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
Summary: A biochemical analysis was undertaken of thermosensitive mutants of Bacillus subtilis 168 harbouring mutations in several tag genes, involved in the synthesis of the major wall teichoic acid, poly(glycerol phosphate), poly(groP). Incorporation of a pulse of [2-3H]glycerol into whole cells, following shift to the restrictive growth temperature, was used to assess synthesis of this polymer and to seek evidence of accumulation of a specific precursor. The rate of incorporation into poly(groP) was strongly decreased in all mutants; glycerol uptake was diminished by 80% or more for a strain harbouring mutation tagB1 (formerly tag-1) and one bearing tagD11 (formerly tag-11). The pool of CDP-glycerol (CDP-gro), a specific precursor of poly(groP), was increased, relative to the wild-type, for all mutations except tagD11, where the pool of CDP-gro was reduced. Cytoplasmic extracts, assayed at the permissive temperature for glycerol-3-phosphate cytidylyltransferase (gro-PCT), the enzyme synthesizing CDP-gro, revealed wild-type activities for all mutations except tagD11. Gro-PCT activity in the latter strain was 100-fold lower and, unlike that in all other mutant strains, highly thermolabile. This thermosensitivity suggests that tagD encodes gro-PCT. The identification, in a gene encoding a poly(groP)-specific enzyme, of a mutation conferring a thermosensitive growth phenotype renders explicit the conclusion that synthesis of this teichoic acid is essential for the growth of B. subtilis.
AndersonR. G.,
DouglasL. J.,
HusseyH.,
BaddileyJ.1973; The control of bacterial cell walls. Interaction in the synthesis of nucleotide precursors. Biochemical Journal136871–876
BandurskyR. S.,
AxelrodB.1952; The chromatographic identification of some biologically important phosphate esters. Journal of Biological Chemistry193405–410
BoylanR. J.,
MendelsonN. H.1969; Initial characterization of a temperature-sensitive rod-mutant of Bacillus subtilis
. Journal of Bacteriology1001316–1321
BoylanR. J.,
MendelsonN. H.,
BrooksD.,
YoungF. E.1972; Regulation of the bacterial cell wall: analysis of a mutant of Bacillus subtilis defective in biosynthesis of teichoic acids. Journal of Bacteriology110281–290
BradfordM. M.1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry72248–254
BrandtC.,
KaramataD.1987; Thermosensitive Bacillus subtilis mutants which lyse at the non-permissive temperature. Journal of General Microbiology1331159–1170
BriehlM.,
PooleyH. M.,
KaramataD.1989; Mutants of Bacillus subtilis 168 thermosensitive for growth and wall teichoic acid synthesis. Journal of General Microbiology1351325–1334
BrooksD.,
MaysL. L.,
HatefiY.,
YoungF. E.1971; Glucosylation of teichoic acid: solubilization and partial characterization of the uridine diphosphoglucose:polyglycerolteichoic acid glucosyl transferase from membranes of Bacillus subtilis
. Journal of Bacteriology107223–229
GlaserL.1965; The synthesis of teichoic acids. IV. On the regulation of cytidine 5’-diphosphateglycerol concentration. Biochimica et Biophysica Ada1016–15
HoneymanA. L.,
StewartG. C.1988; Identification of the protein encoded by rodC, a cell division gene from Bacillus subtilis
. Molecular Microbiology2735–741
KaramataD.,
GrossJ. D.1970; Isolation and genetic analysis of temperature-sensitive mutants of Bacillus subtilis defective in DNA synthesis. Molecular and General Genetics108277–287
KaramataD.,
PooleyH. M.,
MonodM.1987; Expression of heterologous genes for wall teichoic acid in Bacillus subtilis 168. Molecular and General Genetics20773–81
MauëlC.,
YoungM.,
MargotPh.,
KaramataD.1989; The essential nature of teichoic acids in Bacillus subtilis as revealed by insertional mutagenesis. Molecular and General Genetics215388–394
MauëlC.,
YoungM.,
KaramataD.1991; Genes concerned with synthesis of poly(glycerol phosphate), the essential teichoic acid in Bacillus subtilis strain 168, are organized in two divergent transcription units. Journal of General Microbiology137929–941
PooleyH. M.,
PaschoudD.,
KaramataD.1987; The gtaB marker in Bacillus subtilis 168 is associated with a deficiency in UDP-glucose pyrophosphorylase. Journal of General Microbiology1333481–3493
RogersH. J.,
McConnellM.,
BurdettI. D. J.1970; The isolation and characterization of mutants of Bacillus subtilis and Bacillus licheniformis with disturbed morphology and cell division. Journal of General Microbiology61155–171
RogersH. J.,
McConnellM.,
HughesR. C.1971; The chemistry of the cell walls of rod mutants of Bacillus subtilis
. Journal of General Microbiology66297–308
RogersH. J.,
ThurmanP. F.,
TaylorC.,
ReeveJ. N.1974; Mucopeptide synthesis by rod mutants of Bacillus subtilis
. Journal of General Microbiology85335–350
RosenbergerR. F.1976; Control of teichoic and teichuronic acid biosynthesis in Bacillus subtilis 168 trp. Evidence for repression of enzyme synthesis and inhibition of enzyme activity. Biochimica et Biophysica Acta428516–524
RotenC.-A. H.,
BrandtC.,
KaramataD.1991; Genes involved in /weso-diaminopimelate synthesis in Bacillus subtilis: identification of the gene encoding aspartokinase I. Journal of General Microbiology137951–962
SattaG.,
SchitoG. C.,
MeloniG. A.1969; Transizione bastoncinosfera in un ceppo di Klebsiella pneumoniae. Ultrastruttura delle forme coccoidie tipiche ed anormi. Atti del XV° Congresso Nazionale di Microbiologia Torina-Saint Vincent2247–253
ShawD. R. D.,
MirelmanD.,
ChatterjeeA. N.,
ParkJ. T.1970; Ribitol teichoic acid synthesis in bacteriophage-resistant mutants of Staphvlococcus aureus H*. Journal of Biological Chemistry195101–5106
ShiflettM. A.,
BrooksD.,
YoungF. E.1977; Cell wall and morphological changes induced by temperature shift in Bacillus subtilis cell wall mutants. Journal of Bacteriology132681–690
YoungF. E.1967; Requirement of glucosylated teichoic acid for adsorption of phage in Bacillus subtilis 168. Proceedings of the National Academy of Sciences of the United States of America582377–2384
YoungF. E.,
SmithC.,
ReillyB. E.1969; Chromosomal location of genes regulating resistance to bacteriophage in Bacillus subtilis
. Journal of Bacteriology981087–1097
YoungM.,
MauëlC.,
MargotPh.,
KaramataD.1989; Pseudo-allelic relationship between non-homologous genes concerned with biosynthesis of polyglycerol phosphate and polyribitol phosphate teichoic acids in Bacillus subtilis strains 168 and W23. Molecular Microbiology31805–1812
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