Interaction of Concanavalin A with the Cell Wall of Bacillus subtilis

Interactions between concanavalin A and cell wall digests of Bacillus subtilis 168 resulted in insoluble complexes as observed by double gel diffusion, turbidity, and analysis of the precipitate. The macromolecular constituent of the cell walls complexing with concanavalin A was the polyglucosylglycerol phosphate teichoic acid. The complex exhibited two pH optima: 3.1 and 7.4. The complex could be dissociated by saccharides which bind to concanavalin A. In contrast to concanavalin A-neutral polysaccharide complexes, formation of the concanavalin A-wall complex was inhibited by salts. It was subsequently shown that salts induce conformational changes in cell wall digests. The data suggested that for complex formation to occur a rigid rod conformation in the glucosylated teichoic acid is probably necessary. Concanavalin A can be used as a probe to study structural features of bacterial cell walls.     

Involvement of etfA gene during CaCO3 precipitation in Bacillus subtilis biofilm

The eftA gene in Bacillus subtilis has been suggested to be involved in the oxidation/reduction reactions during fatty acid metabolism. Interestingly etfA deletion in B. subtilis results in impairment in CaCO₃ precipitation on the biofilm. Comparisons between the wild type B. subtilis 168 and its etfA mutant during in vitro CaCO₃ crystal precipitation (calcite) revealed changes in phospholipids membrane composition with accumulation of up to 10% of anteiso-C_17:0 and 11% iso-C_17:0 long fatty acids. Ca²⁺ nucleation sites such as dipicolinic acid and teichoic acids seem to contribute to the CaCO₃ precipitation. etfA mutant strain showed up to 40% less dipicolinic acid accumulation compared with B. subtilis 168, while a B. subtilis mutant impaired in teichoic acids synthesis was unable to precipitate CaCO₃. In addition, B. subtilis etfA mutant exhibited acidity production leading to atypical flagella formation and inducing extensive lateral growth on the biofilm when grown on 1.4% agar. From the ecological point of view, this study shows a number of physiological aspects that are involved in CaCO₃ organomineralization on biofilms.     

Monovalent concanavalin A

The preparation of monovalent concanavalin A was achieved by incubating metal-free concanavalin A with trypsin at room temperature for 50 hrs. The digest was subjected to affinity chromatography on a column of ovomucoid-agarose to remove the trypsin and subsequent chromatography on Bio-Gel P-150 to resolve the monovalent fragment. Monovalent concanavalin A bound Mn²⁺, Ca²⁺ and p-nitrophenyl-α-D-mannopyranoside as determined by ultraviolet difference spectroscopy. The modified protein would not precipitate glycogen or agglutinate $\text{Bacillus subtilis}$ cells. The fragment did, however, prevent the agglutination of $\text{B. subtilis}$ by native concanavalin A. Preparation of monovalent concanavalin A could not be achieved unless metals were first removed from the native protein.     

Polyelectrolyte Nature of Bacterial Teichoic Acids

Several physicochemical properties of the teichoic acid of Bacillus subtilis 168 have been determined. The teichoic acid partial specific volume was found to be 0.57 ml/g. The apparent weight-average molecular weight of the polymer was 24,800. Sedimentation was strongly dependent on solvent. The sedimentation coefficient of the teichoic acid was found to have a value of s(20.w) (0) = 1.90S. In dilute buffers and distilled water, the teichoic acid possessed a rigid rod or extended conformation. Salts induced a loss of secondary structure in the polymer, resulting in a random coil configuration. Salt-induced structural changes in the teichoic acid were determined by viscosities, ultraviolet difference spectra, and inhibition of precipitation with concanavalin A. Divalent cations such as Mg(2+) had little effect on the teichoic acid structure. The salt-induced structural changes were reversible, as evidenced by return of the original properties upon dialysis of the teichoic acid against water. Sodium chloride inhibited the adsorption of bacteriophage ?25 to B. subtilis cell walls. Teichoic acid conformation may have a significant influence on the physiology of bacteria.     

Restricted turnover of the cell wall ofBacillus subtilis

Cultures ofBacillus subtilis in balanced growth exhibited a constant rate of turnover of peptidoglycan for 2.5–3.5 generations. Turnover was measured by determining the retention of a labeled precursor of peptidoglycan. When fluorescein-conjugated concanavalin A was used to monitor the fate of cell surface teichoic acid, label disappeared from the cylinders more rapidly than from caps and septa. The results suggest that cell wall poles are partially resistant to turnover.     

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.     

Inhibition of DNA-induced transformation by concanavalin A in Bacillus subtilis.

The use of concanavalin A (Con A) as a probe for studying the role of wall teichoic acid in bacterial transformation was investigated. The transformation of lysozyme-treated and untreated competent cultures of $\text{Bacillus}\text{subtilis}$ strain 168 was found to be inhibited by treatment with Con A. The inhibitory action exerted by Con A was concentration-dependent. The minimum Con A concentration necessary to effect a measurable inhibition of transformation was much lower for the lysozyme-treated than for the untreated bacteria. It was postulated that the wall teichoic acid became more exposed as a result of the lysozyme treatment and, hence, was more accessible to Con A binding. The Con A-mediated inhibition was reversible by α-methyl-D-glucoside.     

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.     

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.     

Control of teichoicand teichuronic acid biosynthesis in Bacillus subitlis 168trp−: Evidence for repression of enzyme synthesis and inhibition of enzyme activity

Phosphate starvatiion induced teichuronic acid synthesis in cells of Bacillus subtilis 168trp^? which had previously been grown with excess phophate. This induction was prevented when protein synthesis was inhibited immediately prior to phosphate starvation and under these conditions cells continued to form teichoic acid. The converse was true when phosphate was added to cells previously grown in phosphate-limited chemostat. The increase in teichoic acid synthesis normally following phosphate addition was prevented by chlorampehnicol or amino acid starvation and cells continued to make teichuronic acid. The suggestion that repression of enzyme synthesis is involved in controlling the type of wall polymer made was supported by the low levels of UDP-glucose dehydrogenase found in cells grown with excess phosphate and of CDP-glycerol pyrophosphorylase in phophate-limited cells. The greater amounts of teichoic acid made under phosphate limitation and of teichuronic acid with excess phosphate when protein synthesis was also inhibited indicated that modulation of enzyme activity occurs. Glycerol starvation of a glycerol-requiring mutant did not derepress teichuronic acid synthesis, indicating that glycerol-containing intermediates do not act as repressors.     

Localization and interactions of teichoic acid synthetic enzymes in Bacillus subtilis

The thick wall of gram-positive bacteria is a polymer meshwork composed predominantly of peptidoglycan (PG) and teichoic acids, both of which have a critical function in maintenance of the structural integrity and the shape of the cell. In Bacillus subtilis 168 the major teichoic acid is covalently coupled to PG and is known as wall teichoic acid (WTA). Recently, PG insertion/degradation over the lateral wall has been shown to occur in a helical pattern. However, the spatial organization of WTA assembly and its relationship with cell shape and PG assembly are largely unknown. We have characterized the localization of green fluorescent protein fusions to proteins involved in several steps of WTA synthesis in B. subtilis: TagB, -F, -G, -H, and -O. All of these localized similarly to the inner side of the cytoplasmic membrane, in a pattern strikingly similar to that displayed by probes of nascent PG. Helix-like localization patterns are often attributable to the morphogenic cytoskeletal proteins of the MreB family. However, localization of the Tag proteins did not appear to be substantially affected by single disruption of any of the three MreB homologues of B. subtilis. Bacterial and yeast two-hybrid experiments revealed a complex network of interactions involving TagA, -B, -E, -F, -G, -H, and -O and the cell shape determinants MreC and MreD (encoded by the mreBCD operon and presumably involved in the spatial organization of PG synthesis). Taken together, our results suggest that, in B. subtilis at least, the synthesis and export of WTA precursors are mediated by a large multienzyme complex that may be associated with the PG-synthesizing machinery.     

Poly(glucosylglycerol phosphate) teichoic acid in the walls of Bacillus stearothermophilus B65.

1. Walls of Bacillus stearothermophilus B65 contain a glycerol teichoic acid in which repeating structures consisting of 1-O-alpha-D-glucopyranosylglycerol phosphate are held together by phosphodiester linkage between the glycerol and glucose moieties of adjacent units. 2. The walls are not agglutinated on incubation with concanavalin A, nor does the isolated teichoic acid form a precipitate with this lectin. 3. No evidence was obtained of the presence of the glucosylated (1 leads to 2)-poly(glycerol phosphate) teichoic acid which has previously been reported to occur in walls of this bacterium.     

The essential nature of teichoic acids in Bacillus subtilis as revealed by insertional mutagenesis

Summary A 30 kb DNA segment from the region of the Bacillus subtilis strain 168 chromosome which contains most, if not all, loci specifically involved in teichoic acid biosynthesis, has been cloned. A restriction map was established to which genetic markers were assigned. Four loci, tagA, tagB, gtaA and gtaD, are located on a DNA segment of about 7 kb, whereas the gtaB locus lies some 10 kb distant. The tagA and tagB loci are apparently transcribed independently. Insertional mutagenesis, using integrational plasmids carrying relevant fragments from the tag region, provides strong evidence that biosynthesis of polyglycerol phosphate [poly(groP)], so far largely considered as a dispensable polymer, is in fact essential for growth.     

Teichoicase from Bacillus subtilis Marburg.

The properties of a teichoic acid degrading enzyme (teichoicase) isolated from Bacillus subtilis Marburg are described. The purified enzyme showed phosphodiesterase activity but not phosphomonoesterase activity, and it had an absolute substrate specificity for alpha-glucosylated glycerol teichoic acid, the endogenous cell wall teichoic acid of the enzyme-producing cell. The substrate was degraded by an exo-mechanism yielding the monomer alpha-D-glucose 1 leads to 2 (sn)glycero-3-phosphate. When B. subtilis Marburg was grown in a rich medium, enzyme activity was detected in extracts from sporulating cells. Teichoicase activity was present in a mutant blocked in stage II of the sporulation process but was absent in a mutant blocked in stage O. It was concluded that teichoicase is active on enzyme-producing cells since the reaction product could be detected in their culture supernatant. Attempts to demonstrate analogous enzyme activity in other Bacillus strains failed. The enzyme could be used for the rapid detection of alpha-glucosylated glycerol teichoic acid and for the controlled alteration of native bacterial cell surfaces exhibiting the appropriate structure.     

The distinct PhoPR mediated responses to phosphate limitation in Bacillus subtilis subspecies subtilis and spizizenii stem from differences in wall teichoic acid composition and metabolism

The PhoPR‐mediated response to phosphate limitation (PHO response) in Bacillus subtilis subsp subtilis is amplified and maintained by reducing the level of Lipid V^G composed of poly(glycerol phosphate), a wall teichoic acid (WTA) biosynthetic intermediate that inhibits PhoR autokinase activity. However, the reduction in Lipid V^G level is effected by activated PhoP～P, raising the question of how the PHO response is first initiated. Furthermore, that WTA is composed of poly(ribitol phosphate) in Bacillus subtilis subsp spizizenii prompted an investigation of how the PHO response is regulated in that bacterium. We report that the PHO responses of B. subtilis subsp subtilis and subsp spizizenii are distinct. The PhoR kinases of the two B. subtilis subspecies are functionally equivalent and are activated either by the TagA/TarA or TagB/TarB enzyme product. However, they are inhibited by Lipid V^G composed of poly(glycerol phosphate) but not by Lipid V^R composed of poly(ribitol phosphate). Therefore, the distinctive PHO responses of these B. subtilis subspecies stem from the differential sensitivity of PhoR kinases to the polyol composition of Lipid V and from the genomic organization of WTA biosynthetic genes and the regulation of their expression.     

Restriction of plasmid-mediated transformation in Bacillus subtilis 168.

Summary When plasmids carrying leucine genes of Bacillus subtilis 168 were isolated from a restriction and modification deficient (r^-m^-) strain and used for transformation of a restricting strain B. subtilis 168 leu recE4, the number of transformants was greatly reduced. Transformation of a rec ⁺ strain (transformation by integration of the donor DNA into the chromosome) with the plasmids was not affected irrespective of whether the recipient carried the r⁺ or r^- phenotype. These results show that the plasmid-mediated transformation is subject to the host controlled restriction and suggest that r^-m^- strains should be used for construction of recombinant DNA molecules in B. subtilis 168.     

Teichoic acids of three type strains of the Bacillus subtilis group, Bacillus mojavensis VKM B-2650, Bacillus amyloliquefaciens subsp. amyloliquefaciens VKM B-2582, and Bacillus sonorensis VKM B-2652

Cell walls of three type strains of the Bacillus subtilis group, Bacillus mojavensis VKM B-2650, Bacillus amyloliquefaciens subsp. amyloliquefaciens VKM B-2582, and Bacillus sonorensis VKM B-2652, are characterized by the individual set of teichoic acids. All strains contained 1,3-poly(glycerol phosphates), unsubstituted, acylated with D-alanine, and glycosylated. The latter differ in the nature of the monosaccharide residue. Teichoic acids of B. mojavensis VKM B-2650^T and B. amyloliquefaciens subsp. amyloliquefaciens VKM B-2582^T contained α-glucopyranose, while those of B. sonorensis VKM B-2652^T contained β-glucopyranose and N-acetyl-α-D-glucosamine. Moreover, cell walls of B. mojavensis VKM B-2650^T contained a teichoic acid of poly(glycosylglycerol phosphate) nature with the following structure of the repeating unit: -4)-α-D-α-D-GlcpNAc-(1 → 3)]-Glcp-(1 → 2)-sn-Gro-(3-P-. The type strains have been characterized according to the composition of cell wall sugars and polyols. Application of teichoic acids (set and structure) as chemotaxonomic characteristics is discussed for six type strains of the Bacillus subtilis group. Polymer structures were determined by chemical and NMR spectroscopic techniques.     

Presence and cellular distribution of soybean agglutinin-binding epitopes in two strains of<Emphasis Type="Italic">Bacillus subtilis</Emphasis>

The cellular location ofN-acetylgalactosamine inBacillus subtilis strains 168 and 170 was examined by electron microscopy using gold-conjugated soybean agglutinin (SBA) as a marker. Post-embedding labeling of sectioned material showed SBA-reactive, galactosamine-containing polymers associated with the cell membrane or the cytoplasm of the two strains. This intracellular location was distinct from the concanavalin A-binding epitopes that were located over the cell wall. Labeling of whole cells (native, fixed in glutaraldehyde, or treated with proteinase K, or Tween 20) before negative staining revealed no galactosamine exposed on the surface of strain 168. On the surface of strain 168 some exposed galactosamine terminal residues were detected; their accessibility to SBA increased when Tween 20 or proteinase K was applied.     

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.     

Soil Strain of Bacillus subtilis Harboring a Large Plasmid That Mediates High-Frequency Conjugal Mobilization

The ability of a soil strain of Bacillus subtilis harboring a large plasmid, p19, to mobilize a small staphylococcal plasmid, pUB110, was studied. The latter plasmid was transferred to the recipient cells of Bacillus subtilis168 at a high frequency (about 10^–2 per recipient cell) both on the filter surface and in liquid medium. Mobilization was initiated 40 min after the beginning of the contact between donor and recipient cells.