Recombination-deficient mutants of Bacillus subtilis.

Two mutant strains of Bacillus subtilis Marburg, NIG43 and NIG45, were isolated. They showed high sensitivities to gamma rays, ultraviolet light (UV), and chemicals. Deficiencies in genetic recombination of these two mutants were shown by the experiments on their capacity in transformation. SPO2 transfection, and PBS1 phage transduction, as well as on their radiation and drug sensitivities and their Hcr+ capacity for UV-exposed phage M2. Some of these characteristics were compared with those of the known strains possessing the recA1 or recB2 alleles. Mapping studies revealed that the mutation rec-43 of strain NIG43 lies in the region of chromosome replication origin. The order was purA dna-8132 rec-43. Another mutation, rec-45, of strain NIG45 was found to be tightly linked to recA1. The mutation rec-43 reduced mainly the frequency of PBS1 transduction. On the other hand, the mutation rec-45 reduced the frequency of recombination involved both in transformation and PBS1 transduction. The mutation rec-43 of strain NIG43 is conditional, but rec-45 of strain NIG45 is not. The UV impairment in cellular survival of strain NIG43 was gradually reverted at higher salt or sucrose concentrations, suggesting cellular possession of a mutated gene produce whose function is conditional. In contrast to several other recombination-deficient strains, SPO2 lysogens of strain NIG43 and NIG45 were not inducible, indicating involvement of rec-43+ or rec-45+ gene product in the development of SPO2 prophage to a vegetative form. The UV-induced deoxyribonucleic acid degradation in vegetative cells was higher in rec-43 and rec-45 strains.     

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.     

Deoxyribonucleoside-requiring mutants of Bacillus subtilis.

A number of deoxyribonucleoside-requiring mutants (dns) of Bacillus subtilis were isolated and their growth characteristics and ribonucleotide reductase activities were compared with those of the wild type and of a dna mutant (tsA13). Both tsA13 and dns mutants required the presence of a mixture of deoxyribonucleosides for growth at 45 degrees C but not at 25 degrees C. All the mutant strains tested contained ribonucleotide reductase activity which showed heat sensitivity similar to that of the enzyme from a wild-type strain. The reductase in B. subtilis seemed to reduce ribonucleoside triphosphates in a similar manner to the enzyme in Lactobacillus leichmannii.     

Glutamine-requiring mutants of Bacillus subtilis.

Two glutamine-requiring (Gln^?) mutants of $\text{Bacillus subtilis}$ SMY were deficient in glutamine synthetase activity $\text{in vitro}$. The Gln^? mutants sporulated poorly unless glutamine was provided at high concentrations. The differential rate of histidase synthesis following induction was 4- to 6-fold higher in the Gln^? mutants than in wild-type cells. In addition, glucose repression of utilization of alternative carbohydrates appeared to be partially relieved in the Gln^? mutants.     

Decadent sporulation mutants of Bacillus subtilis.

In decadent sporulation mutants, sporulating populations are heterogeneous: the cells reach successive chemical and physical resistances with progressively decreasing frequencies. Each decadent mutant can be characterized by the shape and slope of the curve describing the frequency of cells resistant to various agents ('the resistance spectrum'). In some mutants the resistance spectrum decreases progressively from xylene resistance to heat resistance; in other mutants it decreases rapidly between octanol resistance and chloroform resistance. Electron microscopy showed that in two mutants the majority of the cells are blocked at stages III and IV; the number of cells that develop further to reach successive morphological stages falls off progressively. In two other mutants most cells reach stage V. Cortexless spores are also frequent. One of the decadent mutations, SpoL1, was localized between aroD and acf. The phenotype of decadent mutants is discussed in terms of sequential gene activation.     

Catabolite repression-resistant mutants of Bacillus subtilis.

Mutants of Bacillus subtilis that are able to sporulate under the condition of catabolite repression were isolated by a simple selection technique. The mutants used in the present study were able to grow normally on minimal medium with ammonium sulphate as the nitrogen source and glucose as the carbon source. Studies carried out with these mutants show that there is no close relation between catabolite repression of an inducible enzyme, acetoin dehydrogenase, and that of sporulation. Certain mutants are able to sporulate in the presence of all the carbon sources tested but some mutants are resistant only to the carbon source used in isolation. It is suggested that several metabolic steps may be affected in catabolite repression of sporulation.     

Alcohol-resistant sporulation mutants of Bacillus subtilis.

About 80% of Bacillus subtilis cells form spores when grown in nutrient broth. In medium containing various short-chain aliphatic alcohols, the frequency of sporulation was reduced to 0.5%. Mutants sporulated in the presence of alcohols at a frequency of 30 to 40%. Sporulation in the wild-type cells was sensitive to alcohol at the beginning of sporulation (stage zero). Sensitivity to alcohol in the mutants was also at stage zero, even though the sensitivity was considerably reduced. This sensitivity of sporulation to alcohol is the phenotypic expression of a genetic locus designated ssa. Mutations at this locus lead to a decreased sensitivity of sporulation to alcohol without modifying the sensitivity of growth. Genetic analysis by transduction was bacteriophage PBS1 revealed that ssa mutations are near the previously described spo0A locus. ssa mutants also differ from wild-type cells in the composition of membrane phospholipids. The relative amount of phosphatidylglycerol increased, whereas the relative amount of phosphatidylethanolamine and lysylphosphatidylglycerol decreased relative to the proportions in the wild type. The distribution of fatty acids in membrane lipids is the same as in the wild type. No differential sensitivity of phospholipid metabolism to alcohol could be detected in the mutant. This work therefore reveals that the extensive, pleiotropic changes in the membranes of ssa mutants are the phenotypic reflection of alterations at a specific gene locus.     

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.     

Autolytic enzyme-deficient mutants of Bacillus subtilis 168.

Mutants of Bacillus subtilis strain 168 have been isolated that are at least 90 to 95% deficient in the autolytic enzymes N-acetylmuramyl-L-alanine amidase and endo-beta-N-acetylglucosaminidase. These mutants grow at normal rates as very long chains of unseparated cells. The length of the chains is directly related to the growth rates. They are nonmotile and have no flagella, but otherwise appear to have normal cell morphology. Their walls are fully sysceptible to enzymes formed by the wild type and have the same chemical composition as the latter. Cell wall preparations from the mutants lyse at about 10% of the rate of those from the isogenic wild type, with the correspondingly small liberation of both the amino groups of alanine at pH 8.0 and of reducing groups at pH 5.6. Likewise, Microcococcus luteus walls at pH 5.6 and B. subtilis walls at pH 8 are lysed only very slowly by LiCl extracts made from the mutants as compared with rates obtained with wild-type extracts. Thus, the activity of both autolytic enzymes in the mutants is depressed. The frequencies of transformation, the isolation of revertants, and observations with a temperature-sensitive mutant all point to the likelihood that the pleiotropic, phenotypic properties of the strains are due to a single mutation. The mutants did not produce more protease or amylase than did the wild type. They sporulate and the spores germinate normally. The addition of antibiotics to exponentially growing cultures prevents wall synthesis but leads to less lysis than is obtained with the wild type. The bacteriophage PBSX can be induced in the mutants by treatment with mitomycin C.     

Erythromycin resistant mutants of Bacillus subtilis

Summary Erythromycin resistant (ery ^r) mutants were isolated from Bacillus subtilis ATCC 6633. The composition of ribosomal proteins were analyzed for thirteen such ery ^r-mutants with chromatography on a carboxymethyl cellulose (CMC) column. The 50s subunit from all of the ery ^r-mutants was found to contain the altered 50d protein. The ribosomes prepared from the ery ^r-mutants did not show in vitro alteration of the ability to combine with erythromycin.     

Complementation and characterization of chemotaxis mutants of Bacillus subtilis.

A set of chemotaxis mutants of Bacillus subtilis was complemented by using SP beta c2 transducing bacteriophage either containing cloned segments of DNA or derived from abnormal excision of SP beta c2 dl2::Tn917 inserted into the chemotaxis region. Representative mutants were characterized in capillary assays for chemotaxis toward four amino acids and mannitol and in tethered-cell experiments for addition and removal of two attractants and two repellents. Twenty complementation groups were identified, in addition to the cheR previously characterized. All were found to be defective in chemotaxis toward all chemoeffectors. They were assigned the names cheA through cheU. The large number of general chemotaxis genes in B. subtilis, in contrast to the six in Escherichia coli, suggests fundamental differences in the mechanism of chemotaxis in the two species.     

5-Bromouracil-tolerant mutants of Bacillus subtilis

5-Bromouracil (BU)-tolerant mutants of Bacillus subtilis 23 (thy his) have been isolated. Several classes of tolerant mutants were obtained by a sequential selection procedure. The classes can be distinguished by their relative BU tolerance as well as several other phenotypic characteristics. The mutants can grow for an extended period of time in minimal medium supplemented with amino acids and BU, in which the sensitive parental strain (Bu(+)) undergoes rapid cell death. Both mutants But-1 and But-1310 have a greater rate of deoxyribonucleic acid (DNA) synthesis by a factor of two in the presence of BU than Bu(+), But-1 being somewhat faster than But-1310. The preferential incorporation of thymine to BU of But-1 is about half that of the Bu(+) strain during DNA replication in minimal medium supplemented with 10 mug of BU/ml and 1 mug of thymine/ml. It is not known at what step or steps this reduction in selectivity occurs.     

Chloramphenicol resistant mutants of Bacillus subtilis

Summary Telve chloramphenicol resistant (CM ^r)-mutants were isolated from B. subtilis ATCC 6633 and were classified into the following six groups. Group I. No 50s ribosomal protein change was detectable. Ribosomes did not show alteration of the binding ability to CM or to erythromycin in vitro. Group II. A 50s protein, 50a, was altered. Ribosomes did not show alteration of the binding ability to CM or to erythromycin in vitro. The genes specifying the 50a protein was in the cysA-str region on B. subtilis chromosome. Group III. A 50s protein, 50b, was altered. Biological properties of the ribosomes were the same as Group I or II so fas as examined. The genes for 50b protein was in the cysA-str region. Group IV. A 50s protein, 50c, was altered. Ribosomes showed a definite decrease in ability to bind to CM in vitro. The binding of erythromycin to the ribosomes was not impaired. The chromosomal locus of the CM ^r (and for 50c protein) was in the cysA-str region. Group V. A 50s protein, 50e, was changed. The ability of the ribosomes to bind in vitro both to CM and to erythromycin was greatly reduced. The genetic locus of the CM ^r (and for 50e protein) was in the cysA-str region. Group VI. A 50s protein, 50f, was altered. Ribosomes showed a decrease in ability to bind in vitro both to CM and to erythromycin. The genes for 50f protein was in the cysA-str region.The results suggest that the ribosomal resistance to CM may be caused by an independent change of at least several 50s ribosomal protein species. The genetic data shown here and those reported previously show that at least two 30s and seven 50s ribosomal protein genes are situated in the cysA-str region on B. subtilis chromosome.     

Isolation and characterization of uncoupler-resistant mutants of Bacillus subtilis.

Three mutant strains of Bacillus subtilis were isolated on the basis of their ability to grow in the presence of 5 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP). The mutants (AG2A, AG1A3, and AG3A) were also resistant to 2,4-dinitrophenol, and AG2A exhibited resistance to tributyltin and neomycin. The mutants all exhibited (i) elevated levels of membrane ATPase activity relative to the wild type; (ii) slightly elevated respiratory rates, with the cytochrome contents of the membranes being the same as or slightly lower than those of the wild type; (3) a passive membrane permeability to protons that was indistinguishable from that of the wild type in the absence of CCCP and that was increased by addition of CCCP to the same extent as observed with the wild type; and (4) an enhanced sensitivity to valinomycin with respect to the ability of the ionophore to reduce the transmembrane electrical potential. Finally and importantly, starved whole cells of all the mutants synthesized more ATP than the wild type did upon energization in the presence of any one of several agents that lowered the proton motive force. Studies of revertants indicated that the phenotype resulted from a single mutation. Since a mutation in the coupling membrane might produce such pleiotropic effects, an analysis of the membrane lipids was undertaken with preparations made from cells grown in the absence of CCCP. The membrane lipids of the uncoupler-resistant strains differed from those of the wild type in having reduced amounts of monounsaturated C16 fatty acids and increased ratios of iso/anteiso branches on the C15 fatty acids. Correlations between protonophore resistance and the membrane lipid compositions of the wild type, mutants, and revertants were most consistent with the hypothesis that a reduction in the content of monounsaturated C16 fatty acids in the membrane phospholipids is related, perhaps casually, to the ability to synthesize ATP at low bulk transmembrane electrochemical gradients of protons.     

Cortex content of asporogenous mutants of Bacillus subtilis.

A method for the measurement of muramic lactam, which is specifically located in the cortical peptidoglycan of bacterial spores, was developed as a quantitative assay method for spore cortex content. During sporulation of Bacillus subtilis 168, muramic lactam (i.e., spore cortex) began to appear at state IV of sporulation and continued to increase over most of the late stages of sporulation. Spore cortex contents of various spo mutants of B. subitils were surveyed. Cortex was not detected in mutants in which sporulation was blocked earlier than stage II sporulation. Spores of spo IV mutant had about 40% of the cortex content of the wild-type spores. One spo III mutant had a low amount of cortex, but four others had none.     

Growth medium-independent genetic competence mutants of Bacillus subtilis.

The development of competence in Bacillus subtilis is normally dependent on the growth medium. Expression of late competence genes occurs in glucose-minimal salts-based media but not in complex media. Expression is also inhibited when glutamine is added to competence medium and when glycerol is substituted for glucose. Mutations have been identified in two regulatory loci, mecA and mecB, which render competence development independent of these variables. Although in mec mutants the expression of late competence genes, as well as of competence itself, occurred in all media tested, this expression was still growth stage regulated. Thus at least some forms of medium-dependent and growth stage-specific regulation are genetically separable. One of the mecB mutations (mecB31) conferred oligosporogenicity. The mecB mutations were tightly linked by transformation to rif, lpm, and std markers and were located between rif-2103 and cysA14. The mecA42 mutant was linked by transduction to argC4.     

Antibiotic-resistant mutants of Bacillus subtilis conditional for sporulation.

Among spontaneously occurring antibiotic-resistant mutants of Bacillus subtilis 168 we have identified a sub-class that is conditionally sporulative. Mutants in this sub-class are resistant to antibiotic during vegetative growth but are sensitive during sporulation. Mutants conditionally-resistant to erythromycin, kanamycin, spectinomycin, and streptomycin have been isolated and characterized by phase contrast microscopy and with respect to their ability to synthesize heat-resistant endospores or the sporulation-associated enzyme alkaline phosphatase. The results suggest that several entirely different genetic lesions may result in this single phenotype. This group includes mutants whose properties suggest that both th 30S and 50S ribosomal subunits may be altered concomitant with early spore specific metabolism. The blockage imposed by antibiotic may be at or near Stage 2 of sporulation.