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The C(4)-dicarboxylates (C4-DC) l-aspartate and l-malate have been identified as playing an important role in the colonization of mammalian intestine by enteric bacteria, such as Escherichia coli and Salmonella enterica serovar Typhimurium, and succinate as a signaling molecule for host-enteric bacterium interaction. Thus, endogenous and exogenous fumarate respiration and related functions are required for efficient initial growth of the bacteria. l-Aspartate represents a major substrate for fumarate respiration in the intestine and a high-quality substrate for nitrogen assimilation. During nitrogen assimilation, DcuA catalyzes an l-aspartate/fumarate antiport and serves as a nitrogen shuttle for the net uptake of ammonium only, whereas DcuB acts as a redox shuttle that catalyzes the l-malate/succinate antiport during fumarate respiration. The C4-DC two-component system DcuS-DcuR is active in the intestine and responds to intestinal C4-DC levels. Moreover, in macrophages and in mice, succinate is a signal that promotes virulence and survival of S. Typhimurium and pathogenic E. coli. On the other hand, intestinal succinate is an important signaling molecule for the host and activates response and protective programs. Therefore, C4-DCs play a major role in supporting colonization of enteric bacteria and as signaling molecules for the adaptation of host physiology.

Insertion sequencing (INSeq) analysis of Rhizobium leguminosarum bv. viciae 3841 (Rlv3841) grown on glucose or succinate at both 21% and 1% O 2 was used to understand how O 2 concentration alters metabolism. Two transcriptional regulators were required for growth on glucose (pRL120207 [ eryD ] and RL0547 [ phoB ]), five were required on succinate (pRL100388, RL1641, RL1642, RL3427, and RL4524 [ ecfL ]), and three were required on 1% O 2 (pRL110072, RL0545 [ phoU ], and RL4042). A novel toxin-antitoxin system was identified that could be important for generation of new plasmidless rhizobial strains. Rlv3841 appears to use the methylglyoxal pathway alongside the Entner-Doudoroff (ED) pathway and tricarboxylic acid (TCA) cycle for optimal growth on glucose. Surprisingly, the ED pathway was required for growth on succinate, suggesting that sugars made by gluconeogenesis must undergo recycling. Altered amino acid metabolism was specifically needed for growth on glucose, including RL2082 ( gatB ) and pRL120419 ( opaA , encoding omega-amino acid:pyruvate transaminase). Growth on succinate specifically required enzymes of nucleobase synthesis, including ribose-phosphate pyrophosphokinase (RL3468 [ prs ]) and a cytosine deaminase (pRL90208 [ codA ]). Succinate growth was particularly dependent on cell surface factors, including the PrsD-PrsE type I secretion system and UDP-galactose production. Only RL2393 ( glnB , encoding nitrogen regulatory protein PII) was specifically essential for growth on succinate at 1% O 2 , conditions similar to those experienced by N 2 -fixing bacteroids. Glutamate synthesis is constitutively activated in glnB mutants, suggesting that consumption of 2-ketoglutarate may increase flux through the TCA cycle, leading to excess reductant that cannot be reoxidized at 1% O 2 and cell death. IMPORTANCE Rhizobium leguminosarum , a soil bacterium that forms N 2 -fixing symbioses with several agriculturally important leguminous plants (including pea, vetch, and lentil), has been widely utilized as a model to study Rhizobium -legume symbioses. Insertion sequencing (INSeq) has been used to identify factors needed for its growth on different carbon sources and O 2 levels. Identification of these factors is fundamental to a better understanding of the cell physiology and core metabolism of this bacterium, which adapts to a variety of different carbon sources and O 2 tensions during growth in soil and N 2 fixation in symbiosis with legumes.

Previous studies have demonstrated that various tricarboxylic acid (TCA) cycle genes, particularly the succinate dehydrogenase genes ( sdhCAB ), are upregulated in Staphylococcus aureus biofilms. To better study the role of this enzyme complex, an sdhCAB deletion mutant (Δ sdh ) was constructed. Compared to the wild type (wt) the mutant was impaired in planktonic growth under aerobic conditions, excreted acetic acid could not be reused and accumulated continuously, succinate was excreted and found in the culture supernatant, and metabolome analysis with cells grown in chemically defined medium revealed reduced uptake/metabolism of some amino acids from the growth medium. Moreover, the mutant was able to counteract the steadily decreasing extracellular pH by increased urease activity. The addition of fumarate to the growth medium restored the wt phenotype. The mutant showed a small-colony variant (SCV)-like phenotype, a slight increase in resistance to various aminoglycoside antibiotics, and decreased pigmentation. The decreased growth under aerobic conditions is due to the interruption of the TCA cycle (indicated by the accumulation of succinate and acetic acid) with the consequence that many fewer reduction equivalents (NADH and FADH 2 ) can fuel the respiratory chain. The results indicate that the TCA cycle is required for acetate and amino acid catabolism; its upregulation under biofilm conditions is advantageous under such nutrient- and oxygen-limited conditions.

Pseudomonas putida DOT-T1E was used as a model to develop a “phenomics” platform to investigate the ability of P. putida to grow using different carbon, nitrogen, and sulfur sources and in the presence of stress molecules. Results for growth of wild-type DOT-T1E on 90 different carbon sources revealed the existence of a number of previously uncharted catabolic pathways for compounds such as salicylate, quinate, phenylethanol, gallate, and hexanoate, among others. Subsequent screening on the subset of compounds on which wild-type DOT-TIE could grow with four knockout strains in the global regulatory genes Δ crc , Δ crp , Δ cyoB , and Δ ptsN allowed analysis of the global response to nutrient supply and stress. The data revealed that most global regulator mutants could grow in a wide variety of substrates, indicating that metabolic fluxes are physiologically balanced. It was found that the Crc mutant did not differ much from the wild-type regarding the use of carbon sources. However, certain pathways are under the preferential control of one global regulator, i.e., metabolism of succinate and d -fructose is influenced by CyoB, and l - arginine is influenced by PtsN. Other pathways can be influenced by more than one global regulator; i.e., l -valine catabolism can be influenced by CyoB and Crp (cyclic AMP receptor protein) while phenylethylamine is affected by Crp, CyoB, and PtsN. These results emphasize the cross talk required in order to ensure proper growth and survival. With respect to N sources, DOT-T1E can use a wide variety of inorganic and organic nitrogen sources. As with the carbon sources, more than one global regulator affected growth with some nitrogen sources; for instance, growth with nucleotides, dipeptides, d -amino acids, and ethanolamine is influenced by Crp, CyoB, and PtsN. A surprising finding was that the Crp mutant was unable to flourish on ammonium. Results for assayed sulfur sources revealed that CyoB controls multiple points in methionine/cysteine catabolism while PtsN and Crc are needed for N -acetyl- l -cysteamine utilization. Growth of global regulator mutants was also influenced by stressors of different types (antibiotics, oxidative agents, and metals). Overall and in combination with results for growth in the presence of various stressors, these phenomics assays provide multifaceted insights into the complex decision-making process involved in nutrient supply, optimization, and survival.

In bacteria, the uptake of dicarboxylates, such as the tricarboxylic acid (TCA) cycle intermediates succinate, fumarate, and l-malate, is mediated by transporters of different protein families. Whereas Dcu-type transporters facilitate dicarboxylate uptake under anaerobic conditions, the most common aerobic dicarboxylate transporters are members of the dicarboxylate amino acid-cation symporter (DAACS), divalent anion sodium symporter (DASS), tripartite ATP-independent periplasmic (TRAP), and CitMHS transporter families. DAACS transporters are responsible for C4-dicarboxylate uptake under aerobic conditions in various bacteria, e.g., DctA from Escherichia coli, Bacillus subtilis, or Rhizobium leguminosarum, and are involved in different physiological functions (2, 4, 27, 41). The first described member of the TRAP family is the C4-dicarboxylate transporter DctPQM from Rhodobacter capsulatus, which facilitates substrate uptake by the use of an extracytoplasmic solute receptor (8). An example of the DASS family, members of which occur in bacteria, as well in eukaryotes, is the well-characterized transporter SdcS from Staphylococcus aureus (13). Members of the CitHMS family import citrate in symport with the cation Mg2+ or Ca2+. Whereas E. coli possesses one DctA and four different Dcu carriers, no Dcu transporter-encoding genes were found in Corynebacterium glutamicum (16, 19), which is used for the industrial production of amino acids, such as glutamate (33) or l-lysine (39), and is capable of succinate and l-lactate production under oxygen deprivation conditions. A dctA gene was annotated (19); however, C. glutamicum is not able to utilize succinate, malate, or fumarate as a sole carbon source. The uptake systems CitH and TctCBA have been characterized recently as citrate uptake systems (3, 26). Interestingly, we and others have shown that C. glutamicum possesses a DASS family transporter (DccT) for uptake of the C4-dicarboxylates succinate, fumarate, and l-malate (36, 40). Spontaneous mutants showing fast growth in succinate or fumarate minimal medium were isolated and shown to possess promoter-up mutations in the dccT gene (40). In l-malate minimal medium, these spontaneous mutants showed relatively slow growth, and the affinity of DccT for succinate and fumarate was found to be 5- and 12-fold higher than for l-malate, respectively (40). These findings prompted us to search for other uptake systems for l-malate in C. glutamicum. Here, we describe the identification and characterization of the DAACS family protein DctA from C. glutamicum as a proton motive force-driven uptake system for C4-dicarboxylate intermediates of the TCA cycle. Additionally, we compare both uptake systems, DccT and DctA, from C. glutamicum.

Campylobacter jejuni encodes all the enzymes necessary for a complete oxidative tricarboxylic acid (TCA) cycle. Because of its inability to utilize glucose, C. jejuni relies exclusively on amino acids as the source of reduced carbon, and they are incorporated into central carbon metabolism. The oxidation of succinate to fumarate is a key step in the oxidative TCA cycle. C. jejuni encodes enzymes annotated as a fumarate reductase (Cj0408 to Cj0410) and a succinate dehydrogenase (Cj0437 to Cj0439). Null alleles in the genes encoding each enzyme were constructed. Both enzymes contributed to the total fumarate reductase activity in vitro. The frdA :: cat + strain was completely deficient in succinate dehydrogenase activity in vitro and was unable to perform whole-cell succinate-dependent respiration. The sdhA :: cat + strain exhibited wild-type levels of succinate dehydrogenase activity both in vivo and in vitro. These data indicate that Frd is the only succinate dehydrogenase in C. jejuni and that the protein annotated as a succinate dehydrogenase has been misannotated. The frdA :: cat + strain was also unable to grow with the characteristic wild-type biphasic growth pattern and exhibited only the first growth phase, which is marked by the consumption of aspartate, serine, and associated organic acids. Substrates consumed in the second growth phase (glutamate, proline, and associated organic acids) were not catabolized by the the frdA :: cat + strain, indicating that the oxidation of succinate is a crucial step in metabolism of these substrates. Chicken colonization trials confirmed the in vivo importance of succinate oxidation, as the frdA :: cat + strain colonized chickens at significantly lower levels than the wild type, while the sdhA :: cat + strain colonized chickens at wild-type levels.

The rhizobial DctA permease is essential for the development of effective nitrogen-fixing bacteroids, which was correlated with its requirement for growth on C 4 -dicarboxylates. A previously described dctA mutant of Rhizobium tropici CIAT899, strain GA1 ( dctA ), however, was unexpectedly still able to grow on succinate as a sole carbon source but less efficiently than CIAT899. Like other rhizobial dctA mutants, GA1 was unable to grow on fumarate or malate as a carbon source and induced the formation of ineffective nodules. We report an alternative succinate uptake system identified by Tn 5 mutagenesis of strain GA1 that was required for the remaining ability to transport and utilize succinate. The alternative uptake system required a three-gene cluster that is highly characteristic of a dctABD locus. The predicted permease-encoding gene had high sequence similarity with open reading frames encoding putative 2-oxoglutarate permeases (KgtP) of Ralstonia solanacearum and Agrobacterium tumefaciens . This analysis was in agreement with the requirement for this gene for optimal growth on and induction by 2-oxoglutarate. The permease-encoding gene of the alternative system was also designated kgtP in R. tropici . The dctBD -like genes in this cluster were found to be required for kgtP expression and were designated kgtSR . Analysis of a kgtP :: lacZ transcriptional fusion indicated that a kgtSR -dependent promoter of kgtP was specifically induced by 2-oxoglutarate. The expression of kgtPp was found in bacteroids of nodules formed with either CIAT899 or GA1 on roots of Phaseolus vulgaris . Results suggested that 2-oxoglutarate might be transported or conceivably exported in nodules induced by R. tropici on roots of P. vulgaris .

Here, we describe for the first time the Crc ( c atabolite r epression c ontrol) protein from the soil bacterium Acinetobacter baylyi . Expression of A. baylyi crc varied according to the growth conditions. A strain with a disrupted crc gene showed the same growth as the wild type on a number of carbon sources. Carbon catabolite repression by acetate and succinate of protocatechuate 3,4-dioxygenase, the key enzyme of protocatechuate breakdown, was strongly reduced in the crc strain, whereas in the wild-type strain it underwent strong catabolite repression. This strong effect was not based on transcriptional regulation because the transcription pattern of the pca-qui operon (encoding protocatechuate 3,4-dioxygenase) did not reflect the derepression in the absence of Crc. pca-qui transcript abundance was slightly increased in the crc strain. Lack of Crc dramatically increased the mRNA stability of the pca-qui transcript (up to 14-fold), whereas two other transcripts ( pobA and catA ) remained unaffected. p -Hydroxybenzoate hydroxylase activity, encoded by pobA , was not significantly different in the absence of Crc, as protocatechuate 3,4-dioxygenase was. It is proposed that A. baylyi Crc is involved in the determination of the transcript stability of the pca-qui operon and thereby effects catabolite repression.

Many bacteria can utilize C 4 -carboxylates as carbon and energy sources. However, Corynebacterium glutamicum ATCC 13032 is not able to use tricarboxylic acid cycle intermediates such as succinate, fumarate, and l -malate as sole carbon sources. Upon prolonged incubation, spontaneous mutants which had gained the ability to grow on succinate, fumarate, and l -malate could be isolated. DNA microarray analysis showed higher mRNA levels of cg0277, which subsequently was named dccT , in the mutants than in the wild type, and transcriptional fusion analysis revealed that a point mutation in the promoter region of dccT was responsible for increased expression. The overexpression of dccT was sufficient to enable the C. glutamicum wild type to grow on succinate, fumarate, and l -malate as the sole carbon sources. Biochemical analyses revealed that DccT, which is a member of the divalent anion/Na + symporter family, catalyzes the effective uptake of dicarboxylates like succinate, fumarate, l -malate, and likely also oxaloacetate in a sodium-dependent manner.

The attKLM operon encodes a lactonase (AttM) that hydrolyzes acylhomoserine lactone autoinducers, as well as two putative dehydrogenases (AttK and AttL). Here we show that AttK, AttL, and AttM collectively covert gamma-butyrolactone to succinate. Two metabolic intermediates, gamma-hydroxybutyrate and succinic semialdehyde, inactivated the AttJ repressor in vitro and induced attKLM transcription in vivo.

A mutant strain of Bradyrhizobium japonicum USDA110 lacking isocitrate dehydrogenase activity was created to determine whether this enzyme was required for symbiotic nitrogen fixation with soybean ( Glycine max cv. Williams 82). The isocitrate dehydrogenase mutant, strain 5051, was constructed by insertion of a streptomycin resistance gene cassette. The mutant was devoid of isocitrate dehydrogenase activity and of immunologically detectable protein, indicating there is only one copy in the genome. Strain 5051 grew well on a variety of carbon sources, including arabinose, pyruvate, succinate, and malate, but, unlike many microorganisms, was a glutamate auxotroph. Although the formation of nodules was slightly delayed, the mutant was able to form nodules on soybean and reduce atmospheric dinitrogen as well as the wild type, indicating that the plant was able to supply sufficient glutamate to permit infection. Combined with the results of other citric acid cycle mutants, these results suggest a role for the citric acid cycle in the infection and colonization stage of nodule development but not in the actual fixation of atmospheric dinitrogen.

A mutant gyrA allele resulting in an A271E substitution in the DNA gyrase protein generated a strain unable to grow on the C 4 -dicarboxylates succinate, malate, and fumarate. Bacteria harboring gyrA751 displayed decreased negative supercoiling in cells. Expression of the dctA gene, which encodes the C 4 -dicarboxylate transporter, was reduced in a gyrA751 mutant, providing the first evidence that dctA expression is supercoiling sensitive and uncovering a simple metabolic screen for lesions in gyrase that reduce negative supercoiling.

The twin-arginine translocation (TAT) system secretes fully folded proteins that contain a twin-arginine motif within their signal sequence across the cytoplasmic membrane in bacteria. Using a green fluorescent protein fused with a TAT signal sequence, we demonstrated that Mycobacterium smegmatis contains a TAT system. By inactivating individual genes, we showed that three genes ( tatA , tatB , and tatC ) are required for a functional TAT system in M. smegmatis . The tat mutants exhibited a decreased growth rate and altered colony morphology compared to the parent strain. Comparison of the secreted proteins of the Δ tatC and parent strain by two-dimensional polyacrylamide gel electrophoresis revealed an alteration in the secretion of at least five proteins, and one of the major TAT-dependent secreted proteins was identified as β-lactamase (BlaS). The genome of M. smegmatis was analyzed with the TATFIND program, and 49 putative TAT substrates were identified, including the succinate transporter DctP. Because disruption of the TAT secretion system has a direct effect on the physiology of M. smegmatis and homologs of the TAT proteins are also present in the genome of Mycobacterium tuberculosis , the TAT secretion system or its substrates may be good candidates for drug or vaccine development.

The mechanism of fumarate reduction in Geobacter sulfurreducens was investigated. The genome contained genes encoding a heterotrimeric fumarate reductase, FrdCAB, with homology to the fumarate reductase of Wolinella succinogenes and the succinate dehydrogenase of Bacillus subtilis . Mutation of the putative catalytic subunit of the enzyme resulted in a strain that lacked fumarate reductase activity and was unable to grow with fumarate as the terminal electron acceptor. The mutant strain also lacked succinate dehydrogenase activity and did not grow with acetate as the electron donor and Fe(III) as the electron acceptor. The mutant strain could grow with acetate as the electron donor and Fe(III) as the electron acceptor if fumarate was provided to alleviate the need for succinate dehydrogenase activity in the tricarboxylic acid cycle. The growth rate of the mutant strain under these conditions was faster and the cell yields were higher than for wild type grown under conditions requiring succinate dehydrogenase activity, suggesting that the succinate dehydrogenase reaction consumes energy. An orthologous frdCAB operon was present in Geobacter metallireducens , which cannot grow with fumarate as the terminal electron acceptor. When a putative dicarboxylic acid transporter from G. sulfurreducens was expressed in G. metallireducens , growth with fumarate as the sole electron acceptor was possible. These results demonstrate that, unlike previously described organisms, G. sulfurreducens and possibly G. metallireducens use the same enzyme for both fumarate reduction and succinate oxidation in vivo.

The gene ( puhA ) encoding the H subunit of the reaction center (RC) was deleted by site-directed interposon mutagenesis by using a kanamycin resistance cassette lacking transcriptional terminators to eliminate polar effects in both the wild-type strain Rhodospirillum rubrum S1 and the carotenoid-less strain R. rubrum G9. The puhA interposon mutants were incapable of photoheterotrophic growth but grew normally under aerobic chemoheterotrophic conditions. Absorption spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the RCs were absent. In minimal medium and also in modified medium containing succinate and fructose, the light-harvesting 1 complex (LH1) levels of the S1-derived mutants were about 70 to 100% of the wild-type levels in the same media. The correct assembly of LH1 in the membrane and the pigment-pigment interaction were confirmed by near-infrared circular dichroism spectroscopy. LH1 formation was almost absent when the carotenoid-less G9-derived puhA mutants were grown in standard minimal medium, suggesting that carotenoids may stabilize LH1. In the fructose-containing medium, however, the LH1 levels of the G9 mutants were 70 to 100% of the parental strain levels. Electron micrographs of thin sections of R. rubrum revealed photosynthetic membranes in all mutants grown in succinate-fructose medium. These studies indicate that the H subunit of the RC is necessary neither for maximal formation of LH1 nor for photosynthetic membrane formation but is essential for functional RC assembly.

Methylobacterium extorquens AM1, a serine cycle facultative methylotroph, accumulates poly-β-hydroxybutyrate (PHB) as a carbon and energy reserve material during growth on both multicarbon- and single-carbon substrates. Recently, the identification and mutation of the genes involved in the biosynthesis and degradation of PHB have been described for this bacterium, demonstrating that two of the genes of the PHB cycle ( phaA and phaB ) are also involved in C 1 and C 2 metabolism, as part of a novel pathway for glyoxylate regeneration in the serine cycle (N. Korotkova and M. E. Lidstrom, J. Bacteriol. 183:1038-1046, 2001; N. Korotkova, L. Chistoserdova, V. Kuksa, and M. E. Lidstrom, J. Bacteriol. 184:1750-1758, 2002). In this work, three new genes involved in PHB biosynthesis in this bacterium have been investigated via mutation and phenotypic analysis: gap11 , gap20 , and phaR . We demonstrate that gap11 and gap20 encode two major granule-associated proteins (phasins) and that mutants with mutations in these genes are defective in PHB production and also in growth on C 2 compounds, while they show wild-type growth characteristics on C 1 or multicarbon compounds. The phaR mutant shows defects in both PHB accumulation and growth characteristics when grown on C 1 compounds and has defects in PHB accumulation but grows normally on C 3 and C 4 compounds, while both PHB accumulation and growth rate are at wild-type levels during growth on C 2 compounds. Our results suggest that this phenotype is due to altered fluxes of acetyl coenzyme A (CoA), a major intermediate in C 1 , C 2 , and heterotrophic metabolism in M. extorquens AM1, as well as the entry metabolite for the PHB cycle. Therefore, it seems likely that PhaR acts to control acetyl-CoA flux to PHB in this methylotrophic bacterium.

Paracoccus pantotrophuscan express a periplasmic nitrate reductase (Nap) during aerobic growth. A proposed role for this enzyme is the dissipation of excess redox energy during oxidative metabolism of reduced carbon substrates. To investigate the regulation ofnapexpression, a transcriptional fusion between thenappromoter region ofP. pantotrophusand thelacZgene was constructed. When this fusion was used, analyses showed that transcription from thenappromoter increases as the average reduction state of the carbon atoms increases. Thus, β-galactosidase activities increase as the carbon source changes in the order succinate-acetate-butyrate. This result was obtained regardless of which of the three carbon sources was used for culture of the inoculum. If two carbon sources were presented together, the β-galactosidase activity was always the same as it was when the least-reduced carbon source was added alone. This suggests that the regulation is dependent upon metabolism of the more-reduced carbon sources rather than just their presence in the medium. Analysis of culture medium by1H nuclear magnetic resonance showed that for aerobic growthP. pantotrophusstrictly selected its carbon source in the order succinate-acetate-butyrate. This was reflected by diauxic growth kinetics on medium containing mixed carbon substrates. The regulatory mechanism underpinning such a selection is unknown but is likely to be related to the mechanism which controls the transcription of thenapoperon.

In Bacillus subtilis the citM gene encodes the Mg 2+ -citrate transporter. A target site for carbon catabolite repression ( cre site) is located upstream of citM . Fusions of the citM promoter region, including the cre sequence, to the β-galactosidase reporter gene were constructed and integrated into the amyE site of B. subtilis to study catabolic effects on citM expression. In parallel with β-galactosidase activity, the uptake of Ni 2+ -citrate in whole cells was measured to correlate citM promoter activity with the enzymatic activity of the CitM protein. In minimal media, CitM was only expressed when citrate was present. The presence of glucose in the medium completely repressed citM expression; repression was also observed in media containing glycerol, inositol, or succinate-glutamate. Studies with B. subtilis mutants defective in the catabolite repression components HPr, Crh, and CcpA showed that the repression exerted by all these medium components was mediated via the carbon catabolite repression system. During growth on inositol and succinate, the presence of glutamate strongly potentiated the repression of citM expression by glucose. A reasonable correlation between citM promoter activity and CitM transport activity was observed in this study, indicating that the Mg 2+ -citrate uptake activity of B. subtilis is mainly regulated at the transcriptional level.

The dicarboxylate transport (Dct) system of Sinorhizobium meliloti , which is essential for a functional nitrogen-fixing symbiosis, has been thought to transport only dicarboxylic acids. We show here that the permease component of the Dct system, DctA, can transport orotate, a monocarboxylic acid, with an apparent K m of 1.7 mM and a V max of 163 nmol min −1 per mg of protein in induced cells. DctA was not induced by the presence of orotate. The transport of orotate was inhibited by several compounds, including succinamic acid and succinamide, which are not dicarboxylic acids. The dicarboxylic acid maleate ( cis -butenedioic acid) was not an inhibitor of orotate transport, which suggests that it was not recognized by DctA. However, maleate was an excellent inducer of DctA expression. Our evaluation of 17 compounds as inducers and inhibitors of transport suggests that substrates recognized by S. meliloti DctA must have appropriately spaced carbonyl groups and an extended conformation, while good inducers are more likely to have a curved conformation.

Two distinct regions of DNA encode the enzymes needed for phthalate degradation by Burkholderia cepacia DBO1. A gene coding for an enzyme (quinolinate phosphoribosyl transferase) involved in the biosynthesis of NAD + was identified between these two regions by sequence analysis and functional assays. Southern hybridization experiments indicate that DBO1 and other phthalate-degrading B. cepacia strains have two dissimilar genes for this enzyme, while non-phthalate-degrading B. cepacia strains have only a single gene. The sequenced gene was labeled ophE , due to the fact that it is specifically induced by phthalate as shown by lacZ gene fusions. Insertional knockout mutants lacking ophE grow noticeably slower on phthalate while exhibiting normal rates of growth on other substrates. The fact that elevated levels of quinolinate phosphoribosyl transferase enhance growth on phthalate stems from the structural similarities between phthalate and quinolinate: phthalate is a competitive inhibitor of this enzyme and the phthalate catabolic pathway cometabolizes quinolinate. The recruitment of this gene for growth on phthalate thus gives B. cepacia an advantage over other phthalate-degrading bacteria in the environment.

The modified nucleoside 2-methylthio- N -6-isopentenyl adenosine (ms 2 i 6 A) is present in position 37 (adjacent to and 3′ of the anticodon) of tRNAs that read codons beginning with U except tRNA I,V Ser in Escherichia coli . In Salmonella typhimurium , 2-methylthio- N -6-( cis -hydroxy)isopentenyl adenosine (ms 2 io 6 A; also referred to as 2-methylthio cis -ribozeatin) is found in tRNA, most likely in the species that have ms 2 i 6 A in E. coli . Mutants ( miaE ) of S. typhimurium in which ms 2 i 6 A hydroxylation is blocked are unable to grow aerobically on the dicarboxylic acids of the citric acid cycle. Such mutants have normal uptake of dicarboxylic acids and functional enzymes of the citric acid cycle and the aerobic respiratory chain. The ability of S. typhimurium to grow on succinate, fumarate, and malate is dependent on the state of modification in position 37 of those tRNAs normally having ms 2 io 6 A37 and is not due to a second cellular function of tRNA (ms 2 io 6 A37)hydroxylase, the miaE gene product. We suggest that S. typhimurium senses the hydroxylation status of the isopentenyl group of the tRNA and will grow on succinate, fumarate, or malate only if the isopentenyl group is hydroxylated.

Regulation of the katB catalase gene in the anaerobic bacterium Bacteroides fragilis was studied. Northern blot hybridization analyses revealed that katB was transcribed as an approximately 1.6-kb monocistronic mRNA. The levels of katB mRNA increased > 15-fold when anaerobic, mid-logarithmic-phase cultures were exposed to O2, O2 with paraquat, or hydrogen peroxide. Under anaerobic conditions, the low levels of katB mRNA increased in a growth-dependent manner, reaching maximum expression at late logarithmic or early stationary phase, followed by a decrease in stationary phase. Under anaerobic conditions, the expression of katB mRNA was strongly repressed by glucose and to a lesser extent by xylose. However, glucose repression was completely abolished upon exposure to oxygen. The nonfermentable carbon sources fumarate, succinate, acetate, and pyruvate did not significantly affect expression. Phosphate, nitrogen, and hemin limitation did not affect the expression of katB mRNA, suggesting that the nutritional control of katB expression is restricted to carbon and energy sources and not other forms of nutrient limitation. Primer extension analysis revealed that during both oxidative stress and carbon or energy limitation, katB utilized the same promoter region but transcription initiation occurred at two different nucleotides separated by 3 or 4 bases. Interestingly, a 6-bp inverted repeat sequence present in the katB regulatory region was also observed upstream of the B. fragilis superoxide dismutase gene sod. It is possible that this is a recognition site for a DNA binding protein involved in the regulation of oxidative stress genes in this organism.

Mutants lacking the first enzyme in de novo purine synthesis (PurF) can synthesize thiamine if increased levels of pantothenate are present in the culture medium (J. L. Enos-Berlage and D. M. Downs, J. Bacteriol. 178:1476-1479, 1996). Derivatives of purF mutants that no longer required pantothenate for thiamine-independent growth were isolated. Analysis of these mutants demonstrated that they were defective in succinate dehydrogenase (Sdh), an enzyme of the tricarboxylic acid cycle. Results of phenotypic analyses suggested that a defect in Sdh decreased the thiamine requirement of Salmonella typhimurium. This reduced requirement correlated with levels of succinyl-coenzyme A (succinyl-CoA), which is synthesized in a thiamine pyrophosphate-dependent reaction. The effect of succinyl-CoA on thiamine metabolism was distinct from the role of pantothenate in thiamine synthesis.

Enzymes involved in (methyl)phenol degradation of Pseudomonas putida H are encoded by the catabolic operon (phlA-L) on plasmid pPGH1. Transcription of this operon by the sigma54 (RpoN)-containing RNA polymerase is positively controlled by the gene product of the divergently transcribed phlR in response to the availability of the respective substrate. Additionally, phenol degradation is subject to carbon catabolite repression induced by organic acids (e.g., succinate, lactate, and acetate) or carbohydrates (e.g., glucose and gluconate). Analysis of lacZ fusion to the catabolic promoter and quantified primer extension experiments indicate that carbon catabolite repression also occurs at the transcriptional level of the catabolic operon. In this study, it is furthermore shown that carbon catabolite repression is a negative control. Titration of the postulated negative controlling factor was exclusively observed when extra copies of functional phlR gene were present in the cell. We therefore conclude that PhlR is the target and that carbon catabolite repression of phenol degradation occurs by interfering with the activating function of PhlR.

A region of genomic DNA from Clostridium kluyveri was cloned in Escherichia coli by a screening strategy which was based on heterologous expression of the clostridial 4-hydroxybutyrate dehydrogenase gene. The gene region (6,575 bp) contained several open reading frames which encoded the coenzyme A (CoA)- and NADP+-dependent succinate-semialdehyde dehydrogenase (sucD), the 4-hydroxybutyrate dehydrogenase (4hbD), and a succinyl-CoA;CoA transferase (cat1), as analyzed by heterologous expression in E. coli. An open reading frame encoding a putative membrane protein (orfY) and the 5' region of a gene encoding a sigma 54-homologous sigma factor (sigL) were identified as well. Transcription was investigated by Northern (RNA) blot analysis. Protein sequence comparisons of SucD and 4HbD revealed similarities to the adhE (aad) gene products from E. coli and Clostridium acetobutylicum and to enzymes of the novel class (III) of alcohol dehydrogenases. A comparison of CoA-dependent aldehyde dehydrogenases is presented.

The pckA gene of Rhizobium meliloti, encoding phosphoenolpyruvate carboxykinase, was isolated from a genomic cosmid library by complementation of the succinate growth phenotype of a Pck- mutant. The gene region was mapped by subcloning and Tn5 insertion mutagenesis. The DNA sequence for a 2-kb region containing the structural gene and its promoter was determined. The pckA gene encodes as 536-amino-acid protein that shows homology with other ATP-dependent Pck enzymes. The promoter was identified following primer extension analysis and is similar to sigma 70-like promoters. Expression analysis with a pckA::lacZ gene fusion indicated that the pckA gene was strongly induced at the onset of stationary phase in complex medium. When defined carbon sources were tested, the expression level of the pckA gene was found to be high when cells were grown in minimal media with succinate or arabinose as the sole carbon source but almost absent when glucose, sucrose, or glycerol was the sole carbon source. Glucose and sucrose were not found to strongly repress pckA induction by succinate.

To study how changes in the [ATP]/[ADP] ratio affect the level of DNA supercoiling in Escherichia coli, the cellular content of H(+)-ATPase was modulated around the wild-type level. A relatively large drop in the [ATP]/[ADP] ratio from the normal ratio resulted in a small increase in the linking number of our reporter plasmid (corresponding to a small decrease in negative supercoiling). However, when cells depleted their carbon and energy source, the ensuing drop in energy state was accompanied by a strong increase in linking number. This increase was not due to reduced transcription of the DNA in the absence of growth substrate, since rifampin had virtually no effect on the plasmid linking number. To examine whether DNA supercoiling depends more strongly on the cellular energy state at low [ATP]/[ADP] ratios than at high ratios, we used cells that were already at a low energy state after substrate depletion; after the addition of an uncoupler to these cells, the [ATP]/[ADP] ratio decreased further, which resulted in a strong increase in plasmid linking number. Our results suggest that the strong thermodynamic control of DNA supercoiling takes over at low [ATP]/[ADP] ratios, whereas at high ratios homeostatic control mechanisms attenuate thermodynamic control.

The nucleotide sequences of two Escherichia coli genes, dcuA and dcuB (formerly designated genA and genF), have been shown to encode highly homologous products, M(r) 45,751 and 47,935 (434 and 446 amino acid residues) with 36% sequence identity (63% similarity). These proteins have a high proportion (approximately 61%) of hydrophobic residues and are probably members of a new group of integral inner membrane proteins. The locations of the dcu genes, one upstream of the aspartase gene (dcuA-aspA) and the other downstream of the anaerobic fumarase gene (fumB-dcuB), suggested that they may function in the anaerobic transport of C4-dicarboxylic acids. Growth tests and transport studies with mutants containing insertionally inactivated chromosomal dcuA and dcuB genes show that their products perform analogous and mutually complementary roles as anaerobic dicarboxylate carriers. The anaerobic dicarboxylate transport systems (Dcu) are genetically and functionally distinct from the aerobic system (Dct).

Pseudomonas putida PRS2000 is chemotactic to 4-hydroxybenzoate and other aromatic acids. This behavioral response is induced when cells are grown on 4-hydroxybenzoate or benzoate, compounds that are degraded via the beta-ketoadipate pathway. Isolation of a transposon mutant defective in 4-hydroxybenzoate chemotaxis allowed identification of a new gene cluster designated pcaRKF. DNA sequencing, mutational analysis, and complementation studies revealed that pcaR encodes a regulatory protein required for induction of at least four of the enzymes of the beta-ketoadipate pathway and that pcaF encodes beta-ketoadipyl-coenzyme A thiolase, the last enzyme in the pathway. The third gene, pcaK, encodes a transporter for 4-hydroxybenzoate, and this protein is also required for chemotaxis to aromatic acids. The predicted PcaK protein is 47 kDa in size, with a deduced amino acid sequence indicative of membership in the major facilitator superfamily of transport proteins. The protein, expressed in Escherichia coli, catalyzed 4-hydroxybenzoate transport. In addition, whole cells of P. putida pcaK mutants accumulated 4-hydroxybenzoate at reduced rates compared with that in wild-type cells. The pcaK mutation did not impair growth at the expense of 4-hydroxybenzoate under most conditions; however, mutant cells grew somewhat more slowly than the wild type on 4-hydroxybenzoate at a high pH. The finding that 4-hydroxybenzoate chemotaxis can be disrupted without an accompanying effect on metabolism indicates that this chemotactic response is receptor mediated. It remains to be determined, however, whether PcaK itself is a chemoreceptor for 4-hydroxybenzoate or whether it plays an indirect role in chemotaxis. These findings indicate that aromatic acid detection and transport are integral features of aromatic degradation pathways.

Methanobacterium thermoautotrophicum delta H and Marburg were adapted to grow in medium containing up to 0.65 M NaCl. From 0.01 to 0.5 M NaCl, there was a lag before cell growth which increased with increasing external NaCl. The effect of NaCl on methane production was not significant once the cells began to grow. Intracellular solutes were monitored by nuclear magnetic resonance (NMR) spectroscopy as a function of osmotic stress. In the delta H strain, the major intracellular small organic solutes, cyclic-2,3-diphosphoglycerate and glutamate, increased at most twofold between 0.01 and 0.4 M NaCl and decreased when the external NaCl was 0.5 M. M. thermoautotrophicum Marburg similarly showed a decrease in solute (cyclic-2,3-diphosphoglycerate, 1,3,4,6-tetracarboxyhexane, and L-alpha-glutamate) concentrations for cells grown in medium containing > 0.5 M NaCl. At 0.65 M NaCl, a new organic solute, which was visible in only trace amounts at the lower NaCl concentrations, became the dominant solute. Intracellular potassium in the delta H strain, detected by atomic absorption and 39K NMR, was roughly constant between 0.01 and 0.4 M and then decreased as the external NaCl increased further. The high intracellular K+ was balanced by the negative charges of the organic osmolytes. At the higher external salt concentrations, it is suggested that Na+ and possibly Cl- ions are internalized to provide osmotic balance. A striking difference of strain Marburg from strain delta H was that yeast extract facilitated growth in high-NaCl-containing medium. The yeast extract supplied only trace NMR-detectable solutes (e.g., betaine) but had a large effect on endogenous glutamate levels, which were significantly decreased. Exogenous choline and glycine, instead of yeast extract, also aided growth in NaCl-containing media. Both solutes were internalized with the choline converted to betaine; the contribution to osmotic balance of these species was 20 to 25% of the total small-molecule pool. These results indicate that M. thermoautotrophicum shows little changes in its internal solutes over a wide range of external NaCl. Furthermore, they illustrate the considerable differences in physiology in the delta H and Marburg strains of this organism.

A genetic approach was used to study (dissimilatory) ferric iron (Fe3+) reduction in Shewanella putrefaciens 200. Chemical mutagenesis procedures and two rapid plate assays were developed to facilitate the screening of Fe3+ reduction-deficient mutants. Sixty-two putative Fe3+ reduction-deficient mutants were identified, and each was subsequently tested for its ability to grow anaerobically on various compounds as sole terminal electron acceptors, including Fe3+, nitrate (NO3-), nitrite (NO2-), manganese oxide (Mn4+), sulfite (SO3(2-)), thiosulfate (S2O3(2-)), trimethylamine N-oxide, and fumarate. A broad spectrum of mutants deficient in anaerobic growth on one or more electron acceptors was identified. Nine of the 62 mutants (designated Fer mutants) were deficient only in anaerobic growth on Fe3+ and retained the ability to grow on all other electron acceptors. These results suggest that S. putrefaciens expresses at least one terminal Fe3+ reductase that is distinct from other terminal reductases coupled to anaerobic growth. The nine Fer mutants were conjugally mated with an S. putrefaciens genomic library harbored in Escherichia coli S17-1. Complemented S. putrefaciens transconjugants were identified by the acquired ability to grow anaerobically on Fe3+ as the sole terminal electron acceptor. All recombinant cosmids that conferred the Fer+ phenotype appeared to carry a common internal region.

γ-Aminobutyrate (GABA) is metabolized to succinic semialdehyde by GABA aminotransferase (GABA-AT), and the succinic semialdehyde is subsequently oxidized to succinate by succinic semialdehyde dehydrogenase (SSADH). In E scherichia coli , there are duplicate GABA-ATs (GabT and PuuE) and duplicate SSADHs (GabD and YneI). While GabT and GabD have been well studied previously, the characterization and expression analysis of PuuE and YneI are yet to be investigated. By analyzing the amino acid profiles in cells of Δ puuE and/or Δ gabT mutants, this study demonstrated that PuuE plays an important role in GABA metabolism in E. coli cells. The similarity of the amino acid sequences of PuuE and GabT is 67.4%, and it was biochemically demonstrated that the catalytic center of GabT is conserved as an amino acid residue important for the enzymatic activity in PuuE as Lys-247. However, the regulation of expression of PuuE is significantly different from that of GabT. PuuE is induced by the addition of putrescine to the medium and is repressed by succinate and low aeration conditions; in contrast, GabT is almost constitutive. Similarly, YneI is induced by putrescine, while GabD is not. For E. coli , PuuE is important for utilization of putrescine as a sole nitrogen source and both PuuE and YneI are important for utilization of putrescine as a sole carbon source. The results demonstrate that the PuuE-YneI pathway was a putrescine-inducible GABA degradation pathway for utilizing putrescine as a nutrient source.

Diauxic growth was observed upon incubation of Agrobacterium tumefaciens 15955 on a mixture of succinate and mannopine as the carbon source. Diauxic growth was also observed when either fumarate or L-malate was mixed with mannopine. No diauxie was detectable when A. tumefaciens 15955 was grown on a mixture of mannopine and glucose, fructose, sucrose, or L-arabinose. Preferential utilization of succinate was observed in the initial growth phase of diauxie, whereas the final growth phase occurred at the expense of mannopine. Cells harvested during the initial growth phase exhibited a capacity for uptake of [14C]succinate but not of [14C] mannopine. A capacity for [14C]mannopine uptake was expressed during the final growth phase. Extracts from cells grown on a mixture of succinate and mannopine exhibited a low level of mannopine cyclase activity in the initial phase of diauxie. This activity increased substantially in the final phase of growth. Added succinate had no effect on the rate of [14C]mannopine uptake or mannopine cyclase activities of cells previously grown on mannopine. Diauxie was also observed during growth of strain 15955 on a mixture of succinate and octopine.

Sigma 54-dependent transcriptional activators such as Escherichia coli NtrC, Rhizobium meliloti NifA, and Rhizobium leguminosarum DctD share similar central and carboxy-terminal domains but differ in the structure and function of their amino-terminal domains. We have deleted the amino-terminal and carboxy-terminal domains of R. leguminosarum DctD and have demonstrated that the central domain of DctD, like that of NifA, is transcriptionally competent.

Genome sequence information suggests that B 12 -dependent mutases are present in a number of bacteria, including members of the suborder Corynebacterineae like Mycobacterium tuberculosis and Corynebacterium glutamicum . We here functionally identify a methylmalonyl coenzyme A (CoA) mutase in C. glutamicum that is retained in all of the members of the suborder Corynebacterineae and is encoded by NCgl1471, NCgl1472, and NCgl1470. In addition, we observe the presence of methylmalonate in C. glutamicum , reaching concentrations of up to 757 nmol g (dry weight) −1 in propionate-grown cells, whereas in Escherichia coli no methylmalonate was detectable. As demonstrated with a mutase deletion mutant, the presence of methylmalonate in C. glutamicum is independent of mutase activity but possibly due to propionyl-CoA carboxylase activity. During growth on propionate, increased mutase activity has severe cellular consequences, resulting in growth arrest and excretion of succinate. The physiological context of the mutase present in members of the suborder Corynebacterineae is discussed.

The question of whether the promoter ilvEp, located in the coding region of ilvM, the second structural gene in the ilvGMEDA operon, is subject to either amino acid- or growth rate-mediated regulation is examined. The experiments described here were performed with ilvEp-cat and ilvEp-lac fusions carried as single copies on the chromosome. The activity of the ilvEp promoter was found to respond neither to the availability of branched-chain amino acids nor to a wide range of growth rates between 35 to 390 min. In the absence of any known role for the products of the ilvGMEDA operon when repressing levels of branched-chain amino acids are present, there appears to be only a gratuitous role for the transcription at ilvEp.

Escherichia coli ferments l -tartrate under anaerobic conditions in the presence of an additional electron donor to succinate. The carrier for l -tartrate uptake and succinate export and its relation to the general C 4 -dicarboxylate carriers DcuA, DcuB, and DcuC were studied. The secondary carrier TtdT, encoded by the ttdT (previously called ygjE ) gene, is required for the uptake of l -tartrate. The ttdT gene is located downstream of the ttdA and ttdB genes, encoding the l -tartrate dehydratase TtdAB. Analysis of mRNA by reverse transcription-PCR showed that ttdA , ttdB , and ttdT are cotranscribed. Deletion of ttdT abolished growth by l -tartrate and degradation of l -tartrate completely. Bacteria containing TtdT catalyze l -tartrate or succinate uptake and specific heterologous l -tartrate/succinate antiporting. d -Tartrate is not a substrate for TtdT. TtdT operates preferentially in the direction of tartrate uptake and succinate excretion. The Dcu carriers do not support anaerobic growth on l -tartrate or l -tartrate transport. TtdT is related in sequence and function to CitT, which catalyzes heterologous citrate/succinate antiporting in citrate fermentation.

We have cloned and functionally characterized a Na + -coupled dicarboxylate transporter, SdcS, from Staphylococcus aureus . This carrier protein is a member of the divalent anion/Na + symporter (DASS) family and shares significant sequence homology with the mammalian Na + /dicarboxylate cotransporters NaDC-1 and NaDC-3. Analysis of SdcS function indicates transport properties consistent with those of its eukaryotic counterparts. Thus, SdcS facilitates the transport of the dicarboxylates fumarate, malate, and succinate across the cytoplasmic membrane in a Na + -dependent manner. Furthermore, kinetic work predicts an ordered reaction sequence with Na + ( K 0.5 of 2.7 mM) binding before dicarboxylate ( K m of 4.5 μM). Because this transporter and its mammalian homologs are functionally similar, we suggest that SdcS may serve as a useful model for DASS family structural analysis.

The Bacillus subtilis gltAB operon, encoding glutamate synthase, requires a specific positive regulator, GltC, for its expression and is repressed by the global regulatory protein TnrA. The factor that controls TnrA activity, a complex of glutamine synthetase and a feedback inhibitor, such as glutamine, is known, but the signal for modulation of GltC activity has remained elusive. GltC-dependent gltAB expression was drastically reduced when cells were grown in media containing arginine or ornithine or proline, all of which are inducers and substrates of the Roc catabolic pathway. Analysis of gltAB expression in mutants with various defects in the Roc pathway indicated that rocG -encoded glutamate dehydrogenase was required for such repression, suggesting that the substrates or products of this enzyme are the real effectors of GltC. Given that RocG is an enzyme of glutamate catabolism, the main regulatory role of GltC may be prevention of a futile cycle of glutamate synthesis and degradation in the presence of arginine-related amino acids or proline. In addition, high activity of glutamate dehydrogenase was incompatible with activity of TnrA.

Expression of the genes of the alkane degradation pathway encoded in the Pseudomonas putida OCT plasmid are subject to negative and dominant global control depending on the carbon source used and on the physiological status of the cell. We investigated the signals responsible for this control in chemostat cultures under conditions of nutrient or oxygen limitation. Our results show that this global control is not related to the growth rate and responds to two different signals. One signal is the concentration of the carbon source that generates the repressing effect (true catabolite repression control). The second signal is influenced by the level of expression of the cytochome o ubiquinol oxidase, which in turn depends on factors such as oxygen availability or the carbon source used. Since under carbon limitation conditions the first signal is relieved but the second signal is not, we propose that modulation mediated by the cytochrome o ubiquinol oxidase is not classical catabolite repression control but rather a more general physiological control mechanism. The two signals have an additive, but independent, effect, inhibiting induction of the alkane degradation pathway.

Analysis by two-dimensional gel electrophoresis revealed that Mycobacterium avium expresses several proteins unique to an intracellular infection. One abundant protein with an apparent molecular mass of 50 kDa was isolated, and the N-terminal sequence was determined. It matches a sequence in the M. tuberculosis database (Sanger) with similarity to the enzyme isocitrate lyase of both Corynebacterium glutamicum and Rhodococcus fascians . Only marginal similarity was observed between this open reading frame (ORF) (termed icl ) and a second distinct ORF (named aceA ) which exhibits a low similarity to other isocitrate lyases. Both ORFs can be found as distinct genes in the various mycobacterial databases recently published. Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anapleurotic enzyme for growth on acetate and certain fatty acids as carbon source. In this study we express and purify Icl, as well as AceA proteins, and show that both exhibit isocitrate lyase activity. Various known inhibitors for isocitrate lyase were effective. Furthermore, we present evidence that in both M. avium and M. tuberculosis the production and activity of the isocitrate lyase is enhanced under minimal growth conditions when supplemented with acetate or palmitate.

We have evaluated the use of [1,2- 13 C 2 ]propionate for the analysis of propionic acid metabolism, based on the ability to distinguish between the methylcitrate and methylmalonate pathways. Studies using propionate-adapted Escherichia coli MG1655 cells were performed. Preservation of the 13 C- 13 C- 12 C carbon skeleton in labeled alanine and alanine-containing peptides involved in cell wall recycling is indicative of the direct formation of pyruvate from propionate via the methylcitrate cycle, the enzymes of which have recently been demonstrated in E. coli . Additionally, formation of 13 C-labeled formate from pyruvate by the action of pyruvate-formate lyase is also consistent with the labeling of pyruvate C-1. Carboxylation of the labeled pyruvate leads to formation of [1,2- 13 C 2 ]oxaloacetate and to multiply labeled glutamate and succinate isotopomers, also consistent with the flux through the methylcitrate pathway, followed by the tricarboxylic acid (TCA) cycle. Additional labeling of TCA intermediates arises due to the formation of [1- 13 C]acetyl coenzyme A from the labeled pyruvate, formed via pyruvate-formate lyase. Labeling patterns in trehalose and glycine are also interpreted in terms of the above pathways. The information derived from the [1,2- 13 C 2 ]propionate label is contrasted with information which can be derived from singly or triply labeled propionate and shown to be more useful for distinguishing the different propionate utilization pathways via nuclear magnetic resonance analysis.

Expression of the genes for the membrane-bound Fo sector of the Escherichia coli F1Fo proton-translocating ATPase can respond to changes in metabolic conditions, and these changes are reflected in alterations in the subunit stoichiometry of the oligomeric Fo proton channel. Transcriptional and translational lacZ fusions to the promoter and to two Fo genes show that, during growth on the nonfermentable carbon source succinate, transcription of the operon and translation of uncB, encoding the a subunit of Fo, are higher than during growth on glucose. In contrast, translation of the uncE gene, encoding the c subunit of Fo, is higher during growth on glucose than during growth on succinate. Translation rates of both uncB and uncE change as culture density increases, but transcription rates do not. Quantitation of the c stoichiometry shows that more c subunits are assembled into the F1Fo ATPase in cells grown on glucose than in cells grown on succinate. E. coli therefore appears to have a mechanism for regulating the composition and, presumably, the function of the ATPase in response to metabolic circumstances.

The surB gene was identified as a gene product required for Escherichia coli cells to exit stationary phase at 37 degrees C under aerobic conditions. surB was shown to be the same as cydC, whose product is required for the proper assembly and activity of cytochrome d oxidase. Cytochrome d oxidase, encoded by the cydAB operon, is one of two alternate terminal cytochrome oxidases that function during aerobic electron transport in E. coli. Mutations inactivating the cydAB operon also cause a temperature-sensitive defect in exiting stationary phase, but the phenotype is not as severe as it is for surB mutants. In this study, we examined the phenotypes of surB1 delta(cydAB) double mutants and the ability of overexpression of cytochrome o oxidase to suppress the temperature-sensitive stationary-phase-exit defect of surB1 and delta(cydAB) mutants and analyzed spontaneous suppressors of surB1. Our results indicate that the severe temperature-sensitive defect in exiting stationary phase of surB1 mutants is due both to the absence of terminal cytochrome oxidase activity and to the presence of a defective cytochrome d oxidase. Membrane vesicles prepared from wild-type, surB1, and delta(cydAB) strains produced superoxide radicals at the same rate in vitro. Therefore, the aerobic growth defects of the surB1 and delta(cydAB) strains are not due to enhanced superoxide production resulting from the block in aerobic electron transport.

Pyruvate carboxylase (PYC), a biotin-dependent enzyme which catalyzes the conversion of pyruvate to oxaloacetate, was hypothesized to play an important anaplerotic role in the growth of Rhizobium etli during serial subcultivation in minimal media containing succinate (S. Encarnación, M. Dunn, K. Willms, and J. Mora, J. Bacteriol. 177:3058-3066, 1995). R. etli and R. tropici pyc::Tn5-mob mutants were selected for their inability to grow in minimal medium with pyruvate as a sole carbon source. During serial subcultivation in minimal medium containing 30 mM succinate, the R. etli parent and pyc mutant strains exhibited similar decreases in growth rate with each subculture. Supplementation of the medium with biotin prevented the growth decrease of the parent but not the mutant strain, indicating that PYC was necessary for the growth of R. etli under these conditions. The R. tropici pyc mutant grew normally in subcultures regardless of biotin supplementation. The symbiotic phenotypes of the pyc mutants from both species were similar to those of the parent strains. The R. etli pyc was cloned, sequenced, and found to encode a 126-kDa protein of 1,154 amino acids. The deduced amino acid sequence is highly homologous to other PYC sequences, and the catalytic domains involved in carboxylation, pyruvate binding, and biotinylation are conserved. The sequence and biochemical data show that the R. etli PYC is a member of the alpha4, homotetrameric, acetyl coenzyme A-activated class of PYCs.