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Crc (catabolite repression control) protein of Pseudomonas aeruginosa has shown to be involved in carbon regulation of several pathways. In this study, the role of Crc in catabolite repression control has been studied in Pseudomonas putida. The bkd operons of P. putida and P. aeruginosa encode the inducible multienzyme complex branched-chain keto acid dehydrogenase, which is regulated in both species by catabolite repression. We report here that this effect is mediated in both species by Crc. A 13-kb cloned DNA fragment containing the P. putida crc gene region was sequenced. Crc regulates the expression of branched-chain keto acid dehydrogenase, glucose-6-phosphate dehydrogenase, and amidase in both species but not urocanase, although the carbon sources responsible for catabolite repression in the two species differ. Transposon mutants affected in their expression of BkdR, the transcriptional activator of the bkd operon, were isolated and identified as crc and vacB (rnr) mutants. These mutants suggested that catabolite repression in pseudomonads might, in part, involve control of BkdR levels.

The transition from a planktonic (free-swimming) existence to growth attached to a surface in a biofilm occurs in response to environmental factors, including the availability of nutrients. We show that the catabolite repression control (Crc) protein, which plays a role in the regulation of carbon metabolism, is necessary for biofilm formation in Pseudomonas aeruginosa. Using phase-contrast microscopy, we found that a crc mutant only makes a dispersed monolayer of cells on a plastic surface but does not develop the dense monolayer punctuated by microcolonies typical of the wild-type strain. This is a phenotype identical to that observed in mutants defective in type IV pilus biogenesis. Consistent with this observation, crc mutants are defective in type IV pilus-mediated twitching motility. We show that this defect in type IV pilus function is due (at least in part) to a decrease in pilA (pilin) transcription. We propose that nutritional cues are integrated by Crc as part of a signal transduction pathway that regulates biofilm development.

A 729-bp open reading frame (gltR) was identified in Pseudomonas aeruginosa PAO1 that encodes a product homologous to the two-component response regulator family of proteins. Disruption of gltR caused loss of glucose transport activity. Restoration of gltR resulted in wild-type levels of glucose transport. These findings indicate that gltR is required for expression of the glucose transport system in P. aeruginosa.

The gene (crc) responsible for catabolite repression control in Pseudomonas aeruginosa has been cloned and sequenced. Flanking the crc gene are genes encoding orotate phosphoribosyl transferase (pyrE) and RNase PH (rph). New crc mutants were constructed by disruption of the wild-type crc gene. The crc gene encodes an open reading frame of 259 amino acids with homology to the apurinic/apyrimidinic endonuclease family of DNA repair enzymes. However, crc mutants do not have a DNA repair phenotype, nor can the crc gene complement Escherichia coli DNA repair-deficient strains. The crc gene product was overexpressed in both P. aeruginosa and in E. coli, and the Crc protein was purified from both. The purified Crc proteins show neither apurinic/apyrimidinic endonuclease nor exonuclease activity. Antibody to the purified Crc protein reacted with proteins of similar size in crude extracts from Pseudomonas putida and Pseudomonas fluorescens, suggesting a common mechanism of catabolite repression in these three species.

The hexC locus of Pseudomonas aeruginosa PAO1 was localized to a 247-bp segment of chromosomal DNA on the multicopy broad-host-range vector pRO1614. The presence of this plasmid (pPZ196) in strain PAO1 produced the so-called "hexC effect," a two- to ninefold increase in the activities of four carbohydrate catabolism enzymes, glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase. The extent of the hexC effect was restricted, since three independently regulated metabolic enzymes were not affected by the presence of the hexC plasmid. Furthermore, the hexC-containing plasmid did not suppress catabolite repression control. Nucleotide sequence analysis of the segment of DNA encompassing hexC revealed a 128-bp region rich in adenosine-plus-thymine (AT) content separating two divergent open reading frames (ORFs). Transcriptional start sites for these two genes were mapped to the intergenic region, demonstrating that this sequence contained overlapping divergent promoters. The intergenic region contained potential regulatory sequences such as dyad symmetry motifs, polydeoxyadenosine tracts, and a sequence matching the integration host factor recognition site in Escherichia coli. One of the ORFs encoded a 610-amino-acid protein with 55 to 60% identity to 6-phosphogluconate dehydratase from E. coli and Zymomonas mobilis. The second ORF coded for a protein of 335 amino acids that displayed 45 to 60% identity to the NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAP) family of enzymes. The NAD-dependent GAP gene on the P. aeruginosa chromosome was previously unmapped. GAP was found to exhibit the hexC-dependent increase in its basal activity, establishing it as a fifth catabolic enzyme in the multioperonic hex regulon.

Mutants which are defective in catabolite repression control (CRC) of multiple independently regulated catabolic pathways have been previously described. The mutations were mapped at 11 min on the Pseudomonas aeruginosa chromosome and designated crc. This report describes the cloning of a gene which restores normal CRC to these Crc- mutants in trans. The gene expressing this CRC activity was subcloned on a 2-kb piece of DNA. When this 2-kb fragment was placed in a plasmid behind a phage T7 promoter and transcribed by T7 RNA polymerase, a soluble protein with a molecular weight (MW) of about 30,000 was produced in Escherichia coli. A soluble protein of identical size was overproduced in a Crc- mutant when it contained the 2-kb fragment on a multicopy plasmid. This protein could not be detected in the mutant containing the vector without the 2-kb insert or with no plasmid. When a 0.3-kb AccI fragment was removed from the crc gene and replaced with a kanamycin resistance cassette, the interrupted crc gene no longer restored CRC to the mutant, and the mutant containing the interrupted gene no longer overproduced the 30,000-MW protein. Pools of intracellular cyclic AMP and the activities of adenylate cyclase and phosphodiesterase were measured in mutant and wild-type strains with and without a plasmid containing the crc gene. No consistent differences between any strains were found in any case. These results provide original evidence for a 30,000-MW protein encoded by crc+ that is required for wild-type CRC in P. aeruginosa and confirms earlier reports that the mode of CRC is cyclic AMP independent in this bacterium.

Independently controlled, inducible, catabolic genes in Pseudomonas aeruginosa are subject to strong catabolite repression control by intermediates of the tricarboxylic acid cycle. Mutants which exhibited a pleiotropic loss of catabolite repression control of multiple pathways were isolated. The mutations mapped in the 11-min region of the P. aeruginosa chromosome near argB and pyrE and were designated crc. Crc- mutants no longer showed repression of mannitol and glucose transport, glucose-6-phosphate dehydrogenase, glucokinase, Entner-Doudoroff dehydratase and aldolase, and amidase when grown in the presence of succinate plus an inducer. These activities were not expressed constitutively in Crc- mutants but exhibited wild-type inducible expression.

Five of the genes required for phosphorylative catabolism of glucose in Pseudomonas aeruginosa were ordered on two different chromosomal fragments. Analysis of a previously isolated 6.0-kb EcoRI fragment containing three structural genes showed that the genes were present on a 4.6-kb fragment in the order glucose-binding protein (gltB)-glucokinase (glk)-6-phosphogluconate dehydratase (edd). Two genes, glucose-6-phosphate dehydrogenase (zwf) and 2-keto-3-deoxy-6-phosphogluconate aldolase (eda), shown by transductional analysis to be linked to gltB and edd, were cloned on a separate 11-kb BamHI chromosomal DNA fragment and then subcloned and ordered on a 7-kb fragment. The 6.0-kb EcoRI fragment had been shown to complement a regulatory mutation, hexR, which caused noninducibility of four glucose catabolic enzymes. In this study, hexR was mapped coincident with edd. A second regulatory function, hexC, was cloned within a 0.6-kb fragment contiguous to the edd gene but containing none of the structural genes. The phenotypic effect of the hexC locus, when present on a multicopy plasmid, was elevated expression of glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase activities in the absence of inducer.

Glucose may be converted to 6-phosphogluconate by alternate pathways in Pseudomonas aeruginosa. Glucose is phosphorylated to glucose-6-phosphate, which is oxidized to 6-phosphogluconate during anaerobic growth when nitrate is used as respiratory electron acceptor. Mutant cells lacking glucose-6-phosphate dehydrogenase are unable to catabolize glucose under these conditions. The mutant cells utilize glucose as effectively as do wild-type cells in the presence of oxygen; under these conditions, glucose is utilized via direct oxidation to gluconate, which is converted to 6-phosphogluconate. The membrane-associated glucose dehydrogenase activity was not formed during anaerobic growth with glucose. Gluconate, the product of the enzyme, appeared to be the inducer of the gluconate transport system, gluconokinase, and membrane-associated gluconate dehydrogenase. 6-Phosphogluconate is probably the physiological inducer of glucokinase, glucose-6-phosphate dehydrogenase, and the dehydratase and aldolase of the Entner-Doudoroff pathway. Nitrate-linked respiration is required for the anaerobic uptake of glucose and gluconate by independently regulated transport systems in cells grown under denitrifying conditions.

A 6.0-kilobase EcoRI fragment of the Pseudomonas aeruginosa PAO chromosome containing a cluster of genes specifying carbohydrate catabolism was cloned into the multicopy plasmid pRO1769. The vector contains a unique EcoRI site for cloning within a streptomycin resistance determinant and a selectable gene encoding gentamicin resistance. Mutants of P. aeruginosa PAO transformed with the chimeric plasmid pRO1816 regained the ability to grow on glucose, and the following deficiencies in enzyme or transport activities corresponding to the specific mutations were complemented: glcT1, glucose transport and periplasmic glucose-binding protein; glcK1, glucokinase; and edd-1, 6-phosphogluconate dehydratase. Two other carbohydrate catabolic markers that are cotransducible with glcT1 and edd-1 were not complemented by plasmid pRO1816: zwf-1, glucose-6-phosphate dehydrogenase; and eda-9001, 2-keto-3-deoxy-6-phosphogluconate aldolase. However, all five of these normally inducible activities were expressed at markedly elevated basal levels when transformed cells of prototrophic strain PAO1 were grown without carbohydrate inducer. Vector plasmid pRO1769 had no effect on the expression of these activities in transformed mutant or wild-type cells. Thus, the chromosomal insert in pRO1816 contains the edd and glcK structural genes, at least one gene (glcT) that is essential for expression of the glucose active transport system, and other loci that regulate the expression of the five clustered carbohydrate catabolic genes. The insert in pRO1816 also complemented the edd-1 mutation in a glucose-negative Pseudomonas putida mutant but not the eda-1 defect in another mutant. Moreover, pRO1816 caused the expression of high specific activities of glucokinase, an enzyme that is naturally lacking in these strains of Pseudomonas putida.

Mutant strains of Pseudomonas aeruginosa PAO were isolated on the basis of their inability to utilize mannitol as sole carbon source for growth. Four linkage groups (I through IV) among these mutant strains were resolved by two-factor crosses using the general transducing phage F116, and the strains appeared to contain point mutations as evidenced by ability to give rise to spontaneous revertants with wild phenotype on mannitol minimal agar. Group I strains were affected only in ability to grow on mannitol; all were deficient in inducible mannitol dehydrogenase activity, and all but one were deficient in inducible mannitol transport activity. Fructokinase was induced in group I strains and in wild-type bacteria during growth in the presence of mannitol but not fructose, indicating the presence of a pathway specific for endogenously generated fructose. Cells grown on fructose contained phosphoenolpyruvate:fructose-1-phosphotransferase activity, and mannitol-grown cells contained a lower level of this activity. Group II mutants were deficient in constitutive phosphoglucoisomerase, failed to grow on mannitol, grew very slowly on glycerol and fructose, but grew normally on glucose and gluconate. Group III strains were deficient in both nicotinamide adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-linked glucose-6-phosphate dehydrogenase activities that reside in a single enzyme species. 6-Phosphogluconate appeared to be the inductive effector for this enzyme, which was not required for aerobic growth on glucose or gluconate. A single mannitol-negative mutant in group IV also failed to grow on glycerol and glucose, but no biochemical lesion was identified.

Pseudomonas aeruginosa transports and phosphorylates fructose via a phosphoenolpyruvate-dependent fructose phosphotransferase system (PTS). Mutant strains deficient in both PTS activity and glucose-6-phosphate dehydrogenase activity were isolated and were used to select mannitol-utilizing revertant strains singly deficient in PTS activity. These mutants were unable to utilize fructose as a carbon source and failed to accumulate exogenously provided [14C]fructose, and crude cell extracts lacked phosphoenolpyruvate-dependent fructose PTS activity. Thus, the PTS was essential for the uptake and utilization of exogenously provided fructose by P. aeruginosa. Mutations at a locus designated pts, which resulted in a loss of PTS activity, exhibited 57% linkage to argF at 55 min on the chromosome in plasmid R68.45-mediated conjugational crosses. The pts mutations in four independently isolated mutant strains exhibited from 11 to 20% linkage to argF, and one of these mutations exhibited 3% linkage to lys-9015 in phage F116L-mediated transductional crosses.

Cell extracts of Pseudomonas aeruginosa strain PAO were found to contain pyruvate carboxylase activity. Specific activities were minimal when cells were grown on Casamino Acids, acetate, or succinate, but were three- to fourfold higher when cells were grown in glucose, gluconate, glycerol, lactate, or pyruvate minimal media. The reaction in crude cell extracts and in partially purified preparations was dependent on pyruvate, adenosine 5'-triphosphate, and Mg(2+), but was not affected by either the presence or absence of acetyl coenzyme A. Activity was nearly totally inhibited by avidin and this inhibition was substantially blocked by free biotin in incubation mixtures. Cell extracts were shown to fix (14)CO(2) in a reaction that had these same characteristics. Eight pleiotropic, carbohydrate-negative mutant strains of the organism were isolated after nitrosoguanidine mutagenesis. Each mutant strain grew normally in acetate, succinate, and citrate minimal media but failed to utilize glucose, gluconate, 2-ketogluconate, mannitol, glycerol, lactate, and pyruvate as sole sources of carbon and energy. These strains were found by quantitative transductional analysis with phage F116 to form a single linkage group. Cell extracts of each mutant strain were either lacking or severely deficient in pyruvate carboxylase activity. Spontaneous revertants of five of the eight strains were isolated and found to recover simultaneously both pyruvate carboxylase activity and the ability to utilize each of the C(6) and C(3) compounds. A second linkage group of similar mutant strains that grew on the C(3) compounds was found to contain normal levels of pyruvate carboxylase activity, but each strain was deficient in an enzyme of the Entner-Doudoroff pathway.

The initial reactions involved in the catabolism of fructose in Pseudomonas aeruginosa include the participation of a phosphoenolpyruvate:fructose 1-phosphotransferase system (F-PTS). Fractionation of crude extracts of fructose-grown cells revealed that both membrane-associated and soluble components were essential for F-PTS activity. Further resolution of the soluble fraction by both size exclusion and ion-exchange chromatography revealed the presence of only one component, functionally analogous to enzyme I. Enzyme I exhibited a relative molecular weight of 72,000, catalyzed the pyruvate-stimulated hydrolysis of phosphoenolpyruvate to pyruvate, and mediated the phosphorylation of fructose when combined with a source of enzyme II (washed membranes). No evidence for the requirement of a phosphate carrier protein, such as HPr, could be demonstrated. Thus, the F-PTS requires a minimum of two components, a soluble enzyme I and a membrane-associated enzyme II complex, and both were shown to be inducible. Reconstituted F-PTS activity was specific for phosphoenolpyruvate as a phosphate donor (Km, approximately -0.6 mM) and fructose as the sugar substrate (Km, approximately 18 microM). Components of the Pseudomonas F-PTS did not restore activity to extracts of deletion mutants of Salmonella typhimurium deficient in individual proteins of the PTS or to fractionated membrane and soluble components of the F-PTS of Escherichia coli. Similarly, membrane and soluble components of E. coli and S. typhimurium would not cross-complement the F-PTS components from P. aeruginosa.

Citrate synthase [citrate (si)-synthase] (EC 4.1.3.7) was partially purified from extracts of highly purified typhus rickettsiae (Rickettsia prowazekii). Molecular exclusion and affinity column chromatography were used to prepare 200-fold-purified citrate synthase that contained no detectable malate dehydrogenase (EC 1.1.1.37) activity. Rickettsial malate dehydrogenase also was partially purified (200-fold) via this purification procedure. Catalytically active citrate synthase exhibited a relative molecular weight of approximately 62,000 after elution from a calibrated Sephacryl S-200 column. Acetyl coenzyme A saturation of partially purified enzyme was sensitive to strong competitive inhibition with adenylates (ATP greater than ADP much greater than AMP). [beta,gamma-methylene]ATP, dATP, and dADP also caused strong inhibition, but guanosine and cytosine nucleotides were significantly less inhibitory. Adenylates had no effect on oxalacetate saturation kinetics when acetyl coenzyme A was present in high concentration (greater than or equal to 50 microM). Neither NADH nor alpha-ketoglutarate affected the saturation kinetics of rickettsial citrate synthase. Thus, citrate synthase from R. prowazekii exhibits greater similarity to the eucaryotic and gram-positive procaryotic enzymes than to citrate synthase from free-living gram-negative bacteria. These results represent the first characterization of a highly purified key regulatory enzyme from these obligate intracellular parasitic bacteria.

Mutations in carbohydrate-negative mutants of Pseudomonas aeruginosa PAO1 individually deficient in glucose 6-phosphate dehydrogenase (zwf), 6-phosphogluconate dehydratase (edd), or pyruvate carboxylase (pyc) were mapped on the chromosome by plasmid R68.45-mediated conjugation and by bacteriophage F116L-mediated transduction. Loci for all three genes were located in the 45- to 55-min region of the chromosome; both zwf-1 and edd-1 were linked by transduction to nalA, whereas pyc-2 was linked by conjugation to argF10. The zwf-1 mutation exhibited cotransduction frequencies of greater than 95% with both edd-1 and the hex-9001 marker, a mutation reported to prevent growth on hexoses. The latter mutation was shown to cause a specific deficiency in 2-keto-3-deoxy-6-phosphogluconate aldolase activity and was redesignated eda-9001. These results demonstrate tight clustering of the gene loci for glucose 6-phosphate dehydrogenase and for both enzymes unique to the Entner-Doudoroff pathway in P. aeruginosa. Our evidence suggests supraoperonic clustering of these and other inducible carbohydrate catabolic genes in the 45- to 55-min region of the chromosome.

Mutants of Pseudomonas aeruginosa, strain PAO, have been isolated that are unable to grow on mannitol, glucose, gluconate, or 2-ketogluconate, cut that exhibit wild-type growth on pyruvate, lactate, citrate, succinate, or acetate. Although some of these mutants were also unable to grow on glycerol, the mutations formed a single linkage group by quantitative transductional analysis with phage F116 on glucose minimal agar medium. Cell extracts of all mutant strains were either lacking or severely deficient in 6-phosphogluconate dehydratase activity. Glu+ transductants derived from mutant strains that retained the wild-type ability for growth at the expense of glycerol also regained the ability to grow on all C-6 compounds. Although a role for the pentose phosphate pathway in the catabolism of C6 substrates was not found, a functional Entner-Doudoroff pathway appears to be essential for the catabolism of mannitol, glucose, gluconate, and 2-ketogluconate.

A modified Gilman assay was used to determine the concentrations of cyclic adenosine 3',5'-monophosphate (cAMP) in rapidly filtered cells and in the culture filtrates of Pseudomonas aeruginosa, Escherichia coli K-12, and Bacteroides fragilis. In P. aeruginosa cultures, levels of cAMP in the filtrate increased with the culture absorbance (3.5 to 19.8 X 10(-9) M) but did not vary significantly with the carbon source used to support growth. Intracellular concentrations (0.8 to 3.2 X 10(-5) M) were substantially higher and did not vary appreciably during growth or with carbon source. Sodium cAMP (5 mM) failed to reverse the catabolite repression of inducible glucose-6-phosphate dehydrogenase (EC 1.1.1.49) synthesis caused by the addition of 10 mM succinate. Exogenous cAMP also had no discernible effect on the catabolite repression control of inducible mannitol dehydrogenase (EC 1.1.1.67). P. aeruginosa was found to contain both soluble cAMP phosphodiesterase (EC 3.1.4.17) and membrane-associated adenylate cyclase (EC 4.6.1.1) activity, and these were compared to the activities detected in crude extracts of E. coli. B. fragilis crude cell extracts contain neither of these enzyme activities, and little or no cAMP was detected in cells or culture filtrates of this anaerobic bacterium.

Spirillum itersonii ATCC 12639 utilized d-fructose but neither d-glucose nor d-gluconate as a sole source of carbon and energy. The substrate saturation kinetics for d-fructose and d-glucose uptake by whole cells indicated the presence of a carrier-mediated transport system for d-fructose but not for d-glucose. The d-fructose uptake activity was induced (10- to 12-fold increase) during growth on d-fructose-Casamino Acids (CA) or d-glucose-CA medium, but not CA alone. d-Fructose uptake activity was stimulated by Na(+) or Li(+), but was inhibited by KCN, NaN(3), 2,4-dinitrophenol, and p-chloromercuribenzoate. High specific activities of glucokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydratase, and 2-keto-3-deoxy-6-phosphogluconate aldolase were detected in extracts of cells cultured on d-fructose-CA medium. These enzymatic activities were undetectable in extracts of cells grown in CA or succinate-CA medium. No decrease in the maximally induced specific activities of these enzymes occurred after the addition of succinate to cells during exponential growth on d-fructose-CA. Fructose 1,6-diphosphate aldolase and glucose-6-phosphate isomerase specific activities were approximately the same irrespective of cultural conditions. These results indicated that d-glucose was not utilized by cells of S. itersonii because this bacterium was impermeable to this hexose.

Eighteen strains of alginate-producing mucoid Pseudomonas aeruginosa were evaluated with respect to plasmid content and the ability to maintain well-characterized R plasmids. The spontaneous loss of alginate production in these strains varied from 0.01 to 0.7% and was not significantly increased by plasmid curing regimens. Examination of cleared lysates of these strains and their isogenic nonmucoid derivatives by agarose gel electrophoresis failed to reveal plasmid DNA. R-plasmid (P-incompatibility-group) transfer to mucoid P. aeruginosa was unaffected by the presence of the alginate capsule. Maintenance and expression of such plasmids in the mucoid strains were confirmed by agarose gel electrophoresis and by verification of plasmid-linked drug resistance and pilus-specific bacteriophage sensitivity. These studies demonstrate that alginate production does not appear to be plasmid linked and that mucoid P. aeruginosa are capable of receiving and donating certain drug resistance plasmids. Since some of the plasmids used here have been shown to mobilize chromosomal DNA, strains constructed in this study should afford the means for exploring the genetic basis of the mucoid phenotype.

Glucose uptake by whole-cell suspensions of the obligate anaerobe Bacteroides thetaiotaomicron was two- to fourfold higher under aerobic conditions than during incubation under atmospheres of N(2) or H(2) gas. The O(2)-stimulated uptake activity was lost rapidly (>70% in 5 h) when cell suspensions were incubated aerobically, but this loss was prevented by the addition of crude catalase. Catalase had no apparent effect on cell viability during these incubations. Glucose uptake activity was strongly inhibited by a 10-fold excess of mannose or galactose but not by methyl-alpha-d-glucoside, fructose, or lactose. Both glucose and mannose were rapidly incorporated into polyglucose after uptake. The O(2)-stimulated glucose uptake was not inhibited by cyanide, azide, 2,4-dinitrophenol, or 2-N-heptyl-4-hydroxyquinoline-N-oxide. However, p-chloromercuribenzoate, menadione, and sodium fluoride inhibited uptake by 88, 67, and 55%, respectively. All attempts to detect phosphoenolpyruvate-phosphotransferase activity for glucose, methyl-alpha-d-glucoside, and 2-deoxyglucose were negative. The bacteria contained hexokinase activity and a complete glycolytic Embden-Meyerhof pathway.

Mutations causing deficiencies in the inducible, membrane-associated sn-glycerol-3-phosphate dehydrogenase (glpD) and in inducible glucose transport (glcT) were mapped on the Pseudomonas aeruginosa PAO1 chromosome by using the generalized transducing phages F116L and G101. These mutations, in separate catabolic regulatory units, were cotransducible with a previously described cluster of carbohydrate catabolic gene loci (zwf-1 eda-9001 edd-1) that maps at ca. 50 to 53 min on the chromosome. Mutant strain PFB362 (glcT1) did not transport glucose and did not produce a functional, periplasmic, glucose-binding protein that is required for glucose transport. This mutation was cotransducible with zwf-1 (70%), nalA (29%), and phe-2 (19%) but not with glpD1 or leu-10. The glpD1 mutation in strain PRP408 was cotransducible with zwf-1 (5%), eda-9001 (4%), and edd-1 (1%) and also with ami-151 (17%) and phe-2 (33%). These results expand the number of known carbohydrate catabolism genes that are clustered in the 50- to 55-min region of the PAO1 chromosome and allow us to propose the following relative gene order: ami-151 glpD1 phe-2 nalA zwf-1 eda-9001 edd-1 glcT1 leu-10. Three independently obtained nal determinants for high-level resistance to nalidixic acid, which were employed in these studies, exhibited similar cotransduction frequencies with several flanking marker mutations.

Cell extracts of Bacillus licheniformis were found to contain nicotinamide adenine dinucleotide (NAD)-dependent l-alanine dehydrogenase (ADH) (l-alanine: NAD oxidoreductase, EC 1.4.1.1). High specific activities (3.5 to 6.0 IU/mg of protein) were found in extracts of cells throughout growth cycles only when l-alanine served as the primary source of carbon or carbon and nitrogen. Specific activities were minimal (0.02 to 0.04 IU/mg of protein) during growth on glucose, but increased at least sevenfold during the first 5 h of postlogarithmic-phase metabolism. Addition of 10 mM glucose to cultures during logarithmic-phase growth on l-alanine resulted in a rapid decrease in enzyme activity. Addition of 20 mM l-alanine to cells near the completion of log-phase growth on glucose resulted in a 20-fold increase in ADH specific activity during less than one cell generation. Extracts of postlogarithmic-phase cells cultured on glucose, malate, l-glutamate, or Casamino Acids contained intermediate levels of ADH activity. The enzyme was partially purified from crude extracts of B. licheniformis, and apparent kinetic constants were estimated. A role for ADH in the catabolism of l-alanine to pyruvate during vegetative growth on l-alanine and during sporulation of cells cultured on glucose is proposed on the basis of these experimental results.

Cell-free extracts of Bacillus licheniformis and B. cereus were found to contain high specific activities of nicotinamide adenine dinucleotide phosphate (NADP)-dependent-l-glutamate dehydrogenase [EC 1.4.1.4; l-glutamate: NADP oxidoreductase (deaminating)]. Maximum specific activities were found in extracts of cells during the late exponential phase of growth when ammonium ion served as the sole source of nitrogen. Extremely low specific activities were detected throughout the growth cycle when l-glutamate or Casamino Acids served as the source of carbon and nitrogen. The enzyme was purified 55-fold from crude extracts of B. licheniformis, and apparent kinetic constants were determined. Sigmoidal saturation kinetics were not observed, and various adenylates had no effect on the enzyme. Repression of enzyme synthesis during growth on l-glutamate or Casamino Acids was partially overcome by additions of glucose or pyruvate, and this apparent derepression was totally abolished by inhibitors of ribonucleic acid and protein synthesis. Similarly, additions of l-glutamate or Casamino Acids to cells growing on glucose-ammonium ion resulted in strong repression of enzyme synthesis. It is suggested that the enzyme serves an anabolic role in metabolism. Nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase activity was not detected in five species of Bacillus, irrespective of nutritional conditions or of the physiological age of cells.