Harold M. Pooley

Bacillus subtilis α-Phosphoglucomutase Is Required for Normal Cell Morphology and Biofilm Formation

ABSTRACT Mutations designated gtaC and gtaE that affect α-phosphoglucomutase activity required for interconversion of glucose 6-phosphate and α-glucose 1-phosphate were mapped to the Bacillus subtilis pgcA (yhxB) gene. Backcrossing of the two mutations into the 168 reference strain was accompanied by impaired α-phosphoglucomutase activity in the soluble cell extract fraction, altered colony and cell morphology, and resistance to phages φ29 and ρ11. Altered cell morphology, reversible by additional magnesium ions, may be correlated with a deficiency in the membrane glycolipid. The deficiency in biofilm formation in gtaC and gtaE mutants may be attributed to an inability to synthesize UDP-glucose, an important intermediate in a number of cell envelope biosynthetic processes.

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

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.

Expression of heterologous genes for wall teichoic acid in Bacillus subtilis 168.

SummaryA localized region of low DNA sequence homology was revealed in two strains of Bacillus subtilis by a specific 100-fold reduction in transformation by W23 DNA of the tag1 locus, a teichoic acid marker of strain 168. Fifty nine rare recombinants, hybrid at this locus, had all acquired donor-specific phage resistance characters, while losing those specific to the 168 recipient. Chemical analysis of isolated cell walls showed that these modifications are associated with major changes in the wall teichoic acids. Genetic analysis demonstrated that determinants for the ribitol phosphate polymer of strain W23 had been transferred to 168, replacing those for the glycerol phosphate polymer in the recipient. All W23 genes coding for poly(ribitol phosphate) in the hybrids and those specifying anionic wall polymers in strain 168 are clustered near hisA. In addition to tag1, the region exchanged extends just beyond gtaA in some hybrids, whereas in others it may include the more distant gtaB marker, encompassing a region sufficient to contain at least 20 average-sized genes. Surface growth, flagellation, transformability and sporulation all appeared normal in hybrids examined. Recombinants without a major wall teichoic acid from either strain were not found, suggesting that an integral transfer of genes for poly(ribitol phosphate) from W23 had occurred in all hybrids isolated. We interpret these results as indicating an essential role for anionic wall polymers in the growth of B. subtillis.

Characterization of a Bacillus subtilis Thermosensitive Teichoic Acid-Deficient Mutant: Gene mnaA (yvyH) Encodes the UDP-N-Acetylglucosamine 2-Epimerase

The Bacillus subtilis thermosensitive mutant ts-21 bears two C-G-->T-A transitions in the mnaA gene. At the nonpermissive temperature it is characterized by coccoid cell morphology and reduced cell wall phosphate content. MnaA converts UDP-N-acetylglucosamine into UDP-N-acetylmannosamine, a precursor of the teichoic acid linkage unit.

Mutants of Bacillus subtilis 168 thermosensitive for growth and wall teichoic acid synthesis

A protocol designed to isolate mutants with thermosensitive (Ts) synthesis of the bacteriophage o29 receptor, which includes the major wall teichoic acid in Bacillus subtilis 168, yielded a significant enrichment for Ts growth mutants among colonies surviving o29 treatment. Nine mutants, Ts for both o29 susceptibility and cell growth, harboured mutations which were located in the tag locus by PBS1 transduction and recombination index with the tag-1 marker. Physical mapping revealed that they were distributed on a segment of more than 4 kb. Chemical analysis of cell walls showed a marked reduction in phosphate relative to diaminopimelic acid content of all mutants at the non-permissive temperature. Differences between mutants were correlated with the distribution of tag mutations on the genetic map. We conclude (i) that the newly identified markers affect several genes involved in poly(glycerol phosphate) synthesis, and (ii) that on phosphate-rich media, cell growth cannot occur without the synthesis of the latter polymer.

Incorporation of [2-3H]glycerol into cell surface components of Bacillus subtilis 168 and thermosensitive mutants affected in wall teichoic acid synthesis: effect of tunicamycin.

A method is described for measuring the synthesis of poly(glycerol phosphate) [poly(groP)], the major wall teichoic acid (WTA), lipoteichoic acid (LTA) and phospholipid (P-lipid), through fractionation of [2-3H]glycerol ([2-3H]gro)-labelled Bacillus subtilis cells. When cultures of certain temperature-sensitive mutants defective in one of several tag genes, encoding enzymes involved in WTA synthesis, were transferred to the restrictive temperature, the synthesis of WTA underwent a specific, immediate, block, while that of LTA or P-lipid proceeded unimpeded. These results, in addition to confirming the role of tag genes, demonstrated, reciprocally, the specificity of the fractionation procedure used to distinguish label in WTA from that in LTA or P-lipid. Results of analysis of other, less severely affected, tag-deficient mutants, as well as of another genetically unrelated mutant developing comparable morphological phenotypes in non-permissive conditions, are discussed in relation to a possible mechanism generating the latter phenotype. Fractionation of B. subtilis 168 cells labelled either with [2-3H]gro or with [1-14C]N-acetylglucosamine, to which tunicamycin was added at 0.5 microg ml(-1) (the MIC) revealed a specific and marked inhibition of poly(groP) as well as of poly(3-O-beta-D-glucopyranosyl-N-acetylgalactosamine 1-phosphate), the minor WTA. However, for 60 min at least, the syntheses of PG, LTA and P-lipid were barely affected.

Chapter 9 Teichoic acid synthesis in Bacillus subtilis: genetic organization and biological roles

Publisher Summary This chapter discusses the genetic organization and biological roles of teichoic acid synthesis in Bacillus subtilis. The structural characterization of anionic cell wall polymers followed the discovery of their widespread occurrence among Gram-positive organisms. Attempts to understand the biological roles of Wall Teichoic Acid (WTA) followed their discovery, and the realization of their widespread distribution among the Gram-positive bacteria. Their presence in the cell wall was judged to be important, for the negative surface charge, for the binding of divalent cations, for specific affinities for cell autolysins and bacteriophages. Their role as a phosphate reserve and as a component of the cell permeability barrier was also proposed. Until recently, the question of the necessity of WTA synthesis for cell growth had, surprisingly, not been addressed directly. With hindsight, this may be explained by the faculty of certain bacilli to produce a substitute polymer, TU, thereby showing that WTA was not, strictly speaking, indispensable.

Wall Teichoic Acid, Peptidoglycan Synthesis and Morphogenesis in Bacillus Subtilis

Ever since morphological mutants in several rod-shaped bacteria were isolated (Boylan and Mendelson, 1969; Rogers et al., 1970; Satta et al., 1969), the question of cell shape maintenance has been and remains a central one. We examine a possible role in cell morphogenesis of the interdependence of the syntheses of peptidoglycan and other cell wall components.