Molecular analysis of a novel glutamine synthetase of the anaerobe Bacteroides fragilis

Hill, R. T.; Parker, J. R.; Goodman, Heide J. K.; Jones, D. T.; Wood, D. R.

doi:10.1099/00221287-136-4-787

Cited by 21 publications

(43 citation statements)

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“…Nucleotide sequence analysis of the B. fragilis cfiA gene did not reveal any E. col promoter consensus sequences (48). Nucleotide and primer extension analysis of the B. fragilis glnA and recA genes revealed promoters with low homology to the E. coli promoter consensus sequence (12,17).…”

Section: Resultsmentioning

confidence: 99%

Molecular characterization of a fructanase produced by Bacteroides fragilis BF-1

Blatch

Woods

1993

J Bacteriol

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The Bacteroidesfragilis BF-1 fructanase-encoding gene (fruA) was cloned and expressed in Escherichia coli from the recombinant plasmid pBS100. Expression of the fiuA gene was constitutive in E. coli(pBS100) and B. fragilis BF-1. The ratio of sucrase activity to inulinase activity (S/I ratio) was constant for enzyme preparations from E. coli(pBS100), indicating that both activities were associated with the fructanase. For B. fragilis BF-1, the S/I ratio varied considerably depending on the carbon source used for growth, suggesting that a separate sucrase is produced in addition to the fructanase in B. fragilis BF-1. Localization experiments and TnphoA mutagenesis indicated that the fructanase was exported to the periplasm. Sequence analysis of the N-terminal region of the fructanase revealed a putative 30-amino-acid signal peptide. The enzymatic properties of the purified fructanase were investigated. The enzyme was able to hydrolyze sucrose, raffinose, inulin, and levan but not melezitose, indicating that it was a 13-D-fructofuranosidase which was able to hydrolyze 10(2-4)-linked and 0(2-6)-linked fructans.The microbial hydrolysis of fructose-containing disaccharides, trisaccharides, and polymers involves two types of P-D-fructofuranosidase enzymes. The first type is able to efficiently hydrolyze low-molecular-weight, fructose-containing sugars such as sucrose and raffinose, but it has relatively low or nonexistent activity toward fructose polymers. Examples are the Saccharomyces cerevisiae invertase (26) and sucrases of Bacillus subtilis (11), Vbrio alginolyticus (42), Salmonella typhimurium (41), and Klebsiella pneumoniae (46). The second type of enzyme is able to hydrolyze fructose polymers such as levan and inulin in addition to sugars such as sucrose and raffinose. Levan-type fructans consist of P(2-*6)-linked fructosyl units with branching at the 2-1 position, while inulin-type fructans consist exclusively of 3(2-41)-linked fructosyl units with a terminal glucose unit (50). These fructanases (inulinases or levanases) are produced by yeasts (25,37,50), filamentous fungi (50), and bacteria such as Streptococcus mutans (6, 7), Clostridium acetobutylicum (10, 28), and B. subtilis (23, 53). Inulinase (or levanase) activity is often compared with sucrase (or invertase) activity displayed by the same strain or enzyme preparation; the ratio of total sucrase units to total inulinase units is commonly expressed as the S/I ratio (50). S/I values for real sucrases are relatively high numbers ranging from 1,600 to 28,300, while those for real fructanases are relatively low numbers ranging from 0.51 to 18.5.

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Section: Resultsmentioning

confidence: 99%

Molecular characterization of a fructanase produced by Bacteroides fragilis BF-1

Blatch

Woods

1993

J Bacteriol

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“…Primers glnA-1 (59-AGGAATATTTTTTGGTTGACA-39) and glnA-2 (59-AACGATTTCCTGTGAATGC-39), amplifying part of the glutamine synthetase encoding gene, on the alignment of the glnA sequence of B. fragilis [17] and Butyrivibrio ®brisolvens were designed [20]. These primers ampli®ed a 780-bp fragment of the glnA gene in a PCR of 35 cycles, with the following thermal pro®le: 948C for 60 s, 528C for 60 s, 728C for 60 s.…”

Section: Dna Extraction and Pcrmentioning

confidence: 99%

“…The B. fragilis GSIII enzyme is very different from the other GS enzymes (GSI and GSII): its coding region is 2187 bp long (compared with the 1050±1410-bp length of the other two isoforms) and it constitutes a hexamer, not a dodecamer holoenzyme [17]. However, all GS isoforms show at least four regions of amino acid homology [17]. Two of these regions (region II and III) were included in the sequence fragment analysed in the present study.…”

Section: Introductionmentioning

confidence: 99%

recA and glnA sequences separate the Bacteroides fragilis population into two genetic divisions associated with the antibiotic resistance genotypes cepA and cfiA

Gutacker

Valsangiacomo

Bernasconi

et al. 2002

Journal of Medical Microbiology

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The sequences of part of the glutamine synthetase-encoding gene ( glnA) and of the RecA-encoding gene (recA) were determined and aligned for 45 Bacteroides fragilis isolates from different clinical and geographical origin. The patterns of sequence divergence of glnA and recA were very similar. The sequences of a 303-bp fraction of recA showed 45 nucleotide substitutions, 40 of which allowed the separation of B. fragilis into two major divisions, which were not found when the deduced amino acid sequences were considered. The 687-bp sequences analysed for the glnA gene showed 112 nucleotide substitutions, 96 of which separated the population into the same two divisions as those described for recA. In this case, the deduced amino acid sequences showed this subdivision as well: three of the six observed amino acid substitutions were division-speci®c. Within the two divisions, both genes presented a high degree of sequence conservation. Each B. fragilis division was associated with the presence of a different antibiotic resistance gene: cepA encoding a serine-â-lactamase (division I) and c®A encoding a metallo-â-lactamase (division II). No particular clusters associated with geographical or clinical origin, or with the production of an enterotoxin were observed. Sequencing of the c®A gene allowed identi®cation of two different alleles in division II. However, no association of these different c®A alleles with the expression of imipenem resistance was observed. In conclusion, the phylogenetic patterns observed by sequencing recA and glnA are in agreement with those obtained previously by MLEE (multilocus enzyme electrophoresis). Thus, it appears that the evolution of recA and glnA genes is similar to that of the whole chromosome of B. fragilis. Horizontal gene transfer between divisions I and II seems to be low, at best. However, the results of the present study could not clarify de®nitively whether divisions I and II should be considered as two different B. fragilis genospecies.

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“…In both Bacteria and Archaea, GS is an oligomeric enzyme, termed GSI, consisting of twelve identical subunits each with a molecular mass of 50-55 kDa. The only known exception is the anaerobic bacterium Bacteroides fragilis, whose GS is composed of six subunits each of 83 kDa (Hill et al, 1989). The eukaryotic GS, termed GSII, differs from the bacterial and archaeal enzymes in comprising eight subunits, each of 45-48 kDa, whose primary sequence has little similarity to those of the bacterial and archaeal enzymes.…”

Section: Introductionmentioning

confidence: 99%

The glnA gene of the extremely thermophilic eubacterium Thermotoga maritima: cloning, primary structure, and expression in Escherichia coli

Sanangelantoni

Forlani

Ambroselli

et al. 1992

Journal of General Microbiology

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The structural gene (glnA) encoding the glutamine synthetase (GS) of the extremely thermophilic eubacterium Thermotoga maritima has been cloned on a 6.0 kb Hind111 DNA fragment. Sequencing of the region containing the glnA gene (1444 bp) showed an ORF encoding a polypeptide (439 residues) with an estimated mass of 50088 Da, which shared significant homology with the GSI sequences of other Bacteria (Escherichia coli, Bacillus subtilis) and Archaea (Pyrococcus woesei, Sulfolbus solfataricus). The T. maritima glnA gene was expressed in E. coli, as shown by the ability to complement a glnA lesion in the glutamine-auxotrophic strain ET8051. The recombinant GS has been partially characterized with respect to the temperature dependence of enzyme activity, molecular mass and mode of regulation. The molecular mass of the Thermotoga GS (590000 Da), estimated by gel filtration, was compatible with a dodecameric composition for the holoenzyme, as expected for a glutamine synthetase of the GSI type. Comparison of the amino acid sequence of T. maritima GS with those from thermophilic and mesophilic micro-organisms failed to detect any obvious features directly related to thermal stability.

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Molecular analysis of a novel glutamine synthetase of the anaerobe Bacteroides fragilis

Cited by 21 publications

References 0 publications

Molecular characterization of a fructanase produced by Bacteroides fragilis BF-1

Molecular characterization of a fructanase produced by Bacteroides fragilis BF-1

recA and glnA sequences separate the Bacteroides fragilis population into two genetic divisions associated with the antibiotic resistance genotypes cepA and cfiA

The glnA gene of the extremely thermophilic eubacterium Thermotoga maritima: cloning, primary structure, and expression in Escherichia coli

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