The Bacillus subtilis spo0J gene: evidence for involvement in catabolite repression of sporulation.

Previous observations concerning the ability of the Bacillus subtilis bacteriophages SP10 and PMB12 to suppress mutations in spo0J and to make wild-type sporulation catabolite resistant suggested that spo0J had a role in catabolite repression of sporulation. This suggestion was supported in the present report by the ability of the catabolite-resistant sporulation mutation crsF4 to suppress a Tn917 insertion mutation of the B. subtilis spo0J locus (spo0J::Tn917 omega HU261) in medium without glucose. Although crsF4 and SP10 made wild-type B. subtilis sporulation catabolite resistant, neither crsF4 nor SP10 caused a mutant with spo0J::Tn917 omega HU261 to sporulate in medium with glucose. Sequencing the spo0J locus revealed an open reading frame that was 179 codons in length. Disruption of the open reading frame resulted in a sporulation-negative (Spo-) phenotype that was similar to those of other spo0J mutations. Analysis of the deduced amino acid sequence of the spo0J locus indicated that the spo0J gene product contains an alpha-helix-turn-alpha-helix unit similar to the motif found in lambda Cro-like DNA-binding proteins.     

Bacteriophage conversion of spore-negative mutants to spore-positive in Bacillus pumilus.

A pseudolysogenic phage, PMB1, was isolated from soil on the basis of its ability to increase the sporulation frequency of the oligosporogenic Bacillus pumilus strain NRS 576 (sporulation frequency, less than 1%). Several spore-negative mutants (sporulation frequency, less than 10-8) derived from strain NRS 576, which were converted to spore positive by infection with PMB1, were subsequently identified. PMB1 repeatedly grown on a given spore-negative mutant (e.g., GW2) converted GW2 cells to spore positive. Each plaque-forming unit initiated the conversion of a spore-positive clone in semisolid agar overlays. GW2 cells remained spore positive as long as they maintained PMB1. Return of PMB1-converted cells to the orginal spore-negative phenotype correlated with loss of PMB1. In liquid media, PMB1 infection increased the sporulation frequency of mutant GW2 over 106-fold. More than half of the spore-negative mutants we isolated from strain NRS 576 were converted to spore positive by PMB1 infection. PMB1-induced spores of the spore-negative mutant GW2 were somewhat more heat sensitive than uninfected or PMB1-infected spores of the spore positive parent of GW2. PMB1-induced spores of GW2 do not differ from wild-type spores in morphology by phase-contrast microscopy, dipicolinic acid content, or rate of sedimentation through Renografin gradients.     

Bacteriophage PMB12 conversion of the sporulation defect in RNA polymerase mutants of Bacillus subtilis.

The pseudotemperate phage PMB12 was isolated from soil on the basis of its ability to enhance the rate of sporulation of Bacillus subtilis 168. PMB12 was subsequently shown to convert the sporulation defect in two genetically distinct classes of sporulation mutants. One class includes those rifampin-resistant mutants that are also spore-negative (mutated at the rif locus). The other class includes a strain carrying the sporulation mutation spoCM-1. The spoCM-1 mutation is linked to cysA15 by PBS1 transduction but is distinct from the rif locus. Several other sporulation mutants were not converted by PMB12. PMB12 is related to phage PBS1. However, PBS1 did not convert the above sporulation mutants. The replication of PBS2, a clear-plaquing derivative of PBS1, is rifampin insensitive, apparently due to a phage-induced rifampin-insensitive RNA polymerase. PMB12 replication is also rifampin insensitive.     

Characterization of interspecific plasmid transfer mediated by Bacillus subtilis temperate bacteriophage SP02.

Plasmid pPL1010 is a 7.0-kilobase derivative of plasmid pUB110 that harbors the cohesive end site of the bacteriophage SP02 genome. Plasmid pPL1017 is a 6.8-kilobase derivative of plasmid pC194 that contains the immunity region of bacteriophage phi 105 and the cohesive end site of bacteriophage SP02. These plasmids are transducible by bacteriophage SP02 at a frequency of 10(-2) transductants per PFU among mutant derivatives of Bacillus subtilis 168 and have been transferred to other strains of B. subtilis and B. amyloliquefaciens by means of bacteriophage SP02-mediated transduction, with frequencies ranging from 10(-5) to 10(-7) transductants per PFU. The introduced plasmids were stably maintained in nearly all new hosts in the absence of selective pressure. An exception was found in B. subtilis DSM704, which also harbored three cryptic plasmids. Plasmids pPL1010 and pPL1017 were incompatible with a 7.9-kilobase replicon native to strain DSM704. Furthermore, plasmid pPL1017 was processed by strain DSM704 into a approximately 5.3-kilobase replicon that was compatible with the resident plasmid content of strain DSM704. The use of bacteriophage SP02-mediated plasmid transduction has allowed the identification of Bacillus strains that are susceptible to bacteriophage SP02-mediated genetic transfer but cannot support bacteriophage SP02 lytic infection.     

Minimum bacterial density for bacteriophage replication: implications for significance of bacteriophages in natural ecosystems.

Bacteriophage 80 alpha did not increase in number in cultures containing less than about 1.0 X 10(4) to 1.5 X 10(4) CFU of Staphylococcus aureus per ml, but bacteriophage replication did occur when the number of bacteria exceeded this density, either initially or as a result of host cell multiplication. The minimum density of an asporogenous strain of Bacillus subtilis required for an increase in the number of bacteriophage SP beta cI was about 3 X 10(4) CFU/ml. The threshold density of Escherichia coli for the multiplication of bacteriophage T4 was about 7 X 10(3) CFU/ml. In the presence of montmorillonite, bacteriophage T4 did not increase in number until the E. coli population exceeded 10(4) CFU/ml. The mineralization of glucose was not affected in E. coli cultures inoculated with a low number of bacteriophage T4, but it could not be detected in cultures inoculated with a large number of phage. The numbers of bacteriophage T4 and a bacteriophage that lyses Pseudomonas putida declined rapidly after being added to lake water or sewage. We suggest that bacteriophages do not affect the number or activity of bacteria in environments where the density of the host species is below the host cell threshold of about 10(4) CFU/ml.     

Minimum bacterial density for bacteriophage replication: implications for significance of bacteriophages in natural ecosystems

Bacteriophage 80 alpha did not increase in number in cultures containing less than about 1.0 X 10(4) to 1.5 X 10(4) CFU of Staphylococcus aureus per ml, but bacteriophage replication did occur when the number of bacteria exceeded this density, either initially or as a result of host cell multiplication. The minimum density of an asporogenous strain of Bacillus subtilis required for an increase in the number of bacteriophage SP beta cI was about 3 X 10(4) CFU/ml. The threshold density of Escherichia coli for the multiplication of bacteriophage T4 was about 7 X 10(3) CFU/ml. In the presence of montmorillonite, bacteriophage T4 did not increase in number until the E. coli population exceeded 10(4) CFU/ml. The mineralization of glucose was not affected in E. coli cultures inoculated with a low number of bacteriophage T4, but it could not be detected in cultures inoculated with a large number of phage. The numbers of bacteriophage T4 and a bacteriophage that lyses Pseudomonas putida declined rapidly after being added to lake water or sewage. We suggest that bacteriophages do not affect the number or activity of bacteria in environments where the density of the host species is below the host cell threshold of about 10(4) CFU/ml.     

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.     

Interaction of polymyxin B nonapeptide with anionic phospholipids

The interaction of polymyxin B nonapeptide (PMBN) and polymyxin B (PMB) with the anionic phospholipids phosphatidylserine (PS), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidic acid (DPPA), and 1:1 mixtures (w/w) of DPPA and distearoylphosphatidylcholine (DSPC) was studied by calorimetry, electron spin resonance, and fluorescence spectrometry, electron microscopy, and fusion and leakage assays. The phase transition temperatures of DPPA and DPPG were very similar when bound to PMB or PMBN, indicating that the lipids are in a similar state when bound to the cationic peptides. Both PMB and PMBN caused the interdigitation of DPPG bilayers, suggesting that the penetration of hydrophobic side chains from a peptide bound electrostatically on the surface is sufficient to induce this phenomenon. Stopped-flow experiments revealed that PMBN and PMB induced the fusion of small unilamellar PS and large unilamellar DPPA-DSPC vesicles. The aggregation of vesicles was found to be diffusion-controlled process; the subsequent fusion took place with a frequency of 10(2)-(5 X 10(2] s-1 for small vesicles and 1-100 s-1 for large vesicles. The freeze-fracture replicas of the PMB-treated vesicles displayed 12-50-nm depressions on several superimposed bilayers, indicating the formation of stable lipid-PMB domains. Since the incubation with PMBN produced similar depressions only if the specimens were fixed, PMBN-induced domain formation seems to be a reversible rapid process. The differences in the phospholipid-peptide interactions are correlated with the differences in the physiological action of the antibiotic PMB and the nonbactericidal PMBN on the cell envelope of Gram-negative bacteria.     

A study of the role of the hexose monophosphate pathway with respect to fatty acid biosynthesis in sporulation of Saccharomyces cerevisiae

13C NMR was used to study the pattern of label incorporation from [2-13C]acetate into trehalose during sporulation in Saccharomyces cerevisiae. A wild-type strain and a strain homozygous for the zwf1 mutation (which affects glucose-6-phosphate dehydrogenase) were used. In the wild-type it was possible to deduce the cycling of glucose 6-phosphate around the hexose monophosphate pathway whilst in the mutant strain this did not occur. The requirement of the hexose monophosphate pathway for providing NADPH for fatty acid biosynthesis was examined using 13C NMR and GC/MS. The wild-type strain produced a typical profile of fatty acids with palmitoleic acid being the most abundant whereas the mutant contained only one-quarter the amount of total fatty acid. As zwf1 homozygous diploids are able to sporulate this indicates that the large amount of fatty acid biosynthesis observed in sporulation of wild-type strains is not essential to the process.     

IL-12 induction of mRNA encoding substance P in murine macrophages from the spleen and sites of inflammation

Substance P (SP), a neuropeptide, interacts with the neurokinin 1 receptor (NK-1R) on immune cells to help control IFN-gamma production. In murine schistosomiasis mansoni, schistosome worms produce ova that incite focal Th2-type granulomatous inflammation within the liver and intestines. Normal gut is characterized by a controlled state of inflammation. IL-10 knockout mice develop chronic Th1-type colitis spontaneously. Both schistosome granulomas and gut mucosa display an SP immune regulatory circuit. However, the origin and regulation of SP production at these sites of inflammation are poorly understood. Macrophages are a potential source of SP. We therefore studied macrophages (F4/80(+)) from these models of inflammation. SP mRNA (preprotachykinin A (PPT A)) was detected within the schistosome granuloma, spleen, and lamina propria macrophages. Compared with those from wild-type mice, granuloma macrophages from STAT6(-/-) mice had 10-fold higher PPT A mRNA expression, whereas in STAT4(-/-) animals, PPT A mRNA expression was nearly abolished. IL-12 signals via STAT4 to induce Th1-type inflammation. It was demonstrated that IL-12, but not IL-18, induces SP mRNA expression in resting splenic macrophages from Schistosoma-infected mice and in wild-type lamina propria mononuclear cells. Thus, macrophages are a source for SP at these sites of chronic inflammation, and IL-12 and STAT4 are regulators of macrophage SP mRNA expression.     

Combined use of strain construction and affinity chromatography in the rapid, high-yield purification of 6-phosphogluconate dehydrogenase from Escherichia coli.

A rapid, high-yield method for purification of 6-phosphogluconate dehydrogenase from Escherichia coli K-12 is described. Sonic extracts prepared from heat-induced cultures of strain RW184, doubly lysogenic for the specialized transducing bacteriophage lambdacI857St68h80dgndhis and bearing a deletion of the gene for glucose 6-phosphate dehydrogenase, contained levels of 6-phosphogluconate dehydrogenase 15- to 20-fold higher than cultures of wild-type cells. Affinity chromatography on blue dextran-Sepharose with batchwise elution with 1 mM nicotinamide adenine dinucleotide phosphate affected a further 10-fold purification. Enzyme prepared in this manner was homogeneous according to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels and immunoelectrophoresis using antiserum directed against it. Fructose 1,6-diphosphate is an inhibitor of enzyme activity.     

Cloning and expression of bacteriophage SP02 DNAZ polymerase gene L in Bacillus subtilis, using the Staphylococcus aureus plasmid pC194

HindIII restriction endonuclease fragments of DNA from temperate Bacillus subtilis bacteriophage SP02 were cloned in B. subtilis by using the plasmid pC194. Three hybrid plasmids which permit growth of the mutant SP02 susL244 in suppressor-negative bacteria were isolated. SP02 gene L is thought to code for a DNA polymerase essential for autonomous replication of SP02 DNA. Extracts of bacteria carrying one of these hybrid plasmids, pC194-96, had 10- to 30-fold increased DNA polymerase activity. The plasmid-induced DNA polymerase activity differed from that of the known B. subtilis DNA polymerases in several respects. The results of the experiments support the idea that phage SP02 codes for a new DNA polymerase.     

Transfection of Streptococcus pneumoniae with bacteriophage DNA.

It was possible to transfect Streptococcus pneumoniae with DNA obtained from a newly isolated bacteriophage, diplophage-4 (Dp-4). Optimal frequency of transfection (0.9%) required the use of a nuclease-defective mutant; with wild-type bacteria, the transfection frequency was about 100-fold lower. Transfection requires physiological conditions that appear to be similar to the competent state needed for genetic transformation (A. Tomasz, J. Bacteriol. 91:1050--1061, 1966).     

Gene expression in Bacillus subtilis surface biofilms with and without sporulation and the importance of yveR for biofilm maintenance

Five independent DNA microarray experiments were used to study the gene expression profile of a 5-day Bacillus subtilis air-liquid interface biofilm relative to planktonic cells. Both wild-type B. subtilis and its sporulation mutant (DeltaspoIIGB::erm) were investigated to discern the important biofilm genes (in the presence and absence of sporulation). The microarray results indicated that suspension cells were encountering anaerobic conditions, and the air-liquid interface biofilm was metabolically active. For the statistically significant differential expression (P < 0.05), there were 342 genes induced and 248 genes repressed in the wild-type biofilm, whereas 371 genes were induced and 128 genes were repressed in the sporulation mutant biofilm. The microarray results were confirmed with RNA dot blotting. A small portion of cells (1.5%) in the wild-type biofilm formed spores and sporulation genes were highly expressed. In the biofilm formed by the sporulation mutant, competence genes (comGA, srfAA, srfAB, srfAD, and comS) were induced which indicate a role for quorum sensing (bacterial gene expression controlled by sensing their population) in biofilms. There were 53 genes consistently induced in the biofilms of both the wild-type strain and its spoIIGB mutant-those genes have functions for transport, metabolism, antibiotic production-and 26 genes with unknown functions. Besides the large number of genes with known functions induced in the biofilm (121 genes in the wild-type biofilm and 185 genes in the sporulation mutant biofilm), some genes with unknown functions were also induced (221 genes in the wild-type biofilm and 186 genes in the sporulation mutant biofilm), such as the yve operon which appears to be involved in polysaccharide synthesis and the ybc operon which inhibits the growth of competitors for nutrients. A knockout mutant of yveR was constructed, and the mutant showed major defects in biofilm maintenance. Both the wild-type strain and its sporulation mutant formed normal biofilms, suggesting complete sporulation is not necessary for biofilm formation. The expression profiles of these two strains share more repressed genes than induced genes, suggesting that the biofilm cells repress similar pathways in response to starvation and high cell density.