The gdhB gene of Pseudomonas aeruginosa encodes an arginine-inducible NAD(+)-dependent glutamate dehydrogenase which is subject to allosteric regulation.

The NAD(+)-dependent glutamate dehydrogenase (NAD-GDH) from Pseudomonas aeruginosa PAO1 was purified, and its amino-terminal amino acid sequence was determined. This sequence information was used in identifying and cloning the encoding gdhB gene and its flanking regions. The molecular mass predicted from the derived sequence for the encoded NAD-GDH was 182.6 kDa, in close agreement with that determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme (180 kDa). Cross-linking studies established that the native NAD-GDH is a tetramer of equal subunits. Comparison of the derived amino acid sequence of NAD-GDH from P. aeruginosa with the GenBank database showed the highest homology with hypothetical polypeptides from Pseudomonas putida, Mycobacterium tuberculosis, Rickettsia prowazakii, Legionella pneumophila, Vibrio cholerae, Shewanella putrefaciens, Sinorhizobium meliloti, and Caulobacter crescentus. A moderate degree of homology, primarily in the central domain, was observed with the smaller tetrameric NAD-GDH (protomeric mass of 110 kDa) from Saccharomyces cerevisiae or Neurospora crassa. Comparison with the yet smaller hexameric GDH (protomeric mass of 48 to 55 kDa) of other prokaryotes yielded a low degree of homology that was limited to residues important for binding of substrates and for catalytic function. NAD-GDH was induced 27-fold by exogenous arginine and only 3-fold by exogenous glutamate. Primer extension experiments established that transcription of gdhB is initiated from an arginine-inducible promoter and that this induction is dependent on the arginine regulatory protein, ArgR, a member of the AraC/XyIS family of regulatory proteins. NAD-GDH was purified to homogeneity from a recombinant strain of P. aeruginosa and characterized. The glutamate saturation curve was sigmoid, indicating positive cooperativity in the binding of glutamate. NAD-GDH activity was subject to allosteric control by arginine and citrate, which function as positive and negative effectors, respectively. Both effectors act by influencing the affinity of the enzyme for glutamate. NAD-GDH from this organism differs from previously characterized enzymes with respect to structure, protomer mass, and allosteric properties indicate that this enzyme represents a novel class of microbial glutamate dehydrogenases.

Amino acid-mediated induction of the basic amino acid-specific outer membrane porin OprD from Pseudomonas aeruginosa.

Pseudomonas aeruginosa can utilize arginine and other amino acids as both carbon and nitrogen sources. Earlier studies have shown that the specific porin OprD facilitates the diffusion of basic amino acids as well as the structurally analogous beta-lactam antibiotic imipenem. The studies reported here showed that the expression of OprD was strongly induced when arginine, histidine, glutamate, or alanine served as the sole source of carbon. The addition of succinate exerted a negative effect on induction of oprD, likely due to catabolite repression. The arginine-mediated induction was dependent on the regulatory protein ArgR, and binding of purified ArgR to its operator upstream of the oprD gene was demonstrated by gel mobility shift and DNase assays. The expression of OprD induced by glutamate as the carbon source, however, was independent of ArgR, indicating the presence of more than a single activation mechanism. In addition, it was observed that the levels of OprD responded strongly to glutamate and alanine as the sole sources of nitrogen. Thus, that the expression of oprD is linked to both carbon and nitrogen metabolism of Pseudomonas aeruginosa.

Role of ArgR in activation of the ast operon, encoding enzymes of the arginine succinyltransferase pathway in Salmonella typhimurium.

The ast operon, encoding enzymes of the arginine succinyltransferase (AST) pathway, was cloned from Salmonella typhimurium, and the nucleotide sequence for the upstream flanking region was determined. The control region contains several regulatory consensus sequences, including binding sites for NtrC, cyclic AMP receptor protein (CRP), and ArgR. The results of DNase I footprintings and gel retardation experiments confirm binding of these regulatory proteins to the identified sites. Exogenous arginine induced AST under nitrogen-limiting conditions, and this induction was abolished in an argR derivative. AST was also induced under carbon starvation conditions; this induction required functional CRP as well as functional ArgR. The combined data are consistent with the hypothesis that binding of one or more ArgR molecules to a region between the upstream binding sites for NtrC and CRP and two putative promoters plays a pivotal role in modulating expression of the ast operon in response to nitrogen or carbon limitation.

Molecular characterization and regulation of an operon encoding a system for transport of arginine and ornithine and the ArgR regulatory protein in Pseudomonas aeruginosa.

The complete nucleotide sequence for the aot operon of Pseudomonas aeruginosa PAO1 was determined. This operon contains six open reading frames. The derived sequences for four of these, aotJ, aotQ, aotM, and aotP, show high similarity to those of components of the periplasmic binding protein-dependent ABC (ATP binding cassette) transporters of enteric bacteria. Transport studies with deletion derivatives established that these four genes function in arginine-inducible uptake of arginine and ornithine but not lysine. The aotO gene, which encodes a polypeptide with no significant similarity to any known proteins, is not essential for arginine and ornithine uptake. The sixth and terminal gene in the operon encodes ArgR, which has been recently shown to function in regulation of arginine metabolism. Studies with an aotJ::lacZ translational fusion showed that expression of the aot operon is strongly induced by arginine and that this effect is mediated by ArgR. S1 nuclease and primer extension experiments showed the presence of two promoters, P1 and P2. The downstream promoter, P2, is induced by arginine and appears to be subject to carbon catabolite repression. The upstream promoter, P1, is induced by glutamate. Footprinting experiments established the presence of a 44-bp ArgR binding site that overlaps the -35 region for P2, as was shown to be the case for the arginine-inducible aru promoter, and the -10 region for P1, as was shown to be the case for arginine-repressible operons in P. aeruginosa. Sequence alignment confirms the architecture and the consensus sequence of the ArgR binding sites, as was previously reported.

Cloning and characterization of the arginine-specific carbamoyl-phosphate synthetase from Bacillus stearothermophilus.

Bacillus stearothermophilus contains two carbamoyl-phosphate synthetases (CPS), one specific for pyrimidine biosynthesis and the other for arginine biosynthesis. The pyrimidine-specific CPS is repressed by exogenous pyrimidines, and its activity is inhibited by UMP and activated by 5-phospho-alpha-D-ribosyl diphosphate. The arginine-specific CPS is similarly repressed by exogenous arginine but its activity is not sensitive to these or other potential effectors. Each of the two enzymes consist of two unequal subunits, as is the case for other microbial CPS; however, the large subunit for the arginine-specific CPS is smaller than that for the pyrimidine-specific enzyme. Comparison of the derived amino acid sequence for the cloned large subunit of the arginine-specific CPS with those for subunits from pyrimidine-sensitive CPS showed significant similarity throughout the polypeptides except at the carboxy terminus, which was identified by other laboratories to contain the binding site for the pyrimidine effector. Unlike the results previously reported for CPS from an enteric mesophile, the kinetic properties of the arginine-specific CPS were not affected by growth of B. stearothermophilus at temperatures near the minimal growth temperature. Furthermore, calorimetric studies showed that the thermal stability of cloned CPS was identical regardless of the growth temperature of B. stearothermophilus between 42 degrees C and 63 degrees C. The thermal stability of cloned CPS was not affected by expression at 37 C in Bacillus subtilis or Escherichia coli. In contrast, the thermal stabilities for CPS and other proteins were higher in extracts of cells grown at higher temperatures. These results indicate that cellular factors, probably chaperonins, are necessary for thermal stability of proteins at and below the optimal temperature for this thermophile.

Cloning and characterization of argR, a gene that participates in regulation of arginine biosynthesis and catabolism in Pseudomonas aeruginosa PAO1.

Gel retardation experiments indicated the presence in Pseudomonas aeruginosa cell extracts of an arginine-inducible DNA-binding protein that interacts with the control regions for the car and argF operons, encoding carbamoylphosphate synthetase and anabolic ornithine carbamoyltransferase, respectively. Both enzymes are required for arginine biosynthesis. The use of a combination of transposon mutagenesis and arginine hydroxamate selection led to the isolation of a regulatory mutant that was impaired in the formation of the DNA-binding protein and in which the expression of an argF::lacZ fusion was not controlled by arginine. Experiments with various subclones led to the conclusion that the insertion affected the expression of an arginine regulatory gene, argR, that encodes a polypeptide with significant homology to the AraC/XylS family of regulatory proteins. Determination of the nucleotide sequence of the flanking regions showed that argR is the sixth and terminal gene of an operon for transport of arginine. The argR gene was inactivated by gene replacement, using a gentamicin cassette. Inactivation of argR abolished arginine control of the biosynthetic enzymes encoded by the car and argF operons. Furthermore, argR inactivation abolished the induction of several enzymes of the arginine succinyltransferase pathway, which is considered the major route for arginine catabolism under aerobic conditions. Consistent with this finding and unlike the parent strain, the argR::Gm derivative was unable to utilize arginine or ornithine as the sole carbon source. The combined data indicate a major role for ArgR in the control of arginine biosynthesis and aerobic catabolism.

Purification and characterization of an arginine regulatory protein, ArgR, from Pseudomonas aeruginosa and its interactions with the control regions for the car, argF, and aru operons.

Pseudomonas aeruginosa ArgR, a regulatory protein that plays a major role in the control of certain biosynthetic and catabolic arginine genes, was purified to homogeneity. ArgR was shown to be a dimer of two equal subunits, each with a molecular mass of 37,000 Da. Determination of the amino-terminal amino acid sequence showed it to be identical to that predicted from the derived sequence for the argR gene. DNase I footprinting showed that ArgR protects a region of 45 to 47 bp that overlaps the promoters for the biosynthetic car and argF operons, indicating that ArgR exerts its negative control on the expression of these operons by steric hindrance. Studies were also carried out with the aru operon, which encodes enzymes of the catabolic arginine succinyl-transferase pathway. Quantitative S1 nuclease experiments showed that expression of the first gene in this operon, aruC, is initiated from an arginine-inducible promoter. Studies with an aruC::lacZ fusion showed that this promoter is under the control of ArgR. DNase I experiments indicated that ArgR protects two 45-bp binding sites upstream of aruC; the 3' terminus for the downstream binding site overlaps the -35 region for the identified promoter. Gel retardation experiments yielded apparent dissociation constants of 2.5 x 10(-11), 4.2 x 10(-12), and 7.2 x 10(-11) M for carA, argF, and aruC operators, respectively. Premethylation interference and depurination experiments with the car and argF operators identified a common sequence, 5'-TGTCGC-3', which may be important for ArgR binding. Alignment of ArgR binding sites reveals that the ArgR binding site consists of two half-sites, in a direct repeat arrangement, with the consensus sequence TGTCGCN8AAN5.

Identification of greA encoding a transcriptional elongation factor as a member of the carA-orf-carB-greA operon in Pseudomonas aeruginosa PAO1.

A homolog of the transcriptional elongation factor, GreA, was identified in Pseudomonas aeruginosa PAO1. The deduced amino acid sequence for GreA from this organism exhibits 65.2% identity to its counterpart in Escherichia coli K-12. The nucleotide sequence of greA from P. aeruginosa overlaps by four bases the 3' terminus of carB which encodes the large subunit of carbamoylphosphate synthetase. S1 nuclease experiments showed that level of the greA transcript is elevated approximately 10-fold under conditions of pyrimidine limitation, consistent with the conclusion that transcription is initiated from the previously identified pyrimidine-sensitive promoter upstream of the carA-orf-carB-greA operon. Transcriptional fusion experiments showed the presence of an additional weak promoter within the carB sequence. A greA insertional mutant of Pseudomonas aerugionsa was constructed by gene replacement. The mutant derivative grew well in rich medium but did not grow in minimal medium supplemented by arginine and nucleosides. The greA phenotype was suppressed by secondary mutations at a relatively high rate, consistent with the notion of an important physiological role for GreA.

Participation of the purine repressor in control of the carbamoylphosphate synthetase operon in Salmonella typhimurium.

Previous work has shown that the carAB operon of Salmonella typhimurium is transcribed from tandem promoters, P1 and P2, that are negatively controlled by pyrimidines and arginine, respectively. The results reported here show that purines also negatively control expression of carAB and that this effect is absent in a purR::Tn10 derivative. Primer-extension experiments established that the purine effect is exerted at P1, thus redefining this promoter as sensitive to both purines and pyrimidines. The results of gel-retardation experiments as well as DNase I and premethylation footprintings indicate that the purine repressor interacts with a PUR box 85 bp upstream of P1. Modification of this PUR box by site-directed mutagenesis abolishes the repression by purines in a carA::lacZ fusion, confirming that this box functions in vivo in purine control of carAB expression.

Structure and regulation of the carAB operon in Pseudomonas aeruginosa and Pseudomonas stutzeri: no untranslated region exists.

The carAB operons from Pseudomonas aeruginosa PAO1 and Pseudomonas stutzeri JM300 were characterized by Southern and DNA sequence analyses. The results show that the previously reported sequence for carA (S. C. Wong and A. T. Abdelal, J. Bacteriol. 172:630-642, 1990) is derived from P. stutzeri and not P. aeruginosa, as originally reported. Therefore, the amino-terminal sequence of the purified carA product is identical to that derived from the nucleotide sequence in both organisms, P. stutzeri having four additional amino acids. The results also show that while carA and carB are contiguous in P. stutzeri, as is the case in other bacteria, they are surprisingly separated by an open reading frame (ORF) of 216 amino acids in P. aeruginosa. S1 nuclease mapping experiments with RNA extracted under a variety of growth conditions, as well as experiments using different lacZ fusions, indicate that the carA-ORF-carB operon of P. aeruginosa is transcribed from a single promoter. Moreover, these experiments demonstrate that expression of this single transcript is controlled by both arginine and pyrimidines and that variation in arginine levels specifically modulates transcriptional initiation, while pyrimidine regulation is exerted subsequent to transcriptional initiation. Modification of a rho-independent terminator-like structure, which is present upstream of carA in P. aeruginosa, removed all transcriptional sensitivity of a carA::lacZ fusion to pyrimidines. This result, when coupled with the finding that translation of an 18-amino-acid leader polypeptide (associated with this putative rho-independent terminator), is inversely proportional to pyrimidine concentration in the cell, strongly suggests that regulation of carA by pyrimidines is mediated through an attenuation-type mechanism in P. aeruginosa.

The Salmonella typhimurium uracil-sensitive mutation use is in argU and encodes a minor arginine tRNA.

The use gene of Salmonella typhimurium was previously identified by a mutation conferring sensitivity to uracil in glucose minimal medium. The use gene was cloned and identified as an allele of argU encoding a tRNA for a minor arginine codon (CGG). The uracil-sensitive phenotype was shown to result from a base substitution in the anticodon stem of this tRNA.

Complete sequence of the Salmonella typhimurium gene encoding malate dehydrogenase.

The nucleotide (nt) sequence of mdh (encoding malate dehydrogenase) in Salmonella typhimurium is presented. The relative positions of argR (encoding arginine repressor) and mdh on the chromosome of S. typhimurium were determined from analysis of the nt sequence. The homology of the 3'-terminal end with only one of two published mdh sequences in Escherichia coli identifies the correct sequence in this organism.

Characterization of the arginine repressor from Salmonella typhimurium and its interactions with the carAB operator.

Primer extension experiments showed that the argR gene, encoding the arginine repressor in Salmonella typhimurium, is transcribed from a single promoter that is negatively regulated by arginine. A repressor overproducing strain was constructed and the repressor was purified to homogeneity. Gel filtration, sedimentation and cross-linking studies established that the native repressor is a hexamer of identical 17,000 Mr subunits. Gel retardation experiments indicate that the apparent dissociation constant for repressor/carAB operator is 6 x 10(-12) M. These experiments showed that arginine is essential for binding of the repressor to the DNA and that pyrimidine nucleotides have no significant effect on this binding. These results indicate that the effect of pyrimidines on expression of the arginine sensitive "downstream" carAB promoter is not directly mediated by the arginine repressor. These experiments also suggest that a single hexamer binds to the carAB operator, which carries two previously defined "ARG box" sequences that characterize operators for arg genes. Gel retardation experiments with DNA fragments carrying the individual ARG boxes showed that both boxes are required for effective binding of the hexameric repressor to the operator, indicating that the ARG boxes comprise a single binding site for the repressor. Analysis of the potential secondary structure of the arginine repressor does not reveal any of the recognizable structural motifs common to a number of DNA-binding proteins. A combination of DNase I, premethylation interference, depurination and hydroxyl radical footprinting techniques were employed to characterize the interactions of the repressor with the carAB operator, with the results suggesting that the repressor predominantly interacts with A.T residues in this region. Comparative DNA sequence analysis of the known arginine operators of enteric bacteria further indicates that the specificity of interaction may be based more on the precise distance between two defined A.T-rich regions rather than on the specific nucleotide sequence.

Effect of growth temperature on folding of carbamoylphosphate synthetases of Salmonella typhimurium and a cold-sensitive derivative.

The properties of homogeneous preparations of carbamoylphosphate synthetase (CPSase) from wild-type Salmonella typhimurium and a cold-sensitive derivative grown at different growth temperatures were examined. For the cold-sensitive mutant, the affinity for glutamine of the form of CPSase synthesized at 20 degrees C was lower than that of the form of the enzyme synthesized at 37 degrees C, regardless of the assay temperature. Thus, the cold sensitivity of the mutant reflects an effect of temperature on the synthesis of the enzyme rather than the activity of the folded enzyme. The two forms also differed in sensitivities to polyclonal antibodies as well as denaturational enthalpies. The combined results support the hypothesis that carAB mutations conferring cold sensitivity identify amino acid residues that are critical in the folding of CPSase. Quite unexpectedly, certain kinetic properties of cloned parent CPSase were also dependent on the growth temperature, although to a much lesser extent than those of the cold-sensitive mutant. The specific activity of wild-type CPSase synthesized at 15 degrees C was 60% of that synthesized at 37 degrees C. Further, CPSase synthesized at 15 degrees C was less thermostable than the enzyme synthesized at 37 degrees C; the difference in stability (delta G) is estimated to be 4,500 cal mol-1. Thus, variation of temperature within the physiological range for growth influences the folding and consequently the properties of CPSase from wild-type S. typhimurium.

Unorthodox expression of an enzyme: evidence for an untranslated region within carA from Pseudomonas aeruginosa.

The genes encoding carbamoylphosphate synthetase from Pseudomonas aeruginosa PAO1 were cloned in Escherichia coli. Deletion and transposition analysis determined the locations of carA, encoding the small subunit, and carB, encoding the large subunit, on the chromosomal insert. The nucleotide sequence of carA and the flanking regions was determined. The derived amino acid sequence for the small subunit of carbamoylphosphate synthetase from P. aeruginosa exhibited 68% homology with its counterparts in E. coli and Salmonella typhimurium. The derived sequences in the three organisms were essentially identical in the three polypeptide segments that are conserved in glutamine amidotransferases but showed low homology at the amino- and carboxy-terminal regions. The amino-terminal amino acid sequences were determined for the large and small subunits. The first 15 amino acids of the large subunit were identical to those derived from the carB sequence. However, comparison of the derived sequence for carA with the amino-terminal amino acid sequence for the small subunit suggested that codons 5 to 8 are not translated. The DNA sequence for the region encompassing these four codons was confirmed by direct sequencing of chromosomal DNA after amplification by the polymerase chain reaction. The mRNA sequence was also deduced by in vitro synthesis of cDNA, enzymatic amplification, and sequencing, confirming that 12 nucleotides in the 5' terminal of carA are transcribed but are not translated.

Purification and characterization of the anti-Candida toxin of Pichia anomala WC 65.

Pichia anomala WC 65 secretes a toxin that is inhibitory to a variety of yeasts, including strains of the animal pathogen Candida albicans. The toxin was purified to homogeneity by ultrafiltration, ethanol precipitation, ion-exchange chromatography with a Mono Q column, and gel permeation chromatography with a Superose 12 column. The toxin had a molecular weight of 83,300 as determined by electrophoresis on sodium dodecyl sulfate-polyacrylamide gradient gels and a molecular weight of 85,290 as determined by gel permeation chromatography. The isoelectric point of the toxin was pH 5.0. The toxin was stable between pH 2.0 and 5.0. Chemical analysis of the purified toxin indicated that the toxin was a glycoprotein composed of about 86% protein and 14% carbohydrate. At high concentrations, the toxin showed a tendency to aggregate, with loss of biological activity against C. albicans, Pichia bimundalis, and Saccharomycodes ludwigii. Purified toxin expressed killing activity against C. albicans in contrast to the static activity of the crude toxin.

Anti-Candida albicans activity of Pichia anomala as determined by a growth rate reduction assay.

Killer toxin activity of Pichia anomala WC65 appeared fungicidal for P. bimundalis WC38 and fungistatic for Candida albicans RC1. Inhibitory activity against sensitive C. albicans showed a linear relationship between toxin concentrations and the inverse of the reduced growth rates. The plot of toxin concentrations against growth rates was hyperbolic, as is characteristic of saturation kinetics. Sensitivity of C. albicans to the toxin decreased with increased cell age. The measurement of growth rate reduction provided a simple and accurate method for quantitation of toxin.

Carbamoylphosphate synthetase from Pseudomonas aeruginosa. Subunit composition, kinetic analysis and regulation.

Carbamoylphosphate synthetase from Pseudomonas aeruginosa is subject to repression by pyrimidines and significant derepression by limitation of arginine or pyrimidines. Carbamoylphosphate synthetase was purified to homogeneity from a derepressed strain of P. aeruginosa. The molecular weight of the enzyme was estimated to be 168 000 by sucrose gradient ultracentrifugation. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme is composed of two non-identical subunits with molecular weights of 122 000 and 44 000. Cross-linking the enzyme prior to electrophoresis yielded an additional band corresponding to a molecular weight of 165 000, showing that the enzyme is composed of one of each subunit. The enzyme utilized either glutamine (Km 0.15 mM) or NH3 (Km 17 mM) and requires free Mg2+ for maximal activity with the optimal level between 4 mM and 10 mM. Hill plots of MgATP saturation data yielded coefficients of 1.2 and 1.4 at pH 8.0 and 8.5, respectively. A Hill equation was derived on the assumptions that MgATP binds at the same time to two distinct sub-sites as was shown to be the case for carbamoylphosphate synthetase from Escherichia coli and that these sub-sites are strictly non-interacting. The resulting theoretical Hill coefficients correspond very closely to the experimental coefficients. Carbamoylphosphate synthetase activity was subject to activation by ornithine and N-acetylornithine and feedback inhibition by UMP. Carbamoylphosphate synthetase from P. aeruginosa does not associate under all conditions examined, establishing that self-association does not play a role in regulation of enzyme activity as suggested by other workers for the enzyme from E. coli.

Carbamate kinase from Pseudomonas aeruginosa: purification, characterization, physiological role, and regulation.

Pseudomonas aeruginosa PAO1 possessed a carbamate kinase (CKase) distinct from carbamoylphosphate synthetase as well as from a constitutive acetate kinase which also catalyzes the phosphorylation of ADP by carbamoylphosphate. CKase was purified to homogeneity. Polyacrylamide gel electrophoresis of cross-linked CKase in the presence of sodium dodecyl sulfate showed that the enzyme consists of two subunits with identical molecular weights (37,000). The optimal pH of enzyme activity is 7.0. The double-reciprocal plot for carbamoylphosphate was linear at 2 mM ADP, yielding an apparent Km of 5 mM. However, at 0.25 mM ADP, the plot was concave upward, and a Hill plot of the data yielded a coefficient of 1.4. This apparent cooperativity at low ADP concentrations might serve to reduce the extent of catabolism of carbamoylphosphate under growth conditions yielding high energy charge. Experiments on the regulation of synthesis under various growth conditions showed a response to three regulatory signals: CKase was induced to high levels by anaerobiosis, induced to moderate levels by arginine, and repressed by ammonia. Thus, CKase expression is regulated in a manner that allows the enzyme to function as a provider of ammonia under aerobic conditions and of ATP under anaerobic conditions. ATP was an effective inhibitor of CKase activity; this inhibition provides the cell with an effective mechanism for avoiding a futile cycle resulting from the simultaneous operation of CKase and carbamoylphosphate synthetase when cells are grown in the presence of exogenous arginine.

Regulation of carbamylphosphate synthesis in Serratia marcescens.

Serratia marcescens HY possessed a single carbamylphosphate synthase (CPSase) which was subject to cumulative repression by arginine and a pyrimidine. CPSase did not appear to be a part of a multifunctional enzyme complex as is the case for other enzymes of pyrimidine biosynthesis in this organism. CPSase was purified to homogeneity. The molecular weight of the enzyme was estimated to be 167,000 by sucrose density gradient ultracentrifugation. The double-reciprocal plot for magnesium adenosine triphosphate was linear, yielding a Km value of 2.5 mM. The enzyme utilized either glutamine (Km, 0.1 mM) or NH3 (Km, 10.5 mM) as a nitrogen donor in the reaction. CPSase activity was subject to activation by ornithine and feedback inhibition by uridine monophosphate, as is the case for other enteric bacteria. Carbamate kinase activity, detected in crude extracts of S. marcescens, was shown to be due to a constitutive acetate kinase. The absence of carbamate kinase from S. marcescens HY is consistent with the inability of this organism to utilize arginine as a source of energy under anaerobic conditions.