Isolation, characterization, and complementation of Rhizobium meliloti 104A14 mutants that lack glutamine synthetase II activity

Somerville, John; Shatters, Robert G.; Kahn, Michael L.

doi:10.1128/jb.171.9.5079-5086.1989

Cited by 18 publications

(5 citation statements)

References 40 publications

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“…Plasmid pFR101 failed to complement the E. coli AglnA strain YMC11. The glnII genes from B. japonicum and Rhizobium meliloti are similarly not expressed from their own promoters in E. coli (12,41). When pFR12la and pFR12lb were transformed into E. coli YMC11, pFR12la but not pFR12lb complemented the E. coli AglnA mutant when grown on M9 medium, indicating that glnlI expression was being mediated by the lac promoter.…”

Section: Resultsmentioning

confidence: 99%

Molecular cloning, sequencing, and expression of the glutamine synthetase II (glnII) gene from the actinomycete root nodule symbiont Frankia sp. strain CpI1

Rochefort

Benson

1990

J Bacteriol

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In common with other plant symbionts, Frankia spp., the actinomycete N2-fixing symbionts of certain nonleguminous woody plants, synthesize two glutamine synthetases, GSI and GSII. DNA encoding the Bradyrhizobium japonicum gene for GSII (glnII) hybridized to DNA from three Frankia strains. B. japonicum glnII was used as a probe to clone the glnII gene from a size-selected KpnI library of Frankia strain CpI1 DNA. The region corresponding to the Frankia sp. strain CpI1 glnII gene was sequenced, and the amino acid sequence was compared with that of the GS gene from the pea and glnII from B. japonicum. The Frankia glnII gene product has a high degree of similarity with both GSII from B. japonicum and GS from pea, although the sequence was about equally similar to both the bacterial and eucaryotic proteins. The Frankia glnII gene was also capable of complementing an Escherichia coli delta glnA mutant when transcribed from the vector lac promoter, but not when transcribed from the Frankia promoter. GSII produced in E. coli was heat labile, like the enzyme produced in Frankia sp. strain CpI1 but unlike the wild-type E. coli enzyme.

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Section: Resultsmentioning

confidence: 99%

Molecular cloning, sequencing, and expression of the glutamine synthetase II (glnII) gene from the actinomycete root nodule symbiont Frankia sp. strain CpI1

Rochefort

Benson

1990

J Bacteriol

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“…An S. meliloti mutant lacking its two primary GS genes and expressing only its third, minor GS gene exhibited a similar phenotype (35,37). The glnA1 mutant is attenuated for intracellular growth in human THP-1 macrophages.…”

Section: Vol 71 2003 Gs Is Essential For Growth Of M Tuberculosis mentioning

confidence: 89%

Glutamine Synthetase GlnA1 Is Essential for Growth of Mycobacterium tuberculosis in Human THP-1 Macrophages and Guinea Pigs

2003

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To assess the role of glutamine synthetase (GS), an enzyme of central importance in nitrogen metabolism, in the pathogenicity of Mycobacterium tuberculosis, we constructed a glnA1 mutant via allelic exchange. The mutant had no detectable GS protein or GS activity and was auxotrophic for L-glutamine. In addition, the mutant was attenuated for intracellular growth in human THP-1 macrophages and avirulent in the highly susceptible guinea pig model of pulmonary tuberculosis. Based on growth rates of the mutant in the presence of various concentrations of L-glutamine, the effective concentration of L-glutamine in the M. tuberculosis phagosome of THP-1 cells was ϳ10% of the level assayed in the cytoplasm of these cells (4.5 mM), indicating that the M. tuberculosis phagosome is impermeable to even very small molecules in the macrophage cytoplasm. When complemented by the M. tuberculosis glnA1 gene, the mutant exhibited a wild-type phenotype in broth culture and in human macrophages, and it was virulent in guinea pigs. When complemented by the Salmonella enterica serovar Typhimurium glnA gene, the mutant had only 1% of the GS activity of the M. tuberculosis wild-type strain because of poor expression of the S. enterica serovar Typhimurium GS in the heterologous M. tuberculosis host. Nevertheless, the strain complemented with S. enterica serovar Typhimurium GS grew as well as the wild-type strain in broth culture and in human macrophages. This strain was virulent in guinea pigs, although somewhat less so than the wild-type. These studies demonstrate that glnA1 is essential for M. tuberculosis virulence.Glutamine and glutamate are central molecules in nitrogen metabolism. Glutamine is used as the nitrogen donor for many nitrogen-containing molecules in the cell and is synthesized from L-glutamate, ammonia, and ATP by the enzyme glutamine synthetase (GS) (33). The internal L-glutamine pool has been shown to be a sensor of external nitrogen limitation for Salmonella enterica serovar Typhimurium (21). GS is the only known biosynthetic pathway for the synthesis of glutamine and along with glutamate synthetase is responsible for ammonia assimilation under nitrogen-limiting growth conditions. In enteric bacteria, glutamate dehydrogenase can assimilate ammonia directly into glutamate at high concentrations of ammonia. However, for bacteria such as Mycobacterium tuberculosis which lack glutamate dehydrogenase, GS and glutamate synthetase are the sole means of ammonia assimilation. Due to its central role in nitrogen metabolism, GS is subject to varied and complex forms of transcriptional and posttranslational regulation as well as feedback inhibition by several products of glutamine metabolism (7,33).There are at least four major forms of GS (25). In enteric bacteria, a single glnA gene encodes a GS type I (GSI) enzyme, and glnA null mutants are glutamine auxotrophs. Other bacteria have been shown to possess two or three different types of GS. In the case of Sinorhizobium meliloti (formerly Rhizobium meliloti), all three GS genes...

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“…GSI and GSII activities in members of the family Rhizobiaceae are differentially regulated in response to growth conditions (1,14,36,44). However, the function of GSII in symbiosis is unknown, since mutant strains lacking GSII activity exhibit no obvious phenotype and still successfully associate with plants (16,49). Because the occurrence of GSII was thought to be confined to a small group of plant-associated bacteria, the finding of high sequence similarity between GSII from B. japonicum and plant GS enzymes led to the hypothesis of a horizontal GS gene transfer from host plants to their microsymbionts (10).…”

Section: Discussionmentioning

confidence: 99%

Overexpression of a Streptomyces viridochromogenes gene (glnII) encoding a glutamine synthetase similar to those of eucaryotes confers resistance against the antibiotic phosphinothricyl-alanyl-alanine

et al. 1990

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Isolation, characterization, and complementation of Rhizobium meliloti 104A14 mutants that lack glutamine synthetase II activity

Cited by 18 publications

References 40 publications

Molecular cloning, sequencing, and expression of the glutamine synthetase II (glnII) gene from the actinomycete root nodule symbiont Frankia sp. strain CpI1

Molecular cloning, sequencing, and expression of the glutamine synthetase II (glnII) gene from the actinomycete root nodule symbiont Frankia sp. strain CpI1

Glutamine Synthetase GlnA1 Is Essential for Growth of Mycobacterium tuberculosis in Human THP-1 Macrophages and Guinea Pigs

Overexpression of a Streptomyces viridochromogenes gene (glnII) encoding a glutamine synthetase similar to those of eucaryotes confers resistance against the antibiotic phosphinothricyl-alanyl-alanine

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