Division Mutants of Bacillus subtilis: Isolation and PBS1 Transduction of Division-Specific Markers

A procedure for the isolation of Bacillus subtilis mutants that appear to be defective in septum synthesis has been described. Fourteen mutants isolated with this technique were found to be located at four distinct loci on the B. subtilis chromosome. These have been designated divA, divB, divC, and divD. The four mutants in the divA group synthesize septa; however, they do so with a high frequency of error resulting in minicell production. Mapping data were obtained by scoring cotransduction frequencies using PBS1-transducing lysates. Thus, some of the genes apparently involved in septum synthesis and their order on the genome have been established. The results of a study by electron microscope of some of the mutants is also presented.     

Genetic mapping of katA, a locus that affects catalase 1 level in Bacillus subtilis.

Several mutants of Bacillus subtilis deficient in catalase synthesis generated by nitrosoguanidine mutagenesis have been used to map a locus affecting catalase activity. Two- and three-factor bacteriophage PBS1 transductional crosses were used to locate the locus, named katA, between recH and thiA with 98% linkage to thiA at 70 degrees on the B. subtilis genome. The synthesis of catalase 1, found only in vegetative cells, was affected by katA.     

Characteristics of Bacillus stearothermophilus mutants blocked in catabolic function.

With polyacrylamide disc gel electrophoresis and specific staining, it was demonstrated that one mutation involving the alcohol dehydrogenase of a double mutant of Bacillus stearothermophilus 1503 apparently prevented enzyme synthesis, and another lesion in the same organism resulted in synthesis of an inactive form of aconitase. Some properties of the double mutant and two fumarase mutants are discussed in relation to similar mutants derived from Bacillus subtilis.     

Bacillus subtilis mutants defective in bacteriophage phi 29 head assembly.

Virus assembly mutants of asporogenous Bacillus subtilis defective in bacteriophage phi 29 head assembly were detected by the use of antibodies that reacted strongly with the free dodecameric phi 29 portal vertex composed of gene product 10 (gp10) but weakly with the portal vertex assembled into proheads or phage. Phage adsorption and the synthesis of phage proteins, DNA-gene product 3, and prohead RNA were normal in these mutants, but prohead and phage production was greatly reduced. The assembly defect was transferred to competent B. subtilis by transformation and transduction. PBS1 transduction showed that the vam locus was linked to Tn917 located at 317 degrees on the B. subtilis chromosome.     

Teichoic acid is an essential polymer in Bacillus subtilis that is functionally distinct from teichuronic acid

Wall teichoic acids are anionic, phosphate-rich polymers linked to the peptidoglycan of gram-positive bacteria. In Bacillus subtilis, the predominant wall teichoic acid types are poly(glycerol phosphate) in strain 168 and poly(ribitol phosphate) in strain W23, and they are synthesized by the tag and tar gene products, respectively. Growing evidence suggests that wall teichoic acids are essential in B. subtilis; however, it is widely believed that teichoic acids are dispensable under phosphate-limiting conditions. In the work reported here, we carefully studied the dispensability of teichoic acid under phosphate-limiting conditions by constructing three new mutants. These strains, having precise deletions in tagB, tagF, and tarD, were dependent on xylose-inducible complementation from a distal locus (amyE) for growth. The tarD deletion interrupted poly(ribitol phosphate) synthesis in B. subtilis and represents a unique deletion of a tar gene. When teichoic acid biosynthetic proteins were depleted, the mutants showed a coccoid morphology and cell wall thickening. The new wall teichoic acid biogenesis mutants generated in this work and a previously reported tagD mutant were not viable under phosphate-limiting conditions in the absence of complementation. Cell wall analysis of B. subtilis grown under phosphate-limited conditions showed that teichoic acid contributed approximately one-third of the wall anionic content. These data suggest that wall teichoic acid has an essential function in B. subtilis that cannot be replaced by teichuronic acid.     

Regulation of expression of genes coding for small, acid-soluble proteins of Bacillus subtilis spores: studies using lacZ gene fusions.

We constructed in-frame translational fusions of the Escherichia coli lacZ gene with four genes (sspA, sspB, sspD, and sspE) which code for small, acid-soluble spore proteins of Bacillus subtilis, and integrated these fusions into the chromosomes of various B. subtilis strains. With single copies of the fusions in wild-type B. subtilis, beta-galactosidase was synthesized only during sporulation, with the amounts accumulated being sspB much greater than sspE greater than or equal to sspA greater than or equal to sspD. Greater than 97% of the beta-galactosidase was found in the developing forespore, and the great majority was incorporated into mature spores. Less than 2% of the maximum amount of beta-galactosidase was made when these fusions were introduced into B. subtilis strains blocked in stages 0 and II of sporulation, as well as in some stage III mutants. Other stage III mutants, as well as stage IV and V mutants, had no effect on beta-galactosidase synthesis. Increasing the copy number of the sspA-, sspD-, or sspE-lacZ fusions (up to 17-fold for sspE-lacZ) in wild-type B. subtilis resulted in a parallel increase in the amount of beta-galactosidase accumulated (again only in sporulation and with greater than 95% in the developing forespore), with no significant effect on wild-type small, acid-soluble spore protein production. Similarly, the absence of one or more wild-type ssp genes or the presence of multiple copies of wild-type ssp genes had no effect on the expression of the lacZ fusions tested. These data indicate that these ssp-lacZ fusions escape the autoregulation seen for the intact sspA and sspB genes. Strikingly, the kinetics of beta-galactosidase synthesis were identical for all four ssp-lacZ fusions and paralleled those of glucose dehydrogenase synthesis. Similarly, all asporogenous mutants tested had identical effects on both glucose dehydrogenase and ssp-lacZ fusion expression.     

Bacillus subtilis F0F1 ATPase: DNA sequence of the atp operon and characterization of atp mutants.

We cloned and sequenced an operon of nine genes coding for the subunits of the Bacillus subtilis F0F1 ATP synthase. The arrangement of these genes in the operon is identical to that of the atp operon from Escherichia coli and from three other Bacillus species. The deduced amino acid sequences of the nine subunits are very similar to their counterparts from other organisms. We constructed two B. subtilis strains from which different parts of the atp operon were deleted. These B. subtilis atp mutants were unable to grow with succinate as the sole carbon and energy source. ATP was synthesized in these strains only by substrate-level phosphorylation. The two mutants had a decreased growth yield (43 and 56% of the wild-type level) and a decreased growth rate (61 and 66% of the wild-type level), correlating with a twofold decrease of the intracellular ATP/ADP ratio. In the absence of oxidative phosphorylation, B. subtilis increased ATP synthesis through substrate-level phosphorylation, as shown by the twofold increase of by-product formation (mainly acetate). The increased turnover of glycolysis in the mutant strain presumably led to increased synthesis of NADH, which would account for the observed stimulation of the respiration rate associated with an increase in the expression of genes coding for respiratory enzymes. It therefore appears that B. subtilis and E. coli respond in similar ways to the absence of oxidative phosphorylation.     

Viral mutation affecting bacteriophage phi 1 development in Bacillus subtilis 168.

Bacillus subtilis bacteriophage phi 1m, a host-range variant, was isolated after mutagenesis of virulent bacteriophage phi 1. Unlike its wild-type antecedent, phi 1m could not form plaques on lawns of B subtilis 168 at 37 C, although it adsorbed to, penetrated, and killed this bacterium. Experiments conducted in liquid medium at 37 C showed that B. subtilis 168 cells allowed reduced levels of phi 1m development at low multiplicities of infection, whereas high multiplicity infections of this strain by the phage were abortive. Certain mutants, derived originally from B. subtilis 168, were observed to be permissive for phi 1m at 37 C; moreover, their permissive phenotype could be duplicated by growing wild-type B. subtilis 168 cells at temperatures above 47 C. Studies on phi 1m and host nucleic acid synthesis under nonpermissive conditions demonstrated that transciption and DNA synthesis proceeded up to 20 min after infection, after which time there was a cessation of all nucleic acid production. These observations are discussed with respect to other abortive bacteriophage infections in B. subtilis.     

Altered sporulation and respiratory patterns in mutants of Bacillus subtilis induced by acridine orange

Bott, K. F. (The University of Chicago, Chicago, Ill.), and R. Davidoff-Abelson. Altered sporulation and respiratory patterns in mutants of Bacillus subtilis induced by acridine orange. J. Bacteriol. 92:229-240. 1966.-The addition of acridine orange to vegetative cultures of Bacillus subtilis induces the formation of sporulation mutants at a frequency of 20% or greater. These mutants are grouped into seven categories which reflect their different morphological properties. They are altered in their vegetative metabolism, as indicated by abnormal growth on synthetic media. Sporulation of these mutants is impaired at several levels, all of which are stable upon repeated subculturing. The initial stages of sporulation which require no increased metabolic activity (proteolytic enzyme activity and antibiotic production) are functional in all strains, but glucose dehydrogenase activity, an enzyme associated with early synthetic functions in spore synthesis, is significantly reduced. Reduced nicotinamide adenine dinucleotide oxidase is slightly depressed. It is suggested that acridine orange interacts with a cellular constituent controlling respiration and consequently prevents an increased metabolic activity that may be associated with normal spore synthesis.     

Synthesis of thymidine nucleotides in Bacillus subtilis.

To investigate the synthesis of thymidine nucleotides in Bacillus subtilis, mutants that carried various combinations of thyA, thyB, and other mutations affecting pyrimidine metabolism were isolated. It was found that exogenously supplied deoxycytidine was converted to thymidine nucleotides. The present data suggest that deoxycytidine nucleotides are first deaminated to yield deoxyuridine nucleotides which can serve as substrates for both thyA- and thyB-coded synthetases. A deaminase activity for dCDP was found in crude extracts of B. subtilis. A mutant lacking the deaminase activity was unable to convert deoxycytidine nucleotides to thymidine nucleotides.     

Biofilm-defective mutants of Bacillus subtilis

Many bacteria can adopt organized, sessile, communal lifestyles. The gram-positive bacterium, Bacillus subtilis,forms biofilms on solid surfaces and at air-liquid interfaces, and biofilm development is dependent on environmental conditions. We demonstrate that biofilm formation by B. subtilis strain JH642 can be either activated or repressed by glucose, depending on the growth medium used, and that these glucose effects are at least in part mediated by the catabolite control protein, CcpA. Starting with a chromosomal Tn917-LTV3 insertional library, we isolated mutants that are defective for biofilm formation. The biofilm defects of these mutants were observable in both rich and minimal media, and both on polyvinylchloride abiotic surfaces and in borosilicate tubes. Two mutants were defective in flagellar synthesis. Chemotaxis was shown to be less important for biofilm formation than was flagellar-driven motility. Although motility is known to be required for biofilm formation in other bacteria, this had not previously been demonstrated for B. subtilis. In addition, our study suggests roles for glutamate synthase, GltAB, and an aminopeptidase, AmpS. The loss of these enzymes did not decrease growth or cellular motility but had dramatic effects on biofilm formation under all conditions assayed. The effect of the gltAB defect on biofilm formation could not be due to a decrease in poly-gamma-glutamate synthesis since this polymer proved to be nonessential for robust biofilm formation. High exogenous concentrations of glutamate, aspartate, glutamine or proline did not override the glutamate synthase requirement. This is the first report showing that glutamate synthase and a cytoplasmic aminopeptidase play roles in bacterial biofilm formation. Possible mechanistic implications and potential roles of biofilm formation in other developmental processes are discussed.     

Structure of the Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster.

A 10.5-kilobase PstI endonuclease fragment encoding the entire Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster was cloned in Escherichia coli by transformation of a carB strain to uracil-independent growth. The cloned fragment also complemented E. coli pyrB, pyrC, pyrD, pyrE, and pyrF mutants. From the ability of subclones to complement E. coli pyr mutants, the gene order was deduced to be pyrBCADFE. The B. subtilis pyrB gene was shown to be expressed in E. coli, but synthesis of the enzyme was not repressible by the addition of uracil to the growth medium. The approximate molecular weights of the polypeptides encoded by B. subtilis pyrA, pyrB, pyrC, pyrD, pyrE, and pyrF were found to be 110,000, 36,000, 46,000, 34,000, 25,000, and 27,000, respectively.     

Kasugamycin-resistant mutants of Bacillus subtilis.

Kasugamycin-resistant mutants of Bacillus subtilis were isolated and classified into two groups, one of which had resistance to kasugamycin in in vitro protein synthesis and mapped in the ribosomal region. The other group had no resistance to kasugamycin in in vitro protein synthesis and had weak cross-resistance to gentamicin and kanamycin. Neither group could sporulate in the presence of kasugamycin.     

Common Element in the Repression Control of Enzymes of Histidine and Aromatic Amino Acid Biosynthesis in Bacillus subtilus

Single-step mutants of Bacillus subtilis derepressed for enzymes of both aromatic amino acid and histidine biosynthesis were isolated. These mutants occur at a frequency of 10(-6) per cell per generation. All histidine enzymes as well as all enzymes of aromatic acid synthesis which were examined are maximally derepressed. This level cannot be repressed by growth on either histidine or tyrosine. Some of the structural genes which specify the derepressed enzymes are linked to the aromatic cluster; others are unlinked. The significance of these nonrepressible strains is discussed in terms of the mechanism of repression.     

Regulation of Tyrosyl-Transfer Ribonucleic Acid Synthetase in Bacillus subtilis

Derepression of tyrosyl-transfer ribonucleic acid synthetase in Bacillus subtilis was observed in strains grown with limiting tyrosine, but not in regulatory mutants derepressed in biosynthetic enzyme synthesis.     

An enzyme common to histidine and aromatic amino acid biosynthesis in Bacillus subtilis.

Two transaminases exist for tyrosine and phenylalanine synthesis in Bacillus subtilis. One enzyme is also responsible for the transamination of imidazole acetol phosphate to histidinol phosphate, an obligatory reaction in the synthesis of histidine. The gene involved in the synthesis of this enzyme lies in the middle of a cluster of genes, all of which are concerned with the synthesis of the aromatic amino acids. The other gene has not yet been mapped. Mutants have been isolated that lack one or the other enzyme activity. These mutants are prototrophic for tyrosine and phenylalanine. However, both classes of mutants are more sensitive than the wild-type strain to the phenylalanine analogue, fluorophenylalanine, suggesting that each of these mutants synthesizes less phenylalanine than does the wild-type strain. The two enzymes can be separated from one another by ion-exchange chromatography and glycerol-gradient centrifugation. The significance of the observation that an enzyme of histidine synthesis also plays a role in the synthesis of the aromatic acids is considered in light of cross-pathways regulation between the two pathways.     

Actin homolog MreBH governs cell morphogenesis by localization of the cell wall hydrolase LytE

MreB proteins are bacterial actin homologs involved in cell morphogenesis and various other cellular processes. However, the effector proteins used by MreBs remain largely unknown. Bacillus subtilis has three MreB isoforms. Mbl and possibly MreB have previously been shown to be implicated in cell wall synthesis. We have now found that the third isoform, MreBH, colocalizes with the two other MreB isoforms in B. subtilis and also has an important role in cell morphogenesis. MreBH can physically interact with a cell wall hydrolase, LytE, and is required for its helical pattern of extracellular localization. Moreover, lytE and mreBH mutants exhibit similar cell-wall-related defects. We propose that controlled elongation of rod-shaped B. subtilis depends on the coordination of cell wall synthesis and hydrolysis in helical tracts defined by MreB proteins. Our data also suggest that physical interactions with intracellular actin bundles can influence the later localization pattern of extracellular effectors.     

Competence proteins in Bacillus subtilis com mutants

The synthesis of nucleases and proteins specific for competence development have been studied in four different Bacillus subtilis competence-deficient mutants. The nuclease analysis showed that two DNA-binding-deficient mutants were impaired in three nuclease activities involved in binding and entry of donor DNA. The other two strains did not show any reduction in nuclease activities. Two-dimensional gel electrophoresis of the proteins, synthesized during competence development, revealed that all four mutants are lacking several competence-specific polypeptides. Our data show that these com mutations have a strong pleiotropic effect, which could be due to a block in the metabolic pathway leading to competence development.