Localization of prophages of serological group B and F on restriction fragments defined in the restriction map of Staphylococcus aureus NCTC 8325

Abstract Several Staphylococcus aureus strains were lysogenized by the phages of serological group B (phages φ53, φ85) as well as by some of serological group F (phages φ77, φ84) and macrorestriction fragment patterns of genomic DNA were estimated in the lysogenized, non-lysogenic and delysogenized (cured of prophages) strains. It was shown that the integration of phage DNA into chromosome of S. aureus leads to specific changes in restriction fragment pattern in all the lysogenized strains. These changes correlate well with the Sma I restriction map of S. aureus NCTC 8325 since they concern the restriction fragments defined in this map. Phages φ53 and φ85 integrate into Sma I fragment B. On the other hand, phages φ77 and φ84 integrate into Smal fragment E of the S. aureus restriction map. The prophages of strain NCTC 8511 have their integration sites, as follows: the phage designated by us φM integrates in fragment A, whereas the integration site for phage φJ lies in fragment E. Phage φM was estimated to be genetically related to phages of serological group A and phage φJ to those of serological group F. Evidence was given that lysogenization of S. aureus strains by at least four prophages does not cast any doubt upon the estimation of their genetic relatedness based on their similarity in restriction pattern.     

A restriction endonuclease from Staphylococcus aureus.

A specific endonuclease, Sau 3AI, has been partially purified from Staphylococcus aureus strain 3A by DEAE-cellulose chromatography. The enzyme cleaves adenovirus type 5 DNA many times, SV40 DNA eight times but does not cleave double-stranded phi X174 DNA. It recognizes the sequence (see article) and cleaves as indicated by the arrows. Evidence is presented that this enzyme plays a role in the biological restriction-modification system of Staphylococcus aureus strain 3A.     

Two plasmid-determined restriction and modification systems in Streptococcus lactis

Two restriction and modification systems were found in Streptococcus lactis strain IL594 which was found to contain 9 plasmids designated pIL1 to pIL9. On the basis of protoplast-induced curing experiments, we showed that a restriction and modification system was related to the presence of pIL6 or pIL7. The pIL6-determined restriction and modification system was confirmed by cotransfer of the plasmid and of the restriction and modification system to a plasmid-free, nonrestricting, and nonmodifying derivative of S. lactis IL594.     

Accumulation of ppGpp and ppGp in Staphylococcus aureus 8325-4 following nutrient starvation

AIM: To investigate the accumulation of highly phosphorylated guanosine nucleotides in Staphylococcus aureus 8325-4 following nutrient deprivation. METHODS AND RESULTS: Nutrient shiftdown of Staph. aureus, HPLC of nucleotides and Western blotting of cell-free extracts. ppGpp rapidly accumulated when cells were deprived of isoleucine following addition of mupirocin, or after carbon deprivation. In contrast, total amino acid starvation led to delayed production of ppGp, which suggests that Staph. aureus exhibits a unique response to total amino acid deprivation compared with other eubacteria. Intracellular ppGp was observed at high levels under all starvation conditions, which suggests that this nucleotide is linked to nutrient limitation and may therefore be involved in regulating the stringent response in Staph. aureus. pppGpp was not observed under any nutrient-limiting condition. Western blot analysis of whole-cell extracts from Staph. aureus 8325-4, showed that antibodies to RelA and SpoT cross-reacted under conditions that detected these proteins in Escherichia coli. CONCLUSIONS: Staph. aureus produces ppGpp and ppGp following nutrient limitation. Immunological analysis indicates that Staph. aureus contains RelA and SpoT proteins, similar to those produced by E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a new example of the diversity of metabolic regulations in bacteria.     

Molecular characterization and functional analysis of the major autolysin of Staphylococcus aureus 8325/4.

The gene encoding the major autolysin of Staphylococcus aureus 8325/4 has been cloned, sequenced, and insertionally inactivated. The three-domain, 137,384-Da protein has a C-terminal glucosaminidase active site and is involved in cell separation, generalized cell lysis, and release of wall material at the cell surface. Expression occurs throughout growth and is stimulated by low temperatures and in the presence of 1 M NaCl.     

Molecular cloning and expression of the coagulase gene of Staphylococcus aureus 8325-4

The gene coding for coagulase (coa) was cloned from Staphylococcus aureus 8325-4 in a lambda replacement vector in Escherichia coli. Coagulase (plasma-clotting) activity was measured in lambda coa lysates and an immunoreactive protein of 60 kDa was detected by Western immunoblotting with anti-coagulase serum. This protein comigrated with the major immunoreactive protein in supernatants of S. aureus 8325-4. The coa gene was subcloned in pUC vectors. One recombinant expressed a 60 kDa immunoreactive protein and plasma-clotting activity. A putative beta-galactosidase-coagulase fusion protein and truncated peptides were expressed by variants formed by subcloning. These results are consistent with previously published biochemical data that the prothrombin-binding domain of coagulase is located in the N-terminus of the protein. The cloned coa gene was transferred into S. aureus on a shuttle plasmid. Expression of coagulase was higher in a strain with a mutation in the agr locus, which controls the level of several exoproteins in S. aureus, suggesting that agr normally regulates coagulase expression negatively.     

Diverse responses of Staphylococcus aureus NCTC 10442 and some of its variants to methicillin.

The methicillin resistant strain of Staphylococcus aureus NCTC 10442 when aged in a broth culture at 42 degrees C yielded variants showing responses of sensitivity, dependence and indifference to the antibiotic. These responses and that of resistance in the parent cells were displayed in agar dilution and agar diffusion experiments at 30 degrees C. At 42 degrees C, all four organisms were sensitive to methicillin.     

The coagulase of Staphylococcus aureus 8325-4. Sequence analysis and virulence of site-specific coagulase-deficient mutants

The sequence of the coagulase gene (coa) from Staphylococcus aureus strain 8325-4 is reported. The deduced amino acid sequence of the coagulase protein is compared with previously reported sequences of coagulases from strains 213 and BB. The secreted mature forms of coagulase proteins are composed of three distinct segments: (i) the N-terminal 150-270 residues, which are c. 50% identical, (ii) a central region with high (greater than 90%) residue identities, and (iii) a C-terminal region composed of repeated 27-amino-acid residue sequences. The variable N-terminal sequences are probably responsible for antigenic differences among coagulases of different serotype. The region of coagulase which binds to prothrombin and activates it to form staphylothrombin is also located in the N-terminal half of the protein. A site-specific substitution mutation in the coa gene, which abolished plasma clotting activity, was isolated by recombinational allele-replacement in strains 8325-4 and M60. The Coa- mutants did not show diminished virulence in subcutaneous and intramammary infections of mice. No evidence for a role for coagulase in virulence of toxigenic or nontoxigenic strains was obtained. This contradicts findings of several groups using Coa- mutants generated by chemical mutagenesis and suggests that the earlier results were obtained with strains that had suffered additional mutations in virulence-related genes.     

GATC-specific restriction and modification systems in treponemes

AIMS: To investigate the presence of GATC-specific modification and restriction activities in rumen isolates of Treponema sp. METHODS: The presence of N6-methyladenine within GATC (Dam) sequences was analysed using isoschizomeric restriction endonucleases having different sensitivities to the methylation of the target sequence. A fast screening method was used for testing of site-specific endonuclease activities directly in crude cell extracts. Three out of six rumen isolates of Treponema sp. showed restriction activities. Restriction endonucleases were further purified by Heparin-Sepharose chromatography. Using PCR and specific primers, no sequence homologous to the T. pallidum dam gene was found. CONCLUSIONS: Three rumen treponemal strains were documented to possess MboI isoschizomeric restriction-modification systems. SIGNIFICANCE: This is the first report on restriction activity in rumen treponemes.     

Physical and genetic mapping of the protein A gene in the chromosome of Staphylococcus aureus 8325-4

The gene coding for protein A (spa) has been mapped close to nov on the genetic map of the chromosome of Staphylococcus aureus 8325-4. A rapid mapping procedure has been developed which first allowed the region of the chromosome carrying the spa gene to be identified by blot +hybridization of large DNA fragments which had been separated by pulsed-field gel electrophoresis. Restriction endonuclease SmaI fragment G was shown to carry the spa gene. An insertion mutation in spa was constructed by in vitro insertion of a fragment of DNA expressing resistance to kanamycin and neomycin. A spa::Kan(r)Neo(r) mutation was isolated in S. aureus 8325-4 by allele replacement. This provided a selectable marker which allowed the spa gene to be mapped by transformation analysis.     

Co-transfer of vancomycin and other resistance genes from Enterococcus faecalis NCTC 12201 to Staphylococcus aureus

Conjugative transfer, in the apparent absence of plasmid DNA, of high-level vancomycin resistance from Enterococcus faecalis NCTC 12201 to Staphylococcus aureus B111 has been demonstrated in vivo and in vitro. Selection of transconjugants on media containing erythromycin or chloramphenicol may result in the transfer of resistance to erythromycin, chloramphenicol, gentamicin, streptomycin and vancomycin though these are capable of separate transfer. Vancomycin resistance has not been transmitted from staphylococcus to staphylococcus though transfer of erythromycin and of chloramphenicol resistance has been achieved.