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Cloning of the glutamine synthetase gene from group B streptococci.

ABSTRACT: The glnA gene from the human pathogen Streptococcus agalactiae was cloned from a genomic library prepared with the lambda phage vector lambdaDASHII. A 4.6-kb DNA fragment of one of the recombinant phages was subcloned in pUC18. This Escherichia coli clone expressed a 52-kDa protein encoded by a 1,341-bp open reading frame. The nucleotide sequence of the open reading frame and the deduced amino acid sequence shared a significant degree of homology with the sequences of other glutamine synthetases (GS). The highest homology was between our deduced protein and GS of gram-positive bacteria such as Bacillus subtilis, Bacillus cereus, and Staphylococcus aureus. Plasmids with the cloned streptococcal glnA were able to complement E. coli glnA mutants grown on minimal media. Rabbit antisera to streptococcal GS recombinant protein recognized not only the recombinant protein but also a similar-sized band in mutanolysin extracts of all group B streptococcal strains tested, regardless of polysaccharide type or surface protein profile. The amino acid sequence of the deduced protein had similarities to other streptococcal cell-surface-bound proteins. The possible functional role of the immunological features of streptococcal GS is discussed.

SUBMITTER: Suvorov AN

PROVIDER: S-EPMC174575 | biostudies-other | 1997 Jan

REPOSITORIES: biostudies-other

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Cloning of the glutamine synthetase gene from group B streptococci.

Suvorov A N AN Flores A E AE Ferrieri P P

Infection and immunity 19970101 1

The glnA gene from the human pathogen Streptococcus agalactiae was cloned from a genomic library prepared with the lambda phage vector lambdaDASHII. A 4.6-kb DNA fragment of one of the recombinant phages was subcloned in pUC18. This Escherichia coli clone expressed a 52-kDa protein encoded by a 1,341-bp open reading frame. The nucleotide sequence of the open reading frame and the deduced amino acid sequence shared a significant degree of homology with the sequences of other glutamine synthetases ...[more]

PMID: 8975911

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Project description:Group B streptococci (GBS) are a leading cause of neonatal sepsis and meningitis. GBS adhere to fibronectin when it is attached to a solid phase. We isolated a Tn917 transposon mutant, COH1-GT1, which shows decreased adherence to fibronectin. COH1-GT1 also shows decreased adherence to and invasion of respiratory epithelial cells in vitro and decreased virulence in vivo. COH1-GT1 contains a Tn917 insertion in a homolog of glnQ, a gene from Escherichia coli which is required for glutamine transport and codes for a cytoplasmic ATP-binding cassette protein. To confirm that the decreased fibronectin adherence of COH1-GT1 was due to the mutation in glnQ, we constructed COH1-GT2, a strain with a nonpolar site-directed mutation in glnQ. COH1-GT2 showed decreased binding to fibronectin. We also demonstrated that complementation of glnQ in trans restored fibronectin adherence to COH1-GT1. COH1-GT1 shows decreased uptake of radiolabeled glutamine and is resistant to the toxic glutamine analog gamma-L-glutamylhydrazide, demonstrating that the glnQ gene is required for glutamine transport in GBS. glnQ lacks a signal sequence and is a cytoplasmic protein in E. coli and thus is unlikely to act as a fibronectin adhesin. glnQ is transcribed in an operon with a putative glutamine permease gene, glnP, which has a novel predicted structure containing three distinct domains linked in a single gene. The first two domains are putative glutamine binding domains with homology to the E. coli periplasmic glutamine binding gene glnH. The third is a putative permease domain with homology to the E. coli glutamine permease gene glnP. RT-PCR analysis demonstrated that glnP and glnQ are contained within a single transcript. Transcription of scpB, encoding the only known fibronectin-binding adhesin of GBS, is unaffected. We speculate that glnQ may regulate expression of fibronectin adhesins by affecting cytoplasmic glutamine levels and that regulation may be posttranscriptional.

Project description:Streptococcus dysgalactiae subsp. equeisimilis (SDSE) has Lancefield group G or C antigens. Recent epidemiological studies reveal that invasive SDSE infections have been increasing in Asia, Europe and US. Although SDSE possesses similar virulence factors to S. pyogenes including streptolysin S (SLS) and streptolysin O (SLO), some important S. pyogenes virulence factors including active superantigens, SpeB and a hyarulonic acids capsule are missing in SDSE genome. The mechanisms and the key virulence factors for causing invasive diseases by SDSE are poorly understood. Here, we analyzed the transcriptome of SDSE in vivo using the murine sepsis model, revealing the strategy of SDSE to destruct host tissues with the virulence factors and to scavenge depleted nutrients. The expression of SLO operon increased at relatively early stage of infection while the SLS and hyaluronidases upregulated after 4h post infection. Microarray data suggested that SDSE degraded host tissue polysaccharides by streptococcal-secreting poly/oligosaccharide lyases and simultaneously used the Entner-Doudoroff pathway to metabolize acquired carbohydrates. A global negative virulence gene regulator CsrRS of SDSE modulated the expressions of genes encoding SLS and the carbohydrate metabolism enzymes. Moreover, csrS deficient mutant induced sever systemic hemolysis in mice. The most frequently isolated stG6792 strains from invasive disease secreted abundant SLS and SLO rather than other SDSE emm types, indicating the relationship between the SLS and SLO productions and poor outcome by the stG6792 strain infection. Our findings suggest that the concomitant regulation of virulence factors destructing the host tissues and metabolic enzymes play an important role to produce invasive diseases by SDSE. To analyze gene expressions in group G streptococci with the murine infection model, we developed a custom microarray for Streptococcus dysgalactiae subsp. equisimilis (SDSE) based on the genome sequences of three SDSE strains; GGS_124, ATCC12923 and RE378. We intraperitoneally inoculated 10^8 CFU of GGS_124 stain and the csrS deficient mutant into ddY mice. Bacterial cells were collected from the abdominal cavity at 0, 2, 4 and 8 h post infection. GGS_124 cells were also collected from OD600=0.6 culture in brain heart infusion broth as a control.

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Cloning of the glutamine synthetase gene from group B streptococci.

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Cloning of the glutamine synthetase gene from group B streptococci.

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