Cloning of Genes of the Aminopeptidase T Family fromThermus thermophilusHB8 andBacillus stearothermophilusNCIB8924: Apparent Similarity to the Leucyl Aminopeptidase Family

Motoshima, Hidemasa; Minagawa, Etsuo; Tsukasaki, Fuji; Kaminogawa, Shuichi

doi:10.1271/bbb.61.1710

Cited by 19 publications

(12 citation statements)

References 8 publications

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“…CAA15149; 26% identity [2]) and aminopeptidase of Thermus aquaticus (GenBank accession no. P42778; 57% identity [30]), respectively. These genes were designated orf4 and orf5.…”

Section: Nucleotide Sequence Analysismentioning

confidence: 99%

Connection between Poly-β-Hydroxybutyrate Biosynthesis and Growth on C ₁ and C ₂ Compounds in the Methylotroph Methylobacterium extorquens AM1

Korotkova¹,

Lidstrom

2001

J Bacteriol

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Several DNA regions containing genes involved in poly-␤-hydroxybutyrate (PHB) biosynthesis and degradation and also in fatty acid degradation were identified from genomic sequence data and have been characterized in the serine cycle facultative methylotroph Methylobacterium extorquens AM1. Genes involved in PHB biosynthesis include those encoding ␤-ketothiolase (phaA), NADPH-linked acetoacetyl coenzyme A (acetylCoA) reductase (phaB), and PHB synthase (phaC). phaA and phaB are closely linked on the chromosome together with a third gene with identity to a regulator of PHB granule-associated protein, referred to as orf3. phaC was unlinked to phaA and phaB. Genes involved in PHB degradation include two unlinked genes predicted to encode intracellular PHB depolymerases (depA and depB). These genes show a high level of identity with each other at both DNA and amino acid levels. In addition, a gene encoding ␤-hydroxybutyrate dehydrogenase (hbd) was identified. Insertion mutations were introduced into depA, depB, phaA, phaB, phaC, and hbd and also in a gene predicted to encode crotonase (croA), which is involved in fatty acid degradation, to investigate their role in PHB cycling. Mutants in depA, depB, hbd, and croA all produced normal levels of PHB, and the only growth phenotype observed was the inability of the hbd mutant to grow on ␤-hydroxybutyrate. However, the phaA, phaB, and phaC mutants all showed defects in PHB synthesis. Surprisingly, these mutants also showed defects in growth on C 1 and C 2 compounds and, for phaB, these defects were rescued by glyoxylate supplementation. These results suggest that ␤-hydroxybutyryl-CoA is an intermediate in the unknown pathway that converts acetyl-CoA to glyoxylate in methylotrophs and Streptomyces spp.Many prokaryotes, including aerobic methylotrophic bacteria, accumulate poly-␤-hydroxybutyrate (PHB) intracellularly as a carbon and energy reserve material. Methylotrophic bacteria employing the serine cycle for formaldehyde assimilation are able to accumulate up to 80% of PHB by dry weight, while methylotrophic bacteria with the Calvin-Benson-Bassham cycle of C 1 assimilation can synthesize up to 20% of PHB by dry weight (14). In obligate methanol utilizers, which use the ribulose monophosphate pathway for assimilation of reduced C 1 compounds, PHB has not been detected (14).Two pathways for PHB synthesis are known in bacteria. In Ralstonia eutropha, Methylobacterium extorquens, Zoogloea ramigera, and Azotobacter beijerinckii PHB is synthesized from acetyl coenzyme A (acetyl-CoA) as a result of sequential action of three enzymes: ␤-ketothiolase, NADPH-linked acetoacetylCoA reductase, and PHB synthase (5,15,19,33,34) (Fig. 1). In Rhodospirillum rubrum and Methylobacterium rhodezianum PHB synthesis is catalyzed by five enzymes: ␤-ketothiolase, NADH-linked acetoacetyl-CoA reductase, L-(ϩ) and D-(Ϫ)-specific crotonyl-CoA hydratases (crotonases), and PHB synthase (27,28,29).The degradation of PHB in most bacteria is catalyzed by PHB depolymerase, ␤-hydroxybutyrate dehydrogenase, ...

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“…CAA15149; 26% identity [2]) and aminopeptidase of Thermus aquaticus (GenBank accession no. P42778; 57% identity [30]), respectively. These genes were designated orf4 and orf5.…”

Section: Nucleotide Sequence Analysismentioning

confidence: 99%

Connection between Poly-β-Hydroxybutyrate Biosynthesis and Growth on C ₁ and C ₂ Compounds in the Methylotroph Methylobacterium extorquens AM1

Korotkova¹,

Lidstrom

2001

J Bacteriol

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“…In this mutant, the Tn 917 -LTV3 element proved to be inserted in the promoter region of ampS , at the -35 position relative to the transcriptional start site ( fi g. 1 ). ampS is in a monocistronic operon and encodes an aminopeptidase similar to B. stearothermophilus aminopeptidase II [Motoshima et al, 1997;Stoll et al, 1976]. Disruption of the ampS promoter region should lead to a decrease in ampS expression.…”

Section: Phenotype Of the Glutamate Synthase-defective Mutantmentioning

confidence: 99%

Biofilm-Defective Mutants of Bacillus subtilis

Chagneau

Saier²

2004

Microb Physiol

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Many bacteria can adopt organized, sessile, communal lifestyles. The gram-positive bacterium, Bacillus subtilis,forms biofilms on solid surfaces and at air-liquid interfaces, and biofilm development is dependent on environmental conditions. We demonstrate that biofilm formation by B. subtilis strain JH642 can be either activated or repressed by glucose, depending on the growth medium used, and that these glucose effects are at least in part mediated by the catabolite control protein, CcpA. Starting with a chromosomal Tn917-LTV3 insertional library, we isolated mutants that are defective for biofilm formation. The biofilm defects of these mutants were observable in both rich and minimal media, and both on polyvinylchloride abiotic surfaces and in borosilicate tubes. Two mutants were defective in flagellar synthesis. Chemotaxis was shown to be less important for biofilm formation than was flagellar-driven motility. Although motility is known to be required for biofilm formation in other bacteria, this had not previously been demonstrated for B. subtilis. In addition, our study suggests roles for glutamate synthase, GltAB, and an aminopeptidase, AmpS. The loss of these enzymes did not decrease growth or cellular motility but had dramatic effects on biofilm formation under all conditions assayed. The effect of the gltAB defect on biofilm formation could not be due to a decrease in poly-γ-glutamate synthesis since this polymer proved to be nonessential for robust biofilm formation. High exogenous concentrations of glutamate, aspartate, glutamine or proline did not override the glutamate synthase requirement. This is the first report showing that glutamate synthase and a cytoplasmic aminopeptidase play roles in bacterial biofilm formation. Possible mechanistic implications and potential roles of biofilm formation in other developmental processes are discussed.

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“…To date, in addition to bacteria, genes encoding M29 aminopeptidases have been identified in archea (27) and plants (EMBL AJ004922). However, only aminopeptidases from T. aquaticus, Thermus thermophilus, G. stearothermophilus, and S. thermophilus have been conclusively characterized as members of this family (14,36,42). Among the features that justify this classification are thermophilicity, high conservation of C termini, and no significant identity with other known aminopeptidases.…”

Section: Discussionmentioning

confidence: 99%

The Thermophilic, Homohexameric Aminopeptidase ofBorrelia burgdorferiIs a Member of the M29 Family of Metallopeptidases

Bertin¹,

Lozzi²,

Howell³

et al. 2005

Infect Immun

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Proteases are implicated in several aspects of the physiology of microorganisms, as well as in host-pathogen interactions. Aminopeptidases are also emerging as novel drug targets in infectious agents. In this study, we have characterized an aminopeptidase from the spirochete Borrelia burgdorferi, the causative agent of Lyme disease. The aminopeptidolytic activity was identified in cell extracts from B. burgdorferi by using the substrate leucine-7-amido-4-methylcoumarin. A protein displaying this activity was purified from B. burgdorferi by a twostep chromatographic procedure, yielding a ϳ300-kDa homo-oligomeric enzyme formed by monomers of ϳ50 kDa. Gel enzymography experiments showed that enzymatic activity depends on the oligomeric structure of the protease but does not involve interchain disulfide bonds. The enzyme was identified by peptide mass fingerprinting as the putative aminopeptidase II of B. burgdorferi, encoded by the gene BB0069. It shares significant identity to members of the M29/T family of metallopeptidase, is sensitive to bestatin, has a neutral pH optimum, and displays maximal activity at 60°C. Its activity is 1.75-fold higher at the temperature of the mammalian host than at that of the insect host of the pathogen. The activity of this thermophilic aminopeptidase of B. burgdorferi (TAP Bb ) depends on Zn 2؉ , and temperatures over 70°C promoted its inactivation through a transition from the hexameric state to the monomeric state. Since B. burgdorferi is deficient in pathways for amino acid synthesis, TAP Bb could play a role in supplying required amino acids. Alternatively, the enzyme could be involved in peptide and/or protein processing.

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Cloning of Genes of the Aminopeptidase T Family fromThermus thermophilusHB8 andBacillus stearothermophilusNCIB8924: Apparent Similarity to the Leucyl Aminopeptidase Family

Cited by 19 publications

References 8 publications

Connection between Poly-β-Hydroxybutyrate Biosynthesis and Growth on C ₁ and C ₂ Compounds in the Methylotroph Methylobacterium extorquens AM1

Connection between Poly-β-Hydroxybutyrate Biosynthesis and Growth on C ₁ and C ₂ Compounds in the Methylotroph Methylobacterium extorquens AM1

Biofilm-Defective Mutants of <i>Bacillus subtilis</i>

The Thermophilic, Homohexameric Aminopeptidase ofBorrelia burgdorferiIs a Member of the M29 Family of Metallopeptidases

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Cloning of Genes of the Aminopeptidase T Family fromThermus thermophilusHB8 andBacillus stearothermophilusNCIB8924: Apparent Similarity to the Leucyl Aminopeptidase Family

Cited by 19 publications

References 8 publications

Connection between Poly-β-Hydroxybutyrate Biosynthesis and Growth on C 1 and C 2 Compounds in the Methylotroph Methylobacterium extorquens AM1

Connection between Poly-β-Hydroxybutyrate Biosynthesis and Growth on C 1 and C 2 Compounds in the Methylotroph Methylobacterium extorquens AM1

Biofilm-Defective Mutants of <i>Bacillus subtilis</i>

The Thermophilic, Homohexameric Aminopeptidase ofBorrelia burgdorferiIs a Member of the M29 Family of Metallopeptidases

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Connection between Poly-β-Hydroxybutyrate Biosynthesis and Growth on C ₁ and C ₂ Compounds in the Methylotroph Methylobacterium extorquens AM1

Connection between Poly-β-Hydroxybutyrate Biosynthesis and Growth on C ₁ and C ₂ Compounds in the Methylotroph Methylobacterium extorquens AM1