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.     

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.     

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.     

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.     

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.     

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.     

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.     

Characterization of recombination-deficient mutants of Bacillus subtilis

An isogenic set of "prophage-free," DNA repair-proficient and -deficient strains of Bacillus subtilis were characterized phenotypically. The mutant strains were provisionally classified into four categories on the basis of their sensitivity to DNA-damaging agents, their ability to release phage after lysogenization followed by damage to chromosomal DNA, and their impairment in genetic exchange. The properties of double Rec- mutants showed that recF and addA belong to different epistatic groups, whereas recF, recL, and recH fall into the same group. More than one pathway for genetic exchange might be operative in B. subtilis.     

Properties of regeneration mutants of Bacillus subtilis

Abstract Higher regeneration mutants were isolated from Bacillus subtilis . Protoplasts from two out of four mutants regenerated at a 100% frequency on a semi-synthetic hypertonic medium. They conferred less autolytic productivity, and a revertant regained the parental levels of regeneration frequency and autolytic activities. This mutation ( rgn -1) expressed the other pleiotropic properties, i.e., nonmotility, phage PBS1 resistance and different cell morphology.     

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.     

New class of Bacillus subtilis glutamine-requiring mutants

By using genetic analysis, the mutations of eight glutamine-requiring mutants isolated from Bacillus subtilis 168 were all shown to be linked to the thyA marker. A three-factor transduction analysis performed with one of the gln mutations indicated that the gene order in this region of the B. subtilis chromosome was gltA-thyA-gln. On the basis of recombination index values, two closely linked groups were identified. The mutations belonging to one group were assigned to the structural gene for glutamine synthetase, and those belonging to the other group might impair a regulatory locus. The residual glutamine synthetase activities and the cross-reacting materials of the mutants from both recombination groups supported these conclusions.     

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.     

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.     

Plasmid maintenance in Bacillus subtilis recombination-deficient mutants

Summary An isogenic set of 11 recombination-deficient mutant strains of Bacillus subtilis has been constructed. Whereas plasmid pUB110 is stably maintained in such Rec^- cells, the high copy number plasmid pC194 is unstable. Instability in Rec^- strains could be mostly attributed to the deleterious effect of the presence of the plasmid on the Rec^- cells' growth capability. In part, instability of pC194 derivatives could also be correlated with the presence of an unusually high amount of multimeric DNA molecules.