notvere not simply a function of the increase observed even at 100 mM MSX. A likely reasonth rate which accompanied the addition for MSX-induced growth inhibition in this high-onia (or urea) to the linear growth cul- ammonia medium was the inhibition of the low

GS activity present in these cells, resulting indeprivation of the glutamine needed for biosyn-

* ] thesis. This possibility was examined by adding/.2 MSX or MSX plus glutamine or glutamate to

CONTROL 1.2 cultures growing in medium containing 20 mM>. ammonia. Glutamine overcame the MSX-in-

*.0 - duced growth inhibition and allowed growth> equal to that of controls without MSX (Fig. 2).

0.8 - On the other hand, glutamate only partially-*// NH4Cl < overcame the MSX inhibition. Other evidence

ADDITION 0.6 w for this mode of MSX action in S. ruminantium_ was obtained when GS activity in cell extracts0.4 w was measured as a function of MSX concentra-

tion. The addition of 5 mM MSX to assay mix-tures caused 50% inhibition of the GS activity

0.2 present in cell extracts prepared from ammonia-limited, linear-growth cultures (data not shown).

° More than 90% inhibition resulted when 50 mM2 4 6 8 10 12 MSX was added. In contrast, 20 mM MSX didABSORBANC E not affect the activity of GDH, glutamate syn-

thase (25), or urease when added to assay mix-Differential plot of urease and OS total tures of these enzymes.

s in ammonia-limited, linear-growth cultures Sure ase andyGsaented by rapid addition of 20 mM NH4Cl. Sce urease and GS activities were correlatedntical ammonia-limited, linear-growth cul- during growth (Fig. 1), it was of interest to-re grown to an absorbance of 0.7, and then determine whether the MSX inhibition of GSIH4Cl (final concentration) was added to one affected the urease activity of growing cultures.Samples for absorbance and enzyme assays Previously reported results (24) showed that,en at regular intervals. The arrow indicates MSX had a dramatic effect on the urease inof NH4Cl addition. The GS and urease these ammonia-sufficient cultures. Urease activ-

g were measured with whole-cell assays, as ity increased as a function of the MSX concen-d in the text; the total activities were calcu- * * *

mmultiplying the specific activity (nanomoles tration i the medium, up to a maximum of 2.25ct formed per minute per milligram of pro- U/mg of protein at 10 mM MSX. The effects ofthe absorbance at the time the sample was MSX on growth, GS, and urease in ammonia-1). Symbols: A, A, urease; 0, 0, GS. Closed limited, linear-growth cultures were also exam-are control cultures and open symbols are ined. After the addition of 1 mM MSX (finalconia-supplemented cultures. concentration) to these cultures, growth contin-

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UREASE REGULATION IN S. RUMINANTIUM

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2 4 6 8 10

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vent MSX-induced growth inhibition of S. ruminan-tium. Cells were grown to early log phase in 10 ml ofnitrogen-free mineral salts medium supplementedwith 20 mM NH4Cl. The arrow indicates the time ofaddition of 10mMMSX or 10mMMSXplus 50 pg ofglutamate or glutamine (final concentration). Sym-bols: 0, no additions; *, MSX plus glutamine; A,MSX plus glutamate; A, MSX.

ued normally for 1 h and then decreased to asignificantly slower rate (Fig. 3A). The decreasein growth rate was preceded in time by a dra-matic loss in GS activity. The GS activity thenbegan to increase, but at a rate considerablyslower than that observed with the control with-out MSX (Fig. 3B). If both 50 fg ofMSX and 50,ug of glutamine per ml were added simultane-ously, GS activity again decreased, but less so,and no growth inhibition was observed (Fig. 3Aand B). Urease activity in both of the MSX-treated cultures increased twofold or more overthe activity observed with the control (Fig. 3C).

DISCUSSIONThe supply of nitrogen (ammonia) relative to

the biosynthetic requirements of the cell ap-

FIG. 3. Effect of MSX or MSX plus glutamine on

growth and enzyme activities of S. ruminantium am-

monia-limited, linear-growth cultures. At the timeindicated by the arrow, 1 mM MSX or MSXplus 50pg glutamine (Gln) per ml was added to the cultures,and samples were taken at regular intervals for de-termination ofgrowth, GS, and urease. (A) Effect ongrowth; (B) effect on GS activity, as measured inwhole-cell assays; (C) effect on urease activity as

measured in whole-cell assays. Symbols: A, controlculture with no additions; 0, 1 mM MSX plus 50 pgofglutamine added per ml; 0, 1 mMMSX added.

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94 SMITH, HESPELL, AND BRYANT

peared to be an important factor affecting reg-ulation of GS and urease activities in S. rumi-nantium. Culture conditions which resulted innitrogen-limited growth caused dramatic in-creases in GS and urease activities relative tothe low basal levels observed during growth withnonlimiting amounts of ammonia (Table 1). Theextent to which these enzyme activities in-creased varied and appeared to be dependent onthe nitrogen source supplied for growth. Thesevariations were difficult to interpret, since am-monia did not accumulate during growth withany of the other nitrogen sources, and there maybe other factors also involved in the regulationof these enzymes. In this regard, it is interestingthat the levels of GS and urease activities wererelatively low during growth with CasaminoAcids, compared with the other nitrogen-limitedconditions examined (Table 1). The abundanceof different amino acids and potential glutamatesources present in this Casamino Acids-supple-mented medium may have had a cumulativerepressing effect on these enzyme activities.When nitrogen-limited, linear-growth cultures

were supplemented with an excess of ammonia,GS and urease activity increases were decreasedand eventually stopped, compared with those ofthe controls, which greatly increased (Fig. 1).These results show that the two enzymes re-sponded to the increased supply of ammonia.There was, however, no obvious mechanismwhich could rapidly regulate the activities ofthese enzymes, particularly since the adeny-lylation of GS seen in Enterobacteriaceae doesnot occur in S. ruminantium (23). Prolongedgrowth under conditions of excess ammonia re-sulted in low GS and urease activities. This wasquite apparent in the glucose-limited, linear-growth cultures, in which cells were in a state ofenergy limitation and nitrogen excess (Table 2).GS and urease activities remained at low basallevels in the glucose-limited cultures even whenammonia in the medium was maintained at lessthan 0.5 mM. Thus, it appeared that a lowammonia concentration per se did not signifi-cantly affect GS or urease activity, unless it waslow enough to limit growth. For example, whenurea was added in place of ammonia as thenitrogen source to the glucose-limited cultures,both GS and urease activities increased slightly.This stimulation of activity probably resultedfrom a transient nitrogen (ammonia) limitation,but when sufficient ammonia had accumulatedin the media via urease activity, further in-creases in GS or urease activity were inhibited.

In contrast, high levels of GS activity may benecessary for adequate ammonia assimilationand glutamate formation under ammonia-lim-ited growth conditions with S. ruminantium, as

APPL. ENVIRON. MICROBIOL.

is true for many other bacteria (18, 20, 23). Ourobservation that MSX inhibited much of the GSactivity and then inhibited growth in ammonia-limited, linear-growth cultures is consistent withthe idea.

Urease activity, which could supply ammoniafor growth during nitrogen-limited conditions,was highly correlated with the levels of GS ac-tivity. The activities of both enzymes were alsosimultaneously repressed by the addition of ex-cess ammonia to ammonia-limited cultures (Fig.1). Although the supply of ammonia appearedto play a role in the regulation of these enzymes,our results also showed that ammonia was notthe direct repressor of urease activity. In mediacontaining MSX and 20 mM ammonia, ureaseactivity reached high levels which were de-pendent on the concentration of MSX in themedium (24). In ammonia-limited, linear-growthcultures, urease activity increased above controlvalues when MSX was added to the medium.These observations are consistent with the ideathat factors other than simple ammonia limita-tion effect regulation of urease. For example,urease activity may have increased in responseto an MSX-induced glutamine starvation in thesame manner that MSX inhibited GS, but noturease activity. Moreover, since GS and ureaseactivities are coordinately controlled, as our re-sults showed, GS activity may also be regulatedby the intracellular levels of glutamine. Theintracellular levels of glutamine and 2-ketoglu-tarate are known to effect the regulation of GSin Escherichia coli (23). As of now, our data areonly indirect, and further studies are needed toestablish whether a similar regulation exists inS. ruminantium.The levels of GDH activity in S. ruminantium

appear to be less strictly regulated, comparedwith GS or urease activity. There was only abouta fourfold difference between the lowest andhighest GDH activities observed. Maximal GDHactivities were found in cells grown in glucose(energy)-limited, linear-growth cultures (Table2). Under these energy-limited conditions, highGDH activity would maximize ammonia assim-ilation by this non-adenosine trisphosphate-re-quiring pathway. GDH activity was slightlylower when cells were grown in batch cultureswith ammonia; however, because GS activitywas very low, GDH was probably important forammonia assimilation.On the other hand, during ammonia-limited

linear growth, as would occur with K. aerogenes(8) under these conditions, GDH activity in S.ruminantium was not repressed. Since the activ-ity was only about 35% lower than maximum,GDH probably played an important role in am-monia assimilation under these conditions with

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UREASE REGULATION IN S. RUMINANTIUM 95

S. ruminantium. This theory is consistent withdata obtained with E. coli, in which the GDHcan participate in glutamate synthesis evenwhen the ammonia concentration is as low as 75,uM; however, GDH is far less efficient than theGS-glutamate synthase pathway (20). The low-est GDH activities in S. ruminantium were ob-served when amino acids or Casamino Acidswere the source of nitrogen for growth. Duringthese growth conditions, GDH may not be im-portant, since sufficient glutamate could theo-retically be produced via transamination (e.g.,aspartate) or catabolism of the amino acid (e.g.,glutamine).Our data showed that the levels of urease and

GS in S. ruminantium were highly correlatedand responded to the availability of ammonia inthe environment. A similar correlation may existwith other rumen bacteria, as the recent work ofCheng and Wallace (10) has shown that understrictly defined conditions, urease activity ob-served with rumen fluid changes with respect tothe ammonia in the environment. Chalupa et al.(8) have also reported that the urease activity inrumen fluid is lower with animals fed rationswhich produce high ruminal ammonia levels.The concentrations of ammonia which havebeen shown to affect urease activity in vivo are,however, much higher than those we haveshown to affect urease activity in pure culturesof S. ruminantium. One possible explanation forthis apparent discrepancy is that the concentra-tion of ammonia measured in rumen fluid doesnot accurately reflect the amount available tothe bacteria in their microhabitats. For example,clumping and attachment of bacteria to starchgrains or other particles could impede the flowof ammonia to bacteria attached nearest to theparticle. Finally, studies with 15NH4' haveshown that bacterial amide is the most rapidlylabeled nitrogen pool in the ruminal bacteriapopulation over a range of ruminal ammoniaconcentrations (22). These results imply that GSis an important route of ammonia assimilationin the ruminal bacteria population. Thus, itwould follow that urease activity in anaerobicbacteria such as S. ruminantium would also beexpressed in the rumen over a range of ammoniaconcentrations, but definitive in vivo studies tosubstantiate this point have not yet been carriedout.

ACKNOWLEDGMENTS

The assistance of Daniel Schaefer in performing ammoniadeterminations with an ammonia electrode and the very help-ful discussions with Michael Cole are greatly appreciated.

This research was supported by U.S. Department of Agri-culture grant 35-331 and by the Agricultural Experiment Sta-tion of the University of Illinois.

LITERATURE CITED1. Allision, M. J. 1969. Biosynthesis of amino acids by

ruminal microorganisms. J. Anim. Sci. 29:797-807.2. Bender, R. A., K. A. Janssen, A. D. Resnick, M.

Blumenberg, F. Foor, and B. Magasanik. 1977. Bio-chemical parameters of glutamine synthetase fromKlebsiella aerogenes. J. Bacteriol. 129:1001-1009.

3. Brenchley, J. E. 1973. Effect of methionine sulfoximineand methionine sulfone on glutamate synthesis in Kleb-siella aerogenes. J. Bacteriol. 114:666-673.

4. Bryant, M. P. 1972. Commentary on the Hungate tech-nique for culture of anaerobic bacteria. Am. J. Clin.Nutr. 25:1324-1328.

5. Bryant, M. P. 1974. Nutritional feature and ecology ofpredominant anaerobic bacteria of the intestinal tract.Am. J. Clin. Nutr. 84:822-828.

6. Bryant, M. P., and I. M. Robinson. 1962. Some nutri-tional characteristics of predominant culturable rum-inal bacteria. J. Bacteriol. 84:605-614.

7. Caldwell, D. R., and M. P. Bryant. 1966. Mediumwithout rumen fluid for nonselective enumeration andisolation of rumen bacteria. Appl. Microbiol. 14:794-801.

8. Chalupa, W., J. Clark, P. Opliger, and R. Lavker.1970. Ammonia metabolism in rumen bacteria and mu-cosa from sheep fed soy protein or urea. J. Nutr. 100:161-169.

9. Chaney, A. L., and E. P. Marbach. 1962. Modifiedreagents for determination of urea and ammonia. J.Clin. Chem. 8:130-132.

10. Cheng, K.-J., and R. J. Wallace. 1979. The mechanismof passage of endogenous urea through the rumen walland the role of ureolytic epithelial bacteria in the ureaflux. Br. J. Nutr. 42:553-557.

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