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

Summary A 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.     

Changes in wall teichoic acid during the rod-sphere transition of Bacillus subtilis 168.

Wall teichoic acid (WTA) is essential for the growth of Bacillus subtilis 168. To clarify the function of this polymer, the WTAs of strains 168, 104 rodB1, and 113 tagF1 (rodC1) grown at 32 and 42 degrees C were characterized. At the restrictive temperature, the rodB1 and tagF1 (rodC1) mutants undergo a rod-to-sphere transition that is correlated with changes in the WTA content of the cell wall. The amount of WTA decreased 33% in strain 104 rodB1 and 84% in strain 113 tagF1 (rodC1) when they were grown at the restrictive temperature. The extent of alpha-D-glucosylation (0.84) was not affected by growth at the higher temperature in these strains. The degree of D-alanylation decreased from 0.22 to 0.10 in the rodB1 mutant but remained constant (0.12) in the tagF1 (rodC1) mutant at both temperatures. Under these conditions, the degree of D-alanylation in the parent strain decreased from 0.27 to 0.21. The chain lengths of WTA in strains 168 and 104 rodB1 grown at both temperatures were approximately 53 residues, with a range of 45 to 60. In contrast, although the chain length of WTA from the tagF1 (rodC1) mutant at 32 degrees C was similar to that of strains 168 and 104 rodB1, it was approximately eight residues at the restrictive temperature. The results suggested that the rodB1 mutant is partially deficient in completed poly(glycerophosphate) chains. The precise biochemical defect in this mutant remains to be determined. The results for strain 113 tagF1(rodC1) are consistent with the temperature-sensitive defect in the CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase (H. M. Pooley, F.-X. Abellan, and D. Karamata, J. Bacteriol. 174:646-649, 1992).     

Molecular analysis of the tagF gene, encoding CDP-Glycerol:Poly(glycerophosphate) glycerophosphotransferase of Staphylococcus epidermidis ATCC 14990

Staphylococcus epidermidis ATCC 14990 produces a wall-associated glycerol teichoic acid which is chemically identical to the major wall-associated teichoic acid of Bacillus subtilis 168. The S. epidermidis tagF gene was cloned from genomic DNA and sequenced. When introduced on a plasmid vector into B. subtilis 1A486 carrying the conditionally lethal temperature-sensitive mutation tagF1 (rodC1), it expressed an 85-kDa protein which allowed colonies to grow at the restrictive temperature. This showed that the cloned S. epidermidis gene encodes a functional CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase. An amino acid substitution at residue 616 in the recombinant TagF protein eliminated complementation. Unlike B. subtilis, where the tagF gene is part of the tagDEF operon, the tagF gene of S. epidermidis is not linked to any other tag genes. We attempted to disrupt the chromosomal tagF gene in S. epidermidis TU3298 by directed integration of a temperature-sensitive plasmid but this failed, whereas a control plasmid containing the 5' end of tagF on a similarly sized DNA fragment was able to integrate. This suggests that the tagF gene is essential and that the TagF and other enzymes involved in teichoic acid biosynthesis could be targets for new antistaphylococcal drugs.     

Identification of the protein encoded by rodC, a cell division gene from Bacillus subtilis

The rodC1 mutation of Bacillus subtilis is a temperature-sensitive marker which affects the orientation of the plane of cell division. We have cloned the rodC gene and have localized the site of the rodC1 lesion. To identify the rodC gene product, we have subjected several plasmid clones containing B. subtilis chromosomal DNA from the rodC region to maxicell analysis in Escherichia coli. A 68 kiloDalton protein has been identified as the rodC gene product. This is the initial cloning of a cell division gene and the identification of its product from B. subtilis. The rodC gene has also been implicated as being directly associated with the synthesis of glycerol teichoic acid.     

Studies of the genetics, function, and kinetic mechanism of TagE, the wall teichoic acid glycosyltransferase in Bacillus subtilis 168

The biosynthetic enzymes involved in wall teichoic acid biogenesis in gram-positive bacteria have been the subject of renewed investigation in recent years with the benefit of modern tools of biochemistry and genetics. Nevertheless, there have been only limited investigations into the enzymes that glycosylate wall teichoic acid. Decades-old experiments in the model gram-positive bacterium, Bacillus subtilis 168, using phage-resistant mutants implicated tagE (also called gtaA and rodD) as the gene coding for the wall teichoic acid glycosyltransferase. This study and others have provided only indirect evidence to support a role for TagE in wall teichoic acid glycosylation. In this work, we showed that deletion of tagE resulted in the loss of α-glucose at the C-2 position of glycerol in the poly(glycerol phosphate) polymer backbone. We also reported the first kinetic characterization of pure, recombinant wall teichoic acid glycosyltransferase using clean synthetic substrates. We investigated the substrate specificity of TagE using a wide variety of acceptor substrates and found that the enzyme had a strong kinetic preference for the transfer of glucose from UDP-glucose to glycerol phosphate in polymeric form. Further, we showed that the enzyme recognized its polymeric (and repetitive) substrate with a sequential kinetic mechanism. This work provides direct evidence that TagE is the wall teichoic acid glycosyltransferase in B. subtilis 168 and provides a strong basis for further studies of the mechanism of wall teichoic acid glycosylation, a largely uncharted aspect of wall teichoic acid biogenesis.     

Organization of teichoic acid in the cell wall of Bacillus subtilis.

The phytohemagglutinin, concanavalin A (Con A), interacts specifically and reversibly with the polyglucosyl glycerol phosphate teichoic acid of Bacillus subtilis 168 cell walls. Advantage has been taken of this interaction to examine the organization of the surface teichoic acid at the ultrastructural level. Con A-treated whole cells and cell walls contain an irregular, fluffy layer 25 to 60 nm thick which is absent in untreated or alpha-methyl glucoside-treated preparations. This discontinuous layer is present only on the outer profile of Con-A-treated cell walls. The surface teichoic acid is proposed to be oriented perpendicular to the long axis of the cell. Fixation and embedment for electron microscopy result in condensation of this layer which then contributes to the stainable portion of the wall. Con A treatment binds adjacent teichoic acid molecules in their native configuration producing the irregular, fluffy layer visualized.     

Distribution of teichoic acid in the cell wall of Bacillus subtilis.

Hydrolysis of the cell wall of Bacillus subtilis 168 by autolysins or lysozyme resulted in the exposure of glucosylated teichoic acid molecules as evidenced by increased precipitation of [14C] concanavalin A. The number of concanavalin A-reactive sites increased significantly after only limited enzymatic digestion of the walls. Quantitative analyses of [14C] concanavalin A-treated wall or wall hydrolysate complexes indicate that approximately one-half of the teichoic acid molecules are surface-exposed, whereas the remainder are probably embedded within the peptidoglycan matrix. Treatment of the cell walls with sodium dodecyl sulfate or Triton X-100 did not result in new concanavalin A-reactive sites. Partial autolysis diminished the ability of the cell walls to adsorb bacteriophage phi25. Fluorescein-labeled concanavalin A bound intensely over the entire surface of growing B. subtilis 168 cells, suggesting that teichoic acid molecules are located on the total solvent-exposed surface area of the bacteria.     

Control of synthesis of wall teichoic acid in phosphate-starved cultures of Bacillus subtilis W23

CDP-glycerol pyrophosphorylase, CDP-ribitol pyrophosphorylase and poly(ribitol phosphate) synthetase activities have been measured in cultures of Bacillus subtilis W23 as they became phosphate-starved either in batch culture or during changeover from potassium limitation to phosphate limitation in a chemostat. The results indicated that repression of synthesis of all three enzymes occurred at the onset of phosphate starvation and that this was accompanied by inhibition of inactivation of CDP-glycerol pyrophosphorylase and poly(ribitol phosphate) synthetase. These results show that the initial response to phosphate starvation involves more than inhibition of one enzyme as proposed by Glaser and Loewy [Glaser L. and Loewy, A. (1979) J. Biol. Chem. 254, 2184-2186]. Synthesis of both linkage unit and poly(ribitol phosphate) are inhibited independently.     

The tagGH operon of Bacillus subtilis 168 encodes a two-component ABC transporter involved in the metabolism of two wall teichoic acids

We report the nucleotide sequence and the characterization of the Bacillus subtilis tagGH operon. The latter is controlled by a σ^A-dependent promoter and situated in the 308° chromosomal region which contains genes involved in teichoic acid biosynthesis. TagG is a hydrophobic 32.2 kDa protein which resembles integral membrane proteins belonging to polymerexport systems of Gram-negative bacteria. Gene tagH encodes a 59.9 kDa protein whose N-moiety contains the ATP-binding motif and shares extensive homology with a number of ATP-binding proteins, particularly with those associated with the transport of capsular polysaccharides and O-antigens. That the tagGH operon is essential for cell growth was established by the failure to inactivate tagG and the 5′ -moiety of tagH by insertional mutagenesis. During limited tagGH expression, cells exhibited a cocoid morphology while their walls contained reduced amounts of phosphate as well as galactosamine. These observations, revealing impaired metabolism of both wall teichoic acids of B. subtilis 168, i.e. poly(glycerol phosphate), and poly(glucose galactosamine phosphate), combined with sequence homologies, suggest that TagG and TagH are involved in the translocation through the cytoplasmic membrane of the latter teichoic acids or their precursors.     

Cell wall teichoic acid as a reserve phosphate source in Bacillus subtilis.

Although exponential growth of Bacillus subtilis 168 in a phosphate-limited medium halted with the exhaustion of inorganic phosphate, the bacteria continued to grow at a slower rate for a further 3 to 4 h at 37 degrees C. This postexponential growth in the absence of an exogenous phosphate supply was accompanied by a loss of teichoic acid from the cell walls of the bacteria. Quantitative analysis of walls and culture fluids showed that the phosphate loss from the walls could not be accounted for by an increase in phosphate-containing compounds in the medium, which implied that the cells were using their own wall teichoic acids to supply phosphate necessary for growth. Addition of exogenous teichoic acid to phosphate-starved cultures resulted in stimulation of growth and in the simultaneous disappearance of teichoic acid phosphate from the medium. It is proposed that teichoic acids, which can contain more than 30% of the total phosphorus of exponential-phase cells, can be used as a reserve phosphate source when the bacteria are starved for inorganic phosphate.     

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

The resistance spectrum to bacteriophage phi 3T of different Bacillus subtilis 168/W23 strains hybrid for wall teichoic acids suggested that poly(3-O-beta-D-glucopyranosyl-N-acetylgalactosamine 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 phi 3T, 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 PBS1 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 tagB1 (formerly tag-1), a region containing all known genes involved in the synthesis of the major wall teichoic acid, poly(glycerol 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 is a locus named gga. They are likely to affect membrane-bound enzymes involved in the synthesis of the galactosamine-containing teichoic acid. A possible biological role of this polymer is discussed.     

Biosynthesis of the wall teichoic acid in Bacillus licheniformis

1. The biosynthesis of the wall teichoic acid, poly(glycerol phosphate glucose), has been studied with a particulate membrane preparation from Bacillus licheniformis A.T.C.C. 9945. The precursor CDP-glycerol supplies glycerol phosphate residues, whereas UDP-glucose supplies only glucose to the repeating structure of the polymer. 2. Synthesis proceeds through polyprenol phosphate derivatives, and chemical studies and pulse-labelling techniques show that the first intermediate is the phosphodiester, glucose polyprenol monophosphate. CDP-glycerol donates a glycerol phosphate residue to this to give a second intermediate, (glycerol phosphate glucose phosphate) polyprenol. 3. The glucose residue in the lipid intermediates has the beta configuration, and chain extension in the synthesis of polymer occurs by transglycosylation with inversion of anomeric configuration at two stages.     

Membrane synthesis in synchronous cultures of Bacillus subtilis 168

Synthesis of bacterial membranes has been investigated in Bacillus subtilis by examining incorporation of amino acids and glycerol into the protein and lipid of membranes of synchronous cultures. A simple reproducible fractionation scheme divides cellular proteins into three classes (i) truly cytoplasmic, (ii) loosely membrane bound, released by chelating agents, and (iii) tightly membrane bound. These comprise approximately 75, 10, and 15%, respectively, of cellular proteins in this organism. Incorporation of radioactivity into these fractions, using steady-state and pulse labeling has been followed during the cell cycle. Cytoplasmic proteins and the loosely membrane-bound proteins are labeled at an exponential rate throughout the cell cycle. The membrane fraction is labeled discontinuously in the cell cycle, with periods of rapid synthesis over the latter part of the cycle and a period with no net synthesis during the early part of the cycle. Pulse labeling indicates that synthesis of membrane occurs at a linear rate that doubles at a fixed time in each cycle, which coincides with the period of zero net synthesis. Rates of membrane synthesis measured by pulse labeling during the period of rapid membrane synthesis are significantly less than indicated by steady-state labeling. These discrepancies are consistent with the hypothesis that during the cell cycle certain proteins are added to the membrane from the cytoplasm and that during the period of zero net synthesis there is an efflux of proteins from the membrane. Evidence in favor of this has been presented. The activity of succinic dehydrogenase (a representative of class c) varies in a step-wise manner with periods of rapid increase, approximately coincident with bursts of membrane protein synthesis, alternating with periods without any increase in activity. The activities of malate dehydrogenase (class a) and reduced nicotinamide adenine dinucleotide dehydrogenase (class b) increased throughout the cell cycle. Phospholipid synthesis is continuous throughout the cell cycle.     

The major and minor wall teichoic acids prevent the sidewall localization of vegetative DL-endopeptidase LytF in Bacillus subtilis

Cell separation in Bacillus subtilis depends on specific activities of DL-endopeptidases CwlS, LytF and LytE. Immunofluorescence microscopy (IFM) indicated that the localization of LytF depended on its N-terminal LysM domain. In addition, we revealed that the LysM domain efficiently binds to peptidoglycan (PG) prepared by chemically removing wall teichoic acids (WTAs) from the B. subtilis cell wall. Moreover, increasing amounts of the LysM domain bound to TagB- or TagO-depleted cell walls. These results strongly suggested that the LysM domain specifically binds to PG, and that the binding may be prevented by WTAs. IFM with TagB-, TagF- or TagO-reduced cells indicated that LytF-6xFLAG was observed not only at cell separation site and poles but also as a helical pattern along the sidewall. Moreover, we found that LytF was localizable on the whole cell surface in TagB-, TagF- or TagO-depleted cells. These results strongly suggest that WTAs inhibit the sidewall localization of LytF. Furthermore, the helical LytF localization was observed on the lateral cell surface in MreB-depleted cells, suggesting that cell wall modification by WTAs along the sidewall might be governed by an actin-like cytoskeleton homologue, MreB.