Isolation and Characterization of cysK Mutants of Escherichia coli K12

Fimmel, A L; Loughlin, R. E.

doi:10.1099/00221287-103-1-37

Cited by 32 publications

(23 citation statements)

References 25 publications

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“…4A, lane 2), which is consistent with the material observed in extracts prepared from E. coli cysI, cysJ, and cysK mutants (Fig. 4B, lanes 4 to 6) (19,62,63,65 prepared from the mll7575-mll7676 mutant, the introduction of a multicopy plasmid carrying mll7575 and mll7676 (pGTO101) into this mutant resulted in the detection of PAPS, sulfite, and sulfide (Fig. 4A, lane 7).…”

Section: Mesorhizobium Loti Produces Sulfated Polysaccharidessupporting

confidence: 70%

Mesorhizobium lotiProducesnodPQ-Dependent Sulfated Cell Surface Polysaccharides

2006

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Leguminous plants and bacteria from the family Rhizobiaceae form a symbiotic relationship, which culminates in novel plant structures called root nodules. The indeterminate symbiosis that forms between Sinorhizobium meliloti and alfalfa requires biosynthesis of Nod factor, a ␤-1,4-linked lipochitooligosaccharide that contains an essential 6-O-sulfate modification. S. meliloti also produces sulfated cell surface polysaccharides, such as lipopolysaccharide (LPS). The physiological function of sulfated cell surface polysaccharides is unclear, although mutants of S. meliloti with reduced LPS sulfation exhibit symbiotic abnormalities. Using a bioinformatic approach, we identified a homolog of the S. meliloti carbohydrate sulfotransferase, LpsS, in Mesorhizobium loti. M. loti participates in a determinate symbiosis with the legume Lotus japonicus. We showed that M. loti produces sulfated forms of LPS and capsular polysaccharide (KPS). To investigate the physiological function of sulfated polysaccharides in M. loti, we identified and disabled an M. loti homolog of the sulfate-activating genes, nodPQ, which resulted in undetectable amounts of sulfated cell surface polysaccharides and a cysteine auxotrophy. We concomitantly disabled an M. loti cysH homolog, which disrupted cysteine biosynthesis without reducing cell surface polysaccharide sulfation. Our experiments demonstrated that the nodPQ mutant, but not the cysH mutant, showed an altered KPS structure and a diminished ability to elicit nodules on its host legume, Lotus japonicus. Interestingly, the nodPQ mutant also exhibited a more rapid growth rate and appeared to outcompete wild-type M. loti for nodule colonization. These results suggest that sulfated cell surface polysaccharides are required for optimum nodule formation but limit growth rate and nodule colonization in M. loti.Most soils are limited in reduced forms of nitrogen. Thus, plants undergo symbioses with microorganisms that provide the plants with reduced nitrogen. The best studied of these symbioses occur between leguminous plants and a family of gram-negative bacteria known as rhizobia. The symbiosis culminates in the formation of novel structures on the plant root called nodules. To enter and colonize the nodules, the bacteria elicit a series of morphological changes in specialized epidermal cells called root hairs. The bacteria induce curling of the root hairs, trapping bacterial microcolonies in the center of the structure. The bacteria then elicit the formation of a plantderived tubular structure that emanates from the center of the curl, extending through the root hair and ultimately penetrating through the epidermis into the cortical layers of the root. This structure, known as an infection thread, is occupied by the bacteria and allows their entry into the interior of the root. The bacteria are released from the infection thread into the cytoplasm of plant cells within the nodule, where they differentiate into intracellular forms called bacteroids, which then reduce molecular dinitrogen for use...

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Section: Mesorhizobium Loti Produces Sulfated Polysaccharidessupporting

confidence: 70%

Mesorhizobium lotiProducesnodPQ-Dependent Sulfated Cell Surface Polysaccharides

2006

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“…Sulfide is obtained from the transport and reduction of inorganic sulfate or from organic sulfonate compounds such as taurine (61,62). The final step in cysteine biosynthesis is catalyzed by either O-acetylserine (thiol)-lyase A or O-acetylserine (thiol)-lyase B, encoded by the genes cysK and cysM, respectively (19,30,31). The CysK and CysM proteins from Escherichia coli are 43% identical.…”

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Role of a Cysteine Synthase in Staphylococcus aureus

et al. 2004

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The gram-positive human pathogen Staphylococcus aureus is often isolated with media containing potassium tellurite, to which it has a higher level of resistance than Escherichia coli. The S. aureus cysM gene was isolated in a screen for genes that would increase the level of tellurite resistance of E. coli DH5␣. The protein encoded by S. aureus cysM is sequentially and functionally homologous to the O-acetylserine (thiol)-lyase B family of cysteine synthase proteins. An S. aureus cysM knockout mutant grows poorly in cysteine-limiting conditions, and analysis of the thiol content in cell extracts showed that the cysM mutant produced significantly less cysteine than wild-type S. aureus SH1000. S. aureus SH1000 cannot use sulfate, sulfite, or sulfonates as the source of sulfur in cysteine biosynthesis, which is explained by the absence of genes required for the uptake and reduction of these compounds in the S. aureus genome. S. aureus SH1000, however, can utilize thiosulfate, sulfide, or glutathione as the sole source of sulfur. Mutation of cysM caused increased sensitivity of S. aureus to tellurite, hydrogen peroxide, acid, and diamide and also significantly reduced the ability of S. aureus to recover from starvation in amino acid-or phosphate-limiting conditions, indicating a role for cysteine in the S. aureus stress response and survival mechanisms.

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“…Unlike the A isozyme, O-acetylserine (thiol)-lyase B also catalyzes the formation of S-sulfocysteine from 0-acetyl-L-serine and thiosulfate (30, 31) and is required for efficient cysteine biosynthesis during anaerobic growth (12). Both enzymes are presumed to exist in E. coli as well, even though only cysK and its product have been demonstrated in this species (6,13).Genes for the enzymes of cysteine biosynthesis are scattered widely on the bacterial chromosome and are regulated by a system of positive control termed the cysteine regulon. Derepression of the cysteine regulon requires a combination of sulfur starvation, the L-cysteine precursor and coinducer 0-acetyl-L-serine, and the protein encoded by the cysB regulatory gene (21-23).…”

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confidence: 99%

DNA sequences of the cysK regions of Salmonella typhimurium and Escherichia coli and linkage of the cysK regions to ptsH

et al. 1988

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Nucleotide sequences of the cysK regions of SalmoneUa typhimurium and Escherichia coli have been determined. A total of 3,812 and 2,595 nucleotides were sequenced from S. typhimurium and E. coli, respectively. Open reading frames of 323 codons were found in both species and were identified as those of cysK by comparison of deduced amino acid sequences with amino-and carboxyl-terminal amino acid analyses of the S. typhimurium cysK gene product O-acetylserine (thiol)-lyase A. The two cysK DNA sequences were 85% identical, and the deduced amino acid sequences were 96% identical. The major transcription initiation sites for cysK were found to be virtually identical in the two organisms, by using primer extension and Si nuclease protection techniques. The -35 region corresponding to the major transcription start site was TTCCCC in S. typhimurium and TTCCGC in E. coli. The deviation of these sequences from the consensus sequence TTGACA may reflect the fact that cysK is subject to positive control and requires the cysB regulatory protein for expression. Sequences downstream of cysK were found to include ptsH and a portion of ptsl, thus establishing the exact relationship of cysK with these two genes. A 290-codon open reading frame, which may represent the cysZ gene, was identified upstream of cysK.Salmonella typhimurium and Escherichia coli synthesize L-cysteine from O-acetyl-L-serine and sulfide in a reaction catalyzed by O-acetylserine (thiol)-lyase (formerly designated O-acetylserine sulfhydrylase [4,24]). Two such enzymes, termed A and B, are found in S. typhimurium (5) and are coded for by cysK and cysM, respectively (18,19). Both cysK and cysM are closely linked to cysA and to the crr ptsI ptsH cluster located at 49 min on the S. typhimurium chromosome (41) and at 52 min on the E. coli chromosome (3). Native O-acetylserine (thiol)-lyase A has a molecular weight of 68,000, contains 2 molecules of pyridoxal phosphate and is composed of two identical 34-kilodalton subunits (4, 6). The large excess of the A isozyme over the B isozyme during aerobic growth indicates that, under these conditions, the former accounts for the majority of Lcysteine synthesis from O-acetyl-L-serine and sulfide (18,23). Unlike the A isozyme, O-acetylserine (thiol)-lyase B also catalyzes the formation of S-sulfocysteine from 0-acetyl-L-serine and thiosulfate (30, 31) and is required for efficient cysteine biosynthesis during anaerobic growth (12). Both enzymes are presumed to exist in E. coli as well, even though only cysK and its product have been demonstrated in this species (6,13).Genes for the enzymes of cysteine biosynthesis are scattered widely on the bacterial chromosome and are regulated by a system of positive control termed the cysteine regulon. Derepression of the cysteine regulon requires a combination of sulfur starvation, the L-cysteine precursor and coinducer 0-acetyl-L-serine, and the protein encoded by the cysB regulatory gene (21-23). The elucidation of the molecular mechanisms involved in this system of positive regulation...

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Isolation and Characterization of cysK Mutants of Escherichia coli K12

Cited by 32 publications

References 25 publications

Mesorhizobium lotiProducesnodPQ-Dependent Sulfated Cell Surface Polysaccharides

Mesorhizobium lotiProducesnodPQ-Dependent Sulfated Cell Surface Polysaccharides

Role of a Cysteine Synthase in Staphylococcus aureus

DNA sequences of the cysK regions of Salmonella typhimurium and Escherichia coli and linkage of the cysK regions to ptsH

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