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Query: UMLS:C0519030 (Klebsiella)
 21,988 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Although glutamate is a key compound in nitrogen metabolism, little is known about the function or regulation of its two biosynthetic enzymes, glutamate dehydrogenase and glutamate synthase. To begin the characterization of glutamate formation in Salmonella typhimurium, we isolated mutants having altered glutamate dehydrogenase and glutamate synthase activities. Mutants which failed to grow on media with glucose as the carbon source and less than 1 mM (NH(4))(2)SO(4) as the nitrogen source (Asm(-)) had about one-fourth the normal glutamate synthase activity and one-half the glutamine synthetase activity. The asm mutations also prevented growth with alanine, arginine, or proline as nitrogen sources and conferred resistance to methionine sulfoximine. When a mutation (gdh-51) causing the loss of glutamate dehydrogenase activity was transferred into a strain with an asm-102 mutation, the resulting asm-102 gdh-51 mutant had a partial requirement for glutamate. A strain isolated as a complete glutamate auxotroph had a third mutation, in addition to the asm-102 gdh-51 lesions, that further decreased the glutamate synthase activities to 1/20 the normal level. Both the asm-102 and gdh-51 mutations were located on the S. typhimurium linkage map at sites distinct from those found for mutations causing similar phenotypes in Klebsiella aerogenes and Escherichia coli.
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PMID:Salmonella typhimurium mutants with altered glutamate dehydrogenase and glutamate synthase activities. 698 57

For many bacteria Na+ bioenergetics is important as a link between exergonic and endergonic reactions in the membrane. This article focusses on two primary Na+ pumps in bacteria, the Na(+)-translocating oxaloacetate decarboxylase of Klebsiella pneumoniae and the Na(+)-translocating F1Fo ATPase of Propionigenium modestum. Oxaloacetate decarboxylase is an essential enzyme of the citrate fermentation pathway and has the additional function to conserve the free energy of decarboxylation by conversion into a Na+ gradient. Oxaloacetate decarboxylase is composed of three different subunits and the related methylmalonyl-CoA decarboxylase consists of five different subunits. The genes encoding these enzymes have been cloned and sequenced. Remarkable are large areas of complete sequence identity in the integral membrane-bound beta-subunits including two conserved aspartates that may be important for Na+ translocation. The coupling ratio of the decarboxylase Na+ pumps depended on delta muNa+ and decreased from two to zero Na+ uptake per decarboxylation event as delta mu Na+ increased from zero to the steady state level. In P. modestum, delta mu Na+ is generated in the course of succinate fermentation to propionate and CO2. This delta mu Na+ is used by a unique Na(+)-translocating F1Fo ATPase for ATP synthesis. The enzyme is related to H(+)-translocating F1Fo ATPases. The Fo part is entirely responsible for the coupling of ion specificity. A hybrid ATPase formed by in vivo complementation of an Escherichia coli deletion mutant was completely functional as a Na(+)-ATP synthase conferring the E. coli strain the ability of Na(+)-dependent growth on succinate. The hybrid consisted of subunits a, c, b, delta and part of alpha from P. modestum and of the remaining subunits from E. coli. Studies on Na+ translocation through the Fo part of the P. modestum ATPase revealed typical transporter-like properties. Sodium ions specifically protected the ATPase from the modification of glutamate-65 in subunit c by dicyclohexylcarbodiimide in a pH-dependent manner indicating that the Na+ binding site is at this highly conserved acidic amino acid residue of subunit c within the middle of the membrane.
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PMID:Bacterial sodium ion-coupled energetics. 783 94

Aerobic gram-negative methylotrophs oxidize methanol to formaldehyde by using a methanol dehydrogenase that has pyrroloquinoline quinone (PQQ) as a prosthetic group. Seventy-two mutants which are unable to grow on methanol unless the growth medium is supplemented with PQQ have been isolated in the facultative methanol utilizer Methylobacterium extorquens AM1. In addition, 12 previously isolated methanol oxidation mutants of M. extorquens AM1 were shown to be able to grow on methanol in the presence of PQQ. These putative PQQ biosynthesis mutants have been complemented by using previously isolated clones containing M. extorquens AM1 DNA, which were known to contain genes necessary for oxidation of methanol to formaldehyde (mox genes). Subcloning and transposon mutagenesis experiments have assigned these mutants to five complementation groups in two gene clusters. Representatives of each complementation group were shown to lack detectable PQQ in the growth medium and in cell extracts and to contain methanol dehydrogenase polypeptides that were inactive. Therefore, these mutants all appear to be defective in PQQ biosynthesis. PQQ biosynthesis mutants of Methylobacterium organophilum DSM 760 and M. organophilum XX were complemented by using M. extorquens AM1 subclones, and PQQ biosynthesis mutants of M. extorquens AM1 and M. organophilum XX were complemented by using M. organophilum DSM 760 subclones. This analysis suggested that a total of six PQQ biosynthesis complementation groups were present in M. extorquens AM1 and M. organophilum DSM 760. A 2-kb M. extorquens AM1 DNA fragment that complemented the MoxO class of PQQ biosynthesis mutants was sequenced and found to contain two complete open reading frames and the N-terminal sequence of a third. These genes designated pqqDGC, had predicted gene products with substantial similarity to the gene products of corresponding pqq genes in Acinetobacter calcoaceticus and Klebsiella pneumoniae. pqqD encodes a 29-amino-acid peptide which contains a tyrosine residue and glutamate residue that are conserved in the equivalent peptides of K. pneumoniae, PqqA (23 amino acids), and A. calcoaceticus, PqqIV (24 amino acids), and are thought to be the precursors for PQQ biosynthesis. The organizations of a cluster of five PQQ biosynthetic genes appear to be similiar in four different bacteria (M. extorquens AM1, M. organophilum DSM 760, K. pneumoniae, and A. calcoaceticus). Our results show that a total of seven pqq genes are present in M. extorquens AM1, and these have been designated pqqDGCBA and pqqEF.
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PMID:Isolation, phenotypic characterization, and complementation analysis of mutants of Methylobacterium extorquens AM1 unable to synthesize pyrroloquinoline quinone and sequences of pqqD, pqqG, and pqqC. 813 70

The signal sequence of the Klebsiella oxytoca puIG gene product, which is required for extracellular secretion of the enzyme pullulanase, is similar in many respects to the corresponding segment of the precursors of type IV (me-Phe) pilins. The significance of this similarity is confirmed by the observation that the puIO gene product processes prePuIG at the consensus type IV prepilin peptidase cleavage site at the amino-terminal end of the PuIG signal sequence. Like most type IV pilins, processed PuIG was found to have a methylated amino-terminal phenylalanine residue. Site-directed mutagenesis was used to replace amino acids in prePuIG that correspond to residues shown by others to be essential for processing, methylation and assembly of type IV pilins. The glycine residue on the amino-terminal side of the prePuIG cleavage site is absolutely required for processing and for pullulanase secretion. The glutamate residue at position 11(+5) is also required for pullulanase secretion but not for processing or methylation. This result contrasts with that reported for corresponding variants of Pseudomonas aeruginosa type IV prepilin, which were processed but only inefficiently N-methylated. Cleavage of prePuIG and pullulanase secretion were both unaffected by replacement of the phenylalanine residue on the carboxy-terminal side of the cleavage site by leucine, isoleucine or valine, by a conservative substitution within the hydrophobic core of the prePuIG signal sequence, or by a glutamine to proline substitution within the processed segment. However, replacement of the same glutamine residue by arginine abolished secretion without affecting either processing or methylation.
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PMID:Processing and methylation of PuIG, a pilin-like component of the general secretory pathway of Klebsiella oxytoca. 841 82

Klebsiella aerogenes strains with reduced levels of D-amino acid dehydrogenase not only fail to use alanine as a growth substrate but also become sensitive to alanine in minimal media supplemented with glucose and ammonium. The inability of these mutant strains to catabolize the alanine provided in the medium interferes with both pathways of glutamate production. Alanine derepresses the nitrogen regulatory system (Ntr), which in turn represses glutamate dehydrogenase, one pathway of glutamate production. Alanine also inhibits the enzyme glutamine synthetase, the first enzyme in the other pathway of glutamate production. Therefore, in the presence of alanine, strains with mutations in dadA (the gene that codes for a subunit of the dehydrogenase) exhibit a glutamate auxotrophy when ammonium is the sole source of nitrogen. The alanine catabolic operon of Klebsiella aerogenes, dadAB, was cloned, and its DNA sequence was determined. The clone complemented the alanine defects of dadA strains. The operon has a high similarity to the dadAB operon of Salmonella typhimurium and the dadAX operon of Escherichia coli, each of which codes for the smaller subunit of D-amino acid dehydrogenase and the catabolic alanine racemase. Unlike the cases for E. coli and S. typhimurium, the dad operon of K. aerogenes is activated by the Ntr system, mediated in this case by the nitrogen assimilation control protein (NAC). A sequence matching the DNA consensus for NAC-binding sites is located centered at position -44 with respect to the start of transcription. The promoter of this operon also contains consensus binding sites for the catabolite activator protein and the leucine-responsive regulatory protein.
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PMID:Alanine catabolism in Klebsiella aerogenes: molecular characterization of the dadAB operon and its regulation by the nitrogen assimilation control protein. 945 58

An aminotransferase which catalyzes the final step in methionine recycling from methylthioadenosine, the conversion of alpha-ketomethiobutyrate to methionine, has been purified from Klebsiella pneumoniae and characterized. The enzyme was found to be a homodimer of 45-kDa subunits, and it catalyzed methionine formation primarily using aromatic amino acids and glutamate as the amino donors. Histidine, leucine, asparagine, and arginine were also functional amino donors but to a lesser extent. The N-terminal amino acid sequence of the enzyme was determined and found to be almost identical to the N-terminal sequence of both the Escherichia coli and Salmonella typhimurium tyrosine aminotransferases (tyrB gene products). The structural gene for the tyrosine aminotransferase was cloned from K. pneumoniae and expressed in E. coli. The deduced amino acid sequence displayed 83, 80, 38, and 34% identity to the tyrosine aminotransferases from E. coli, S. typhimurium, Paracoccus denitrificans, and Rhizobium meliloti, respectively, but it showed less than 13% identity to any characterized eukaryotic tyrosine aminotransferase. Structural motifs around key invariant residues placed the K. pneumoniae enzyme within the Ia subfamily of aminotransferases. Kinetic analysis of the aminotransferase showed that reactions of an aromatic amino acid with alpha-ketomethiobutyrate and of glutamate with alpha-ketomethiobutyrate proceed as favorably as the well-known reactions of tyrosine with alpha-ketoglutarate and tyrosine with oxaloacetate normally associated with tyrosine aminotransferases. The aminotransferase was inhibited by the aminooxy compounds canaline and carboxymethoxylamine but not by substrate analogues, such as nitrotyrosine or nitrophenylalanine.
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PMID:Tyrosine aminotransferase catalyzes the final step of methionine recycling in Klebsiella pneumoniae. 1007 65

The ability of Klebsiella pneumoniae (NCTC, CL687/80) to produce and, in turn, excrete glutamate has been equated with the presence of a large indigenous plasmid with an apparent molecular mass in the region of 96 +/- 2 kbp (n = 6). Unlike mitomycin C, novobiocine and ethidium bromide (curing agents), the use of sodium dodecyl sulphate (SDS) proved very effective in curing the plasmid with a relatively high frequency (6.25 x 10(-4)). Furthermore, the absence of isocitrate dehydrogenase (ICDH) activity in the cured strain strongly suggests that the structural gene encoding ICDH in this organism, in sharp contrast to all known ICDHs, is plasmid-encoded. Moreover, the SDS-based protocol reported for the isolation of the K. pneumoniae indigenous plasmid has proven successful with other organisms including Pseudomonad and Corynebacteria, as well as in recombinant strains of Escherichia coli.
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PMID:Isolation and curing of the Klebsiella pneumoniae large indigenous plasmid using sodium dodecyl sulphate. 1086 47

Internal pool sizes of glutamine and glutamate in Klebsiella pneumoniae grown under nitrogen limitation or nitrogen sufficiency were measured to study the signal transduction of external nitrogen limitation. K. pneumoniae cells were grown in an anaerobic, ammonium-limited chemostat culture. At a growth rate of 0.217 h(-1), the steady state ammonium concentration in the culture was 55 microm, correlating with repression of the nitrogen fixation (nif) genes. At growth rates below 0.138 h(-1), the ammonium concentration in the culture dropped below 0.5 microm and the nif genes became derepressed. During the transition from nitrogen sufficiency to nitrogen limitation, the internal glutamine pool in K. pneumoniae decreased by a factor of approximately 6. The glutamate pool, however, remained stable. Similarly, in anaerobic batch cultures with different limiting nitrogen sources, the glutamine pool generally decreased by a factor of 7 to 9 when nif gene derepression was achieved. All the limiting nitrogen sources used resulted in decreased growth rates compared with growth under nitrogen excess, suggesting an inverse relationship between glutamine pool size and doubling time. These studies indicate that K. pneumoniae perceives external nitrogen limitation as internal glutamine limitation.
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PMID:Internal glutamine and glutamate pools in Klebsiella pneumoniae grown under different conditions of nitrogen availability. 1101 74

Experiments were performed to evaluate the comparative growth and physiology of Klebsiella oxytoca grown attached to granular activated carbon particles (GAC) and in liquid medium. Laboratory studies showed that when this organism attached to GAC, the growth rate was enhanced more than 10 times in the presence of glutamate, a substrate that adsorbed to the surface. No differences were observed if the substrate was glucose, which did not adsorb to GAC. Cellular [3H]thymidine uptake was used to estimate DNA biosynthesis. Attached bacteria grown in a minimal nutrient medium containing 20.0 mg/liter glutamate took up 5 times more [3H]thymidine than cells grown in suspension. [3H]uridine was used as a measure of RNA turnover. Attached cells were shown to assimilate 11 times more [3H]uridine than cells in liquid media. Cell size measurements were performed by differential filtration. Cells grown in a minimal medium with 20.0 mg/liter glutamate decreased in size over time, with 62% of the total number passing through a 1.0 micron filter after 9 days incubation. In the same period, 39% of a cell population that was grown on GAC passed through a 1.0 micron filter. These studies indicate that GAC provides an interfacial environment for the enhanced growth of K. oxytoca when glutamate is the substrate.
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PMID:Growth and comparative physiology of Klebsiella oxytoca attached to granular activated carbon particles and in liquid media. 1153 3

Mutants of Escherichia coli and Klebsiella aerogenes that are deficient in glutamate synthase (glutamate-oxoglutarate amidotransferase [GOGAT]) activity have difficulty growing with nitrogen sources other than ammonia. Two models have been proposed to account for this inability to grow. One model postulated an imbalance between glutamine synthesis and glutamine degradation that led to a repression of the Ntr system and the subsequent failure to activate transcription of genes required for the use of alternative nitrogen sources. The other model postulated that mutations in gltB or gltD (which encode the subunits of GOGAT) were polar on a downstream gene, gltF, which is necessary for proper activation of gene expression by the Ntr system. The data reported here show that the gltF model is incorrect for three reasons: first, a nonpolar gltB and a polar gltD mutation of K. aerogenes both show the same phenotype; second, K. aerogenes and several other enteric bacteria lack a gene homologous to gltF; and third, mutants of E. coli whose gltF gene has been deleted show no defect in nitrogen metabolism. The argument that accumulated glutamine represses the Ntr system in gltB or gltD mutants is also incorrect, because these mutants can derepress the Ntr system normally so long as sufficient glutamate is supplied. Thus, we conclude that gltB or gltD mutants grow slowly on many poor nitrogen sources because they are starved for glutamate. Much of the glutamate formed by catabolism of alternative nitrogen sources is converted to glutamine, which cannot be efficiently converted to glutamate in the absence of GOGAT activity. Finally, GOGAT-deficient E. coli cells growing with glutamine as the sole nitrogen source increase their synthesis of the other glutamate-forming enzyme, glutamate dehydrogenase, severalfold, but this is still insufficient to allow rapid growth under these conditions.
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PMID:Roles of glutamate synthase, gltBD, and gltF in nitrogen metabolism of Escherichia coli and Klebsiella aerogenes. 1167 31


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