A conditional-lethal mutant of Bacillus subtilis 168 with a thermosensitive glycerol-3-phosphate cytidylyltransferase, an enzyme specific for the synthesis of the major wall teichoic acid

Pooley, Harold M.; Abellan, François-Xavier; Karamata, Dimitri

doi:10.1099/00221287-137-4-921

Cited by 66 publications

(53 citation statements)

References 34 publications

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“…However, the level of expression of a transcriptional fusion to tagC in strain L4578, barely detectable in the absence of SOS induction, increased rapidly upon MMC treatment (Fig. 5b) (Pooley et al, 1992) respectively, while the identification of the enzymes encoded by operon tagAB remained elusive (see below).…”

Section: Sos Inductionmentioning

confidence: 99%

Analysis of Bacillus subtilis tag gene expression using transcriptional fusions

Mauël¹,

Young²,

Monsutti-Grecescu³

et al. 1994

Microbiology

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Five of the genes known to encode the synthesis of poly(glycero1 phosphate), the major teichoic acid of Bacillus subtilis 168, are organized in two divergently transcribed operons (a divergon), denoted tagAB and tagDEF. To monitor their expression, the 399 bp intergenic region separating the first structural genes of these operons was fused, in both orientations, to a lacZ reporter gene, allowing measurement of promoter activity under specific physiological conditions. Under all experimental conditions, tagA and tagD appeared coordinately expressed, the level of tagD being always higher than that of tagA. No influence of the chromosomal context was observed. Phosphate limitation was accompanied by reduced tag gene expression. Following the onset of sporulation, expression of tag genes diminished rapidly and was essentially abolished by stage II. During germination, the activity of tag genes was detectable before the rise in culture turbidity associated with spore outgrowth. In contrast to tagC (dinC), the expression of which is DNA-damageinducible, the induction of 505 functions had no effect on tagA and tagD gene expression. The biological significance of these results is discussed.

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Section: Sos Inductionmentioning

confidence: 99%

Analysis of Bacillus subtilis tag gene expression using transcriptional fusions

Mauël¹,

Young²,

Monsutti-Grecescu³

et al. 1994

Microbiology

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“…M broth, M agar and M soft-agar (Yehle & Doi, 1967) supplemented as described by Yasbin et al (1973) were used to assay phage adsorption and infection. L medium, TS plates and SAT medium as well as media for transformation and transduction were as previously described (Karamata & Gross, 1970;Pooley & Karamata, 1984;Karamata et al, 1987). Minimal medium was supplemented with amino acids (20 pg ml-I) and bases (100 pg ml-l) when necessary.…”

Section: Met Hodsmentioning

confidence: 99%

“…Mutations in gtaC and gtaB confer resistance to 43T and serologically related phages, whereas gtaA mutations allow infection (Estrela et al, 1986). While mutations in all the gta markers prevent glucosylation of the major cell wall teichoic acid, poly(groP), those in gtaB and gtaC are associated, unlike gtaA, with greatly reduced amounts of N-acetylgalactosamine (GalNAc) in cell walls (Young, 1967;Pooley et al, 1987).…”

Section: Introductionmentioning

confidence: 99%

“…Genetic analysis of conditional lethal mutants deficient in poly(groP) synthesis (Boylan et al, 1972;Karamata et al, 1972;Pooley et al, 1987;Briehl et al, 1989;, and in particular the more recent nucleotide sequencing studies (Honeyman & Stewart, 1989;Mauel et al, 1991) have led to the identification of six genes likely to be specifically involved in the synthesis of glucosylated poly(groP). Putative enzyme activities for the encoded products of two of these genes have been proposed (Young, 1967 ; Pooley et al, 199 1).…”

Section: Introductionmentioning

confidence: 99%

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Genetic and biochemical characterization of Bacillus subtilis 168 mutants specifically blocked in the synthesis of the teichoic acid poly(3-O- -D-glucopyranosyl-N-acetylgalactosamine 1-phosphate): gneA, a new locus, is associated with UDP-N-acetylglucosamine 4-epimerase activity

Estrela¹,

Pooley²,

Lencastre³

et al. 1991

Journal of General Microbiology

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Journal of General Microbiology (1991), 137, 943-950. Printed in Great Britain 943Genetic and biochemical characterization of Bacillus subtifis 168 mutants specifically blocked in the synthesis of the teichoic acid poly(3-O-p-~-glucopyranosyl-N-acetylgalactosamine 1-phosphate) : gneA, a new locus, is associated with UDP-N-acetylglucosamine 4-epimerase activity The resistance spectrum to bacteriophage 43T of different Bacillus subtilis 168/W23 strains hybrid for wall teichoic acids suggested that poly(3-O-~-D-glucopyranosyl-~-acety~galac~osam~e 1-phosphate), a so-called minor teichoic acid of strain 168, forms part of the receptor for this phage, and a serologically related group of phages. A representative sample of 25 mutants specifically resistant to 43T, obtained from a mutagenized culture by direct selection, were all found to have a greatly reduced galactosamine content. Relevant mutations in these strains were shown by PBSl transduction and transformation to belong to two linkage groups; a minority, associated with an atypical colony morphology, were localized between sacA and purA, whereas the majority mapped between gtaB and tagBl (formerly tag-1), a region containing all known genes involved in the synthesis of the major wall teichoic acid, poly(glycero1 phosphate). The former mutations mapped in a new locus, gneA, characterized by a deficiency in UDP-N-acetylglucosamine 4-epimerase, while the latter ones, as well as the previously identified pha-3 (Estrela et al., 1986, Journal of General Microbiology 132,411-415), map in a locus named gga. They are likely to affect membrane-bound enzymes involved in the synthesis of the galactosaminecontaining teichoic acid. A possible biological role of this polymer is discussed. IntroductionCell walls of many Gram-positive bacteria contain relatively large amounts of anionic polymers (Baddiley, 1970; Archi bald, 1974). In Bacillus subtilis 168, several such polymers have been identified (Burger & Glaser, 1964;Shibaev et al., 1973); the phosphate-rich poly(g1y-cerol phosphate) [poly(groP)], glucosylated poly(groP) and poly (3-O-fl-D-glucopyranosyl-N-ace tylgalac tosamine 1-phosphate) [poly(Glc-GalNAc 1-P)], present under conditions of saturating phosphate, are replaced by teichuronic acid (Janczura et al., 1961 ;Ward, 1981) Abbreviations: DMAB, p-dimethylaminobenzaldehyde; GalN, galactosamine; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; MMC, mitomycin C ; MNNG, N-methyl-N'-nitro-Nnitrosoguanidine; ND, nephelometric density; 43TR, 43T resistant; 43TS, 43T sensitive; poly(G1c-GalNAc 1-P), pOly(3-O-P-D-glUCOpyranosyl-N-acetylgalactosamine 1-phosphate) ; poly(groP), poly(glycero1 phosphate).under phosphate-limiting growth conditions. Genetic analysis of conditional lethal mutants deficient in poly(groP) synthesis (Boylan et al., 1972;Karamata et al., 1972;Pooley et al., 1987;Briehl et al., 1989;, and in particular the more recent nucleotide sequencing studies (Honeyman & Stewart, 1989;Mauel et al., 1991) have led to the identification of six genes likely ...

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“…CDPglycerol then serves as a principal precursor required for biosynthesis of poly(glycerol phosphate), the major teichoic acid found in the bacterial cell wall (1,2). GCT appears to be a member of a cytidylyltransferase family that includes CTP: phosphocholine cytidylyltransferases (CCT), a key regulatory enzyme in the CDP-choline pathway for phosphatidylcholine biosynthesis in higher eukaryotes (3)(4)(5).…”

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Identification of Functional Conserved Residues of CTP:glycerol-3-phosphate Cytidylyltransferase

Park¹,

Gee²,

Sanker³

et al. 1997

Journal of Biological Chemistry

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The CTP:glycerol-3-phosphate cytidylyltransferase (GCT) of Bacillus subtilis has been shown to be similar in primary structure to the CTP:phosphocholine cytidylyltransferases of several organisms. To identify the residues of this cytidylyltransferase family that function in catalysis, the conserved hydrophilic amino acid residues plus a conserved tryptophan of the GCT were mutated to alanine. The most dramatic losses in activity occurred with H14A and H17A; these histidine residues are part of an HXGH sequence similar to that found in class I aminoacyl-tRNA synthetases. The k cat values for H14A and H17A were decreased by factors of 5 ؋ 10 ؊5 and 4 ؋ 10 ؊4 , respectively, with no significant change in K m values. Asp-11, which is found near the HXGH sequence in the cytidylyltransferases but not aminoacyltRNA synthetases, was also important for activity, with the D11A mutation decreasing activity by a factor of 2 ؋ 10 ؊3 . Several residues found in the sequence RTEGISTT, a signature sequence for this cytidylyltransferase family, as well as other isolated residues were also shown to be important for activity, with k cat values decreasing by factors of 0.14 -4 ؋ 10 ؊4 . The K m values of three mutant enzymes, D38A, W74A, and D94A, for both CTP and glycerol-3-phosphate were 6 -130-fold higher than that of the wild-type enzyme. Mutant enzymes were analyzed by two-dimensional NMR to determine if the overall structures of the enzymes were intact. One of the mutant enzymes, D66A, was defective in overall structure, but several of the others, including H14A and H17A, were not. These results indicate that His-14 and His-17 play a role in catalysis and suggest that their role is similar to the role of the His residues in the HXGH sequence in class I aminoacyl-tRNA synthetases, i.e. to stabilize a pentacoordinate transition state.The CTP:glycerol-3-phosphate cytidylyltransferase (GCT) 1 from Bacillus subtilis catalyzes the formation of CDP-glycerol and pyrophosphate from CTP and glycerol-3-phosphate. CDPglycerol then serves as a principal precursor required for biosynthesis of poly(glycerol phosphate), the major teichoic acid found in the bacterial cell wall (1, 2). GCT appears to be a member of a cytidylyltransferase family that includes CTP: phosphocholine cytidylyltransferases (CCT), a key regulatory enzyme in the CDP-choline pathway for phosphatidylcholine biosynthesis in higher eukaryotes (3-5). Fig. 1 shows a comparison of the deduced amino acid sequences of several cytidylyltransferases: presumed GCTs from Staphylococcus aureus and Streptomyces wedmorensis, GCT from B. subtilis, CCTs from a variety of organisms, and ethanolamine phosphate cytidylyltransferase (ECT) from yeast. This comparison reveals a number of residues that are conserved among these three enzymes (6). These conserved amino acids are contained within the catalytic core of the CCTs (7). Conservation of these amino acids suggests that they may be required for functions such as catalysis, recognition of substrates, structural integrity, or association ...

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A conditional-lethal mutant of Bacillus subtilis 168 with a thermosensitive glycerol-3-phosphate cytidylyltransferase, an enzyme specific for the synthesis of the major wall teichoic acid

Cited by 66 publications

References 34 publications

Analysis of Bacillus subtilis tag gene expression using transcriptional fusions

Analysis of Bacillus subtilis tag gene expression using transcriptional fusions

Genetic and biochemical characterization of Bacillus subtilis 168 mutants specifically blocked in the synthesis of the teichoic acid poly(3-O- -D-glucopyranosyl-N-acetylgalactosamine 1-phosphate): gneA, a new locus, is associated with UDP-N-acetylglucosamine 4-epimerase activity

Identification of Functional Conserved Residues of CTP:glycerol-3-phosphate Cytidylyltransferase

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