Genetic analysis of spo0A and spo0C mutants of Bacillus subtilis with a phi 105 prophage merodiploid system.

An 8.0-kilobase chromosomal fragment of Bacillus subtilis which contained an intact spo0A gene was recloned onto temperate phage phi 105 from the rho 11dspo0A+-1 transducing phage. A specialized transducing phage, phi 105-dspo0A+-1, was constructed and used to transduce the spo0A12 mutant strain 1S9. A Spo+ transductant which was a single lysogen of the phi 105dspo0A+-1 transducing phage was isolated. From competent cells of this Spo+ transductant was isolated a Spo- (Spo0A) strain which was immune to phi 105. It was used to prepare a lysate of the phi 105dspo0A12 phage. Transduction of the spo0C9V recE4 strain with the phi 105dspo0A12 and phi 105dspo0A+-1 phages was carried out. The phi 105dspo0A+-1 phage gave rise to a large number of heat-resistant cells, but the phi 105dspo0A12 phage formed no heat-resistant cells. These results indicate that the spo0A12 and spo0C9V mutant genes do not complement each other in the ability to sporulate and that the spo0C9V mutation is located within the spo0A gene. Although the spo0C9V strain was completely asporogenous, the spo0C9V/spo0C9V diploid strain produced heat-resistant cells at a frequency of ca. 10(-3) in the sporulation medium. This result indicates that two copies of the spo0C9V mutant gene partially restore the ability of these cells to sporulate.     

Early-blocked asporogenous mutants of Bacillus subtilis are lysogenized at reduced frequency by temperate bacteriophages.

The establishment of lysogeny in early-blocked asporogenous (Spo-) mutants of Bacillus subtilis 168, which were also defective in the production of antibiotics (Abs-), by temperate phage phi105 or SPO2 was studied. It was found that the frequency of lysogenization of Spo-Abs-mutants was 10 to 20% that of the wild-type bacteria. There was no difference in the efficiency of plating and the burst size of phi105 between wild-type and mutant strains. Phi105 lysogens of mutant strains were as stable as those of the wild type. Several rifampin-resistant mutants defective in the production of antibiotics were isolated. They were also defective in spore formation and lysogenized by phi105 at reduced frequency.     

Formation of virulent antigen-modified mutants (Fra-, Fra-Tox-) of plague bacteria resistant to rifampicin and quinolones]

Experiments were performed with two strains of plague bacteria--231 (isolated from marmot) and 358 (isolated from human) and their isogenic variants with Fra- and Fra-Tox- phenotype. Mutants resistant to rifampicin (Rifr) and nalidixic acid (Nalr) appeared independently of pathogen phenotype and genotype with frequency n.10(-8)-n.10(-9), subsequently. Rifr mutation influenced on virulence manifestation at albino mice and antigendeficient variants with Fra- and Fra-Tox- phenotype. In every group of strains highly virulent subcultures were registered. Resistance to nalidixic acid mainly was not associated with virulence loss. Nalr mutants of parent and antigenmodified mutants were cross resistant to fluoroqinolones (ciprofloxacin, ofloxacin, pefloxacin, lomefloxacin). LD50 for untreated albino mice did not differ from LD50, for mice treated with rifampicin (when mice were infected with strain resistant to rifampicin) or with nalidixic acid and fluoroquinolones (when animals were infected with Nalr mutants). Antigenmodified strains of plague bacteria and their Rifr, Nalr mutants were able to overcome specific immune reaction. The drugs should be used in synergic combinations (with aminoglycosides or cephalosporines of III generation) to prevent appearance of virulent strains resistant to rifampicin and fluroquinolones.     

Glycosylation of a Streptomyces coelicolor A3(2) cell envelope protein is required for infection by bacteriophage phi C31

Mutants of Streptomyces coelicolor A3(2) J1929 (Delta pglY) were isolated that were resistant to the Streptomyces temperate phage phi C31. These strains could be transfected with phi C31 DNA, but could not act as infective centres after exposure to phage. Thus, it was concluded that infection was blocked at the adsorption/DNA injection step. The mutants fell into three classes. Class I mutants were complemented by a gene, SCE87.05, isolated from the cosmid library of S. coelicolor A3(2). The product of SCE87.05 had good overall homology to a Mycobacterium tuberculosis hypothetical protein and regions with similarity to dolichol phosphate-D-mannose:protein O-D-mannosyltransferases. Concanavalin A (ConA) inhibited phi C31 infection of S. coelicolor J1929, and this could be partially reversed by the addition of the sugar, alpha-D-methyl-pyranoside. Moreover, glycosylated proteins from J1929, but not from the class I mutant DT1017, were detected using ConA as a probe in Western blots. Class I and II mutants were sensitive to phi C31hc, a previously isolated phage exhibiting an extended host range phenotype, conferred by h. A phage with the same phenotype, phi DT4002, was isolated independently, and a missense mutation was found in a putative tail gene. It is proposed that the phi C31 receptor is a cell wall glycoprotein, and that the phi C31h mutation compensates for the lack of glycosylation of the receptor.     

Factors involved in the electroporation-induced transformation of Clostridium perfringens

The following factors were found to improve the efficiency of transformation of Clostridium perfringens 3624A Rifr Strr: (1) a reduction in cuvette sample volume (DNA and cell suspension) to 0.8 ml, (2) use of a 1 microgram/ml concentration of transforming DNA, (3) use of late-logarithmic phase cells, (4) 3-fold concentration of cell density (3.0 x 10(8) CFU/ml), and (5) a reduction in the pH of the expression and selective plating medium to 6.4. Application of the improved conditions resulted in transformation efficiencies for C. perfringens 3624A Rifr Strr ranging from 7.1 transformants/microgram DNA for plasmic pIP401 to 9.2 x 10(4) transformants per microgram DNA for plasmid pAK201. The greatest transformation efficiency obtained using pAK201 was 9.8 x 10(6) transformants/micrograms DNA for C. perfringens strain 13. Using the improved protocol, pAM beta 1 was transformed at a 42-fold greater level when compared with the values reported earlier [1]. In addition to C. perfringens 3624A Rifr Strr, strains 13, 10543A, 3628C, NTG-4, and 3624A were successfully transformed. Nuclease does not appear to be a factor in the C. perfringens strain-specific electro-transformation protocol.     

Structural alterations in the Bacillus subtilis Spo0A regulatory protein which suppress mutations at several spo0 loci

Secondary site mutations that restore sporulation to sporulation-defective spo0F or spo0B deletion mutants were found to reside in the spo0A gene. Sequence analysis of 23 such sof mutants showed that the sof mutations fell into six classes of missense codon changes, primarily in the conserved amino-terminal domain of the response regulator Spo0A protein. Changes were observed in codons 12, 14, 60, 92, and 121. The residues affected were predominantly located in the potential turn regions at one end of the amino-terminal conserved domain on the same topological face as the active site aspartate residues. The ability of sof mutations to suppress deficiencies in the transmitter kinases, KinA and KinB, of two-component regulatory systems was tested. All of the sof mutations suppressed the sporulation deficiency of kinA mutants but only two classes among five tested suppressed kinB mutations. sof mutants segregated Spo- colonies at high frequency. Five of these Spo- mutants were found to result from mutations in the spo0A locus that reversed the effect of the sof mutatation. One of these was sequenced and found to have the original sof mutation and a new mutation, sos, at codon 105. The accumulation of sos mutations in sof strains suggested that the sof mutations have a subtle, yet deleterious, effect on the growth of the cell. The results suggested that the sof mutations increase the avidity for or reactivity with transmitter kinases in an allele-specific manner, although in some cases it is possible that the sof mutations obviate the need for phosphorylation to activate the Spo0A protein. An alternative hypothesis is presented in which the sof mutations play the role of bypass mutations for kinases.     

Production of asporogenous mutants of Bacillus sphaericus 2362 in continuous culture

AIMS: To report the production of asporogenous mutants (Spo-) of Bacillus sphaericus 2362 in continuous culture. METHODS AND RESULTS: Microbial culture samples were taken at 0.05 h-1 dilution rate and plated out on nutrient agar plates. Translucent colonies were obtained with vegetative morphology under phase contrast microscope. Heat resistance evaluations at different temperature settings showed that the Spo- mutants had lower heat resistance than the Spo+ wild type. Western blots analyses carried out on both wild type and the mutants indicated the presence of binary protein toxins of 42 and 51 kDa in both. Bioassays carried out on the wild type and the Spo- mutants against mosquitoes showed the mutants to be 100-fold less toxic in comparison to the wild type. CONCLUSION: Existence and production of asporogenous mutants of Bacillus sphaericus 2362 in continuous culture at low dilution rates is demonstrated by this study. The organism's ability to produce toxins appears to be significantly reduced by the mutational process. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of asporogenous mutants had not been reported previously among strains of Bacillus sphaericus. The present report on the toxigenic capability of asporogenous mutants also raises the possibility of using continuous culture to significantly improve the productivity of toxin production in future.     

Factors Affecting Competence for Transformation in Staphylococcus aureus

A chemically defined medium has been developed for isolation of amino acid-requiring mutants of Staphylococcus aureus strain 8325, and for use as a selective medium in transformation assays. Variables affecting transformation of both plasmid and chromosomal markers have been studied. The optimal pH and temperature for transformation are 6.75 to 7.0 and 30 C, respectively. Ca ions are required for transformation, and only cells lysogenic for the phage phi11 can be transformed. Superinfection of competent cells with phi11 does not increase the transformation frequency. Maximal number of transformants is obtained after 20 min of contact between cells and deoxyribonucleic acid. The transformation frequencies for the plasmid marker erythromycin resistance (ero) and the chromosomal markers trp, thy, and cyt are of the same order of magnitude, whereas the frequency for the chromosomal marker tyr is approximately one order of magnitude lower.     

Spo0A Differentially Regulates Toxin Production in Evolutionarily Diverse Strains of Clostridium difficile

Clostridium difficile is an important pathogen of humans and animals, representing a significant global healthcare problem. The last decade has seen the emergence of epidemic BI/NAP1/027 and ribotype 078 isolates, associated with the onset of more severe disease and higher rates of morbidity and mortality. However, little is known about these isolates at the molecular level, partly due to difficulties in the genetic manipulation of these strains. Here we report the development of an optimised Tn916-mediated plasmid transfer system, and the use of this system to construct and complement spo0A mutants in a number of different C. difficile strain backgrounds. Spo0A is a global regulator known to control sporulation, but may also be involved in the regulation of potential virulence factors and other phenotypes. Recent studies have failed to elucidate the role of Spo0A in toxin A and toxin B production by C. difficile, with conflicting data published to date. In this study, we aimed to clarify the role of Spo0A in production of the major toxins by C. difficile. Through the construction and complementation of spo0A mutants in two ribotype 027 isolates, we demonstrate that Spo0A acts as a negative regulator of toxin A and toxin B production in this strain background. In addition, spo0A was disrupted and subsequently complemented in strain 630Δerm and, for the first time, in a ribotype 078 isolate, JGS6133. In contrast to the ribotype 027 strains, Spo0A does not appear to regulate toxin production in strain 630Δerm. In strain JGS6133, Spo0A appears to negatively regulate toxin production during early stationary phase, but has little effect on toxin expression during late stationary phase. These data suggest that Spo0A may differentially regulate toxin production in phylogenetically distinct C. difficile strain types. In addition, Spo0A may be involved in regulating some aspects of C. difficile motility.     

Identification of Four Phage Resistance Plasmids from Lactococcus lactis subsp. cremoris HO2

The bacteriophage-host sensitivity patterns of 16 strains of Lactococcus lactis originally isolated from a mixed strain Cheddar cheese starter culture were determined. Using phages obtained from cheese factory whey, four of the strains were found to be highly phage resistant. One of these isolates, Lactococcus lactis subsp. cremoris HO2, was studied in detail to determine the mechanisms responsible for the phage insensitivity phenotypes. Conjugal transfer of plasmid DNA from strain HO2 allowed a function to be assigned to four of its six plasmids. A 46-kb molecule, designated pCI646, was found to harbor the lactose utilization genes, while this and plasmids of 58 kb (pCI658), 42 kb (pCI642), and 4.5 kb (pCI605) were shown to be responsible for the phage resistance phenotypes observed against the small isometric-headed phage 712 (936 phage species) and the prolate-headed phage c2 (c2 species). pCI658 was found to mediate an adsorption-blocking mechanism and was also responsible for the fluffy pellet phenotype of cells containing the molecule. pCI642 and pCI605 were both shown to be required for the operation of a restriction-modification system.     

Effect of Elevated Temperature on Deoxyribonucleic Acid Synthesis in Bacteriophage φ29-Infected Bacillus amyloliquefaciens

Deoxyribonucleic acid (DNA) synthesis in bacteriophage phi29-infected Bacillus amyloliquefaciens was studied at 37 and 45 C. Infectious intracellular particles appear at the same time at both temperatures, but the average burst size is reduced 45 to 50% at 45 C. There is a transient inhibition of cellular mass increase at 45 C which is not observed at the lower temperature. In addition, the rate of host DNA synthesis is reduced and the onset of viral-specific DNA replication is delayed for 6 to 9 min at 45 C. These findings allowed us to screen phage phi29 mutants which are sensitive to growth at 45 C for their ability to synthesize phi29 DNA in the absence of host DNA replication. We obtained mutants which make no viral DNA, reduced levels of DNA, or normal quantities of DNA under nonpermissive conditions. Pulse-labeled viral DNA which sediments more rapidly than mature phi29 DNA molecules was observed after gentle cell lysis and zone sedimentation. This DNA is not a precursor of normally sedimenting phi29 DNA and apparently consists of mature phi29 DNA molecules aggregated with large pieces of bacterial DNA.     

Identification of the lipopolysaccharide core region as the receptor site for a cytotoxin-converting phage, phi CTX, of Pseudomonas aeruginosa.

A temperate phage, phi CTX, is a cytotoxin-converting phage of Pseudomonas aeruginosa. In this study, we characterized the lipopolysaccharide (LPS) structures of phi CTX-resistant mutants derived from phi CTX-sensitive strains. phi CTX infectivity was neutralized by LPS preparations derived from sensitive strains but not by those from resistant strains. phi CTX-resistant mutants had lower-molecular-weight rough (R)-type LPS than the parental strains and lacked the reactivity of some anti-LPS core monoclonal antibodies. Some LPS core components were lacking or significantly decreased in the resistant mutants. These results suggested that a receptor site of the cytotoxin-converting phage phi CTX was the LPS core region and that especially L-rhamnose and D-glucose residues in the outer core were involved in phage binding. The host range of phi CTX was nearly O-serotype dependent, probably because of the diversity of the LPS core structure among P. aeruginosa strains. phi CTX bound to most strains of Homma serotypes A, G, and I but not to strains of serotypes B and E. Furthermore, we found that a genetic locus specifying phi CTX sensitivity (and consequently participating in the biosynthesis of part of the LPS core) existed in or near the locus participating in the determination of O-serotype specificity (somA), which has been mapped between leu-10 and eda-9001. phi CTX, as well as anti-LPS core monoclonal antibodies, will be a good tool for structural characterization of the P. aeruginosa LPS core region.     

Characterization of a Generalized Transducing Phage of Poly-(gamma)-Glutamic Acid-Producing Bacillus subtilis and Its Application for Analysis of Tn917-LTV1 Insertional Mutants Defective in Poly-(gamma)-Glutamic Acid Production

A generalized transducing phage, (phi)BN100, was isolated from a Bacillus subtilis (natto) strain producing poly-(gamma)-glutamic acid ((gamma)PGA). Transduction frequencies for a given marker ranged from 3.8 x 10(sup-8) to 1.6 x 10(sup-6) per phage particle. The genome size of the phage was approximately 42 kb. (phi)BN100 was used successfully to identify bona fide Tn917-LTV1 transpositional mutants defective in (gamma)PGA production.     

Bacteriophage and bacteriophage-like structures carried by Bacillus medusa and their effect on sporulation.

Bacillus medusa was found to carry three phages or phagelike structures named phi med-1, phi med-2, and phi med-3. phi med-1 is a minute, 25-nm-diameter particle without a tail. It was extracted from the sporulation lysate of a phi med-2-minus strain of B. medusa and purified by differential centrifugation. The nucleic acid from this structure was shown to be orcinol positive, alkali sensitive, RNase sensitive, and DNase resistant. An RNase-resistant core of nucleic acid was not found, indicating that it was single-stranded RNA. A host strain has not yet been found for phi med-1. Phage phi med-3 was induced with mitomycin C or UV light and consisted of empty heads of 57 nm in diameter, whereas phi med-2 induced with mitomycin was a phage of 60-nm head diameter and 220-nm tail length. The sporulation sequence proceeded faster in those mutants lacking phi med-2, and when the phage was reintroduced to B. medusa the extended wild-type sporulation sequence was observed. B. thuringiensis var. schwetzova was sensitive to phi med-2 and yielded small turbid plaques. B. medusa produced small numbers of phi med-2 during growth. The other phage may be produced at the same time but were not detected. Phi med-1 was found in sporulating cells by electron microscopy techniques. Its release from these was demonstrated by both electron microscopy techniques and a radioactive assay. It appears to participate in the formation of a surface layer on the parasporal inclusion of B. medusa.     

Molecular basis for the exploitation of spore formation as survival mechanism by virulent phage phi29

Phage phi29 is a virulent phage of Bacillus subtilis with no known lysogenic cycle. Indeed, lysis occurs rapidly following infection of vegetative cells. Here, we show that phi29 possesses a powerful strategy that enables it to adapt its infection strategy to the physiological conditions of the infected host to optimize its survival and proliferation. Thus, the lytic cycle is suppressed when the infected cell has initiated the process of sporulation and the infecting phage genome is directed into the highly resistant spore to remain dormant until germination of the spore. We have also identified two host-encoded factors that are key players in this adaptive infection strategy. We present evidence that chromosome segregation protein Spo0J is involved in spore entrapment of the infected phi29 genome. In addition, we demonstrate that Spo0A, the master regulator for initiation of sporulation, suppresses phi29 development by repressing the main early phi29 promoters via different and novel mechanisms and also by preventing activation of the single late phi29 promoter.     

Autocide AMI rescues development in dsg mutants of Myxococcus xanthus

Low concentrations of autocide AMI rescued aggregation and sporulation in the dsg mutant class of Myxococcus xanthus but were incapable of rescuing asg, bsg, or csg mutants. AMI-induced spores of dsg mutants were resistant to heat and sonication and germinated when plated on nutrient-rich agar. AMI accelerated aggregation and sporulation and increased the final spore number in submerged cultures of a wild-type strain of M. xanthus. Development of M. xanthus was accompanied by release of a fluorescent material (emission maximum, 438 nm) into the supernatant fluid. The release of this material began early and continued throughout development. All Spo- mutant strains tested released significantly reduced levels of this material. These levels were increased in the presence of AMI in all Spo- mutant classes, most dramatically in the dsg mutants.