Whole-genome sequencing of Staphylococcus aureus strain RN4220, a key laboratory strain used in virulence research, identifies mutations that affect not only virulence factors but also the fitness of the strain

Staphylococcus aureus RN4220, a cloning intermediate, is sometimes used in virulence, resistance, and metabolic studies. Using whole-genome sequencing, we showed that RN4220 differs from NCTC8325 and contains a number of genetic polymorphisms that affect both virulence and general fitness, implying a need for caution in using this strain for such studies.     

Effect of the Prophage and Penicillinase Plasmid of the Recipient Strain Upon the Transduction and the Stability of Methicillin Resistance in Staphylococcus aureus

Transduction of a methicillin-resistance determinant (mec) in Staphylococcus aureus RN450 was dependent on its prior lysogenization with an appropriate temperate phage. In addition, an appropriate transduced penicillinase plasmid was usually required. Some phage 80-resistant variants of RN450 or of its parental lysogenic strain, NCTC 8325, were also effective recipients for transduction of mec. Elimination of prophage from RN450 abrogated its effectiveness as a transductional recipient of mec. Elimination of prophage from a methicillin-resistant transductant of RN450 reduced resistance to undetectable levels in six of seven phage-eliminated strains. In four of these a variable number of clones again became phenotypically resistant after lysogenization alone or lysogenization combined with reintroduction of a penicillinase plasmid. In two prophage-eliminated strains, no evidence of residual mec could be adduced. The establishment, expression, or stability of the transduced mec in strain RN450 appeared to depend on some function determined by a prophage or a prophage and a penicillinase plasmid.     

Interstrain transfer of the prophage ϕNM2 in staphylococcal strains

Staphylococcus aureus is a successful pathogen in part because the bacterium can adapt rapidly to selective pressures imparted by the external environment. Horizontal gene transfer (HGT) plays an integral role in the evolution of bacterial genomes, and phage transduction is likely to be the most common and important HGT mechanism for S. aureus. Phage can transfer not only its own genome DNA but also host bacterial DNA with or without pathogenicity islands to other bacteria. Here, we demonstrate that the staphylococcal prophage ?NM2 could transfer between strains Newman and NCTC8325/NCTC8325-4 by simulating a natural situation in laboratory without mitomycin C or ultra-violet light treatment. This transference may be caused by direct contact between Newman and NCTC8325/NCTC8325-4 instead of phage particles released in Newman culture’s supernatant. The rates of successful horizontal genetic transfer in recipients NCTC8325 and NCTC8325-4 were 2.1% and 1.8%, respectively. Prophage ?NM2 was integrated with one direction at an intergenic region between rpmF and isdB in all 17 lysogenic isolates. Phage particles were spontaneously released from lysogenic strains again and had no noticeable influence on the growth of host cells. The results reported herein provide insight into how mobile genetic elements such as prophages can lead to the emergence of genetic diversity among S. aureus strains.     

Enhancement of the viability of irradiated bacteria by chromosome transfer

The effect of x-irradiating recipient cells of Escherichia coli K-12 before mating on the formation of recombinants and on the distribution of parental genetic material among recombinants was investigated in both the wild-type (Rec+) and a recombination-deficient (Rec-) mutant. In crosses involving Rec- recipients, recombinants selected for a late donor marker were formed in almost normal numbers. Rec- cells exposed to otherwise lethal doses of x-rays were still able to form viable recombinants for a distal male marker. These recombinants had inherited almost all the transferred donor chromosome, as evidenced by the preponderance of male markers in the recombinants. In contrast, the recombinant-forming ability was about as x-ray-sensitive as the colony-forming ability in Rec+ cells. No preference for donor chromosomal material was observed in recombinants from crosses involving x-irradiated Rec+ cells.     

Comparative genomics of Staphylococcus aureus musculoskeletal isolates

Much of the research aimed at defining the pathogenesis of Staphylococcus aureus has been done with a limited number of strains, most notably the 8325-4 derivative RN6390. Several lines of evidence indicate that this strain is unique by comparison to clinical isolates of S. aureus. Based on this, we have focused our efforts on two clinical isolates (UAMS-1 and UAMS-601), both of which are hypervirulent in our animal models of musculoskeletal infection. In this study, we used comparative genomic hybridization to assess the genome content of these two isolates relative to RN6390 and each of seven sequenced S. aureus isolates. Our comparisons were done by using an amplicon-based microarray from the Pathogen Functional Genomics Resource Center and an Affymetrix GeneChip that collectively represent the genomes of all seven sequenced strains. Our results confirmed that UAMS-1 and UAMS-601 share specific attributes that distinguish them from RN6390. Potentially important differences included the presence of cna and the absence of isaB, sarT, sarU, and sasG in the UAMS isolates. Among the sequenced strains, the UAMS isolates were most closely related to the dominant European clone EMRSA-16. In contrast, RN6390, NCTC 8325, and COL formed a distinct cluster that, by comparison to the other four sequenced strains (Mu50, N315, MW2, and SANGER-476), was the most distantly related to the UAMS isolates and EMRSA-16.     

Molecular cloning and mapping of 16S-23S rRNA gene complexes of Staphylococcus aureus.

Staphylococcus aureus BB255, a derivative of NCTC8325, had six rRNA operons, and each operon contained two SmaI sites about 3 kb apart. By molecular cloning and pulsed-field gel electrophoresis, all operons were mapped at the junctions of SmaI fragments in the published map of NCTC8325 except one, which was connected to a previously unidentified 23-kb SmaI fragment.     

Staphylococcus aureus SH1000 and 8325‐4: comparative genome sequences of key laboratory strains in staphylococcal research

Aims: To provide comparative genome sequence data for two related model strains of Staphylococcus aureus (SH1000 and 8325‐4) that are used extensively in laboratory research. Methods and Results: Comparative genome sequencing was used to identify genetic differences between Staph. aureus SH1000 and the fully genome‐sequenced ancestral strain, Staph. aureus NCTC 8325. PCR amplification and DNA sequencing were employed to determine which of the genetic polymorphisms identified were also present in Staph. aureus 8325‐4, a direct derivative of 8325 and the parent strain of SH1000. Aside from known genetic differences between these strains, Staph. aureus SH1000 harboured 15 single‐nucleotide polymorphisms compared with 8325 (of which 12 were also found in 8325‐4), and a 63‐bp deletion upstream of the spa gene not present in either 8325 or 8325‐4. Conclusions: Staphylococcus aureus SH1000 and 8325‐4 contain a number of genetic polymorphisms relative to the progenitor strain of the lineage (8325) and to each other. Significance and Impact of the Study: The comparative genome sequences of SH1000 and 8325‐4 presented here define the genotypes of two key strains in staphylococcal laboratory research and reveal genetic polymorphisms that may impact their phenotypic properties.     

Changes in Staphylococcus aureus susceptibility to bactericidal action of teicoplanin in the presence of different prophages in the bacterial genome

The influence exerting by lysogeny state on the susceptibility of Staphylococcus aureus to bactericidal action of teicoplanin was studied. In this aim the standard, non-lysogenic, bacteriophage-free S. aureus NCTC 8325-4 strain was lysogenized with 10 different, bacteriophages obtained in our laboratory. All bacteriophages were derived from multiresistant S. aureus strains and all were able to convert staphylokinase. For all derivatives MBCs and MICs of teicoplanin were determined. In the case of four strains the ratio MBC/MIC showed the presence of tolerance to teicoplanin (MBC/MIC > or = 32) and was significantly higher than in the case of the parent strain NCTC8325-4. In the case of two strains this ratio was smaller than for parent strain. Only small correlation with our previous results obtained for vancomycin was observed.     

The interleukin-1 receptor antagonist gene and the inhibitor of kappa B-like protein gene polymorphisms are not associated with myocardial infarction in Slovene population with type 2 diabetes

In this study we investigated the association of the interleukin-1 receptor antagonist gene variable number tandem repeat (IL1RN VNTR) polymorphism and of the inhibitor of kappa B-like protein (IKBL) gene polymorphism with myocardial infarction (MI) in a group of patients with type 2 diabetes. The IL1RN VNTR and the IKBL+ 738T > C gene polymorphisms were tested in 374 Caucasians: 151 cases with MI and 223 subjects with no history of coronary artery disease. The IL1RN VNTR polymorphism was not a risk factor for MI in Caucasians with type 2 diabetes (genotype 22 vs. the rest: odds ratio (OR) 1.6; 95% confidence interval (CI) = 0.8-3.5; p = 0.2). We also failed to demonstrate that IKBL+ 738T > C gene polymorphism was associated with MI in patients with type 2 diabetes (OR = 0.9; 95% CI = 0.3-2.6; p = 0.9). We provide evidence that the IL1RN VNTR and the IKBL + 738T > C gene polymorphisms are not risk factors for MI in Caucasians with type 2 diabetes.     

Chromosomal mapping of a gene affecting enterotoxin A production in Staphylococcus aureus

In a previous study, transformation demonstrated that a gene governing enterotoxin A production (entA+) in Staphylococcus aureus strain S-6 was located on the chromosome between the purB110 and ilv-129 markers; in contrast, the entA+ gene of strain FRI-196E was shown not to be located in the same position. In the current study, 54 enterotoxin A-producing strains of S. aureus were examined to locate the entA+ gene. Conventional transformation procedures and a series of multiply marked derivatives of NCTC 8325 were used as recipients for chromosomal mapping. Of the 54 strains tested, 23 were found to contain the entA+ gene at the original locus between the purB110 and ilv-129 markers. Twenty-seven strains could not be analyzed either because their DNA was genetically ineffective in transforming strain 8325 (23 strains), or Pur+ Ilv+ transformants could not be recovered (four strains). Four other strains contained an entA+ gene that could not be located in any of the chromosomal linkage groups. A new insertion site for Tn551 was located within the hla+ gene involved in alpha-toxin production. It eliminated alpha-toxin production and was used to separate the entA+ gene from the hla+ marker in the purB110-ilv-129 region. This segment of the chromosome is shown to consist of the purB110, entA+, hla+, and ilv-129 markers in that order.     

Proline transport in Staphylococcus aureus: a high-affinity system and a low-affinity system involved in osmoregulation.

L-Proline enhanced the growth of Staphylococcus aureus in high-osmotic-strength medium, i.e., it acted as an osmoprotectant. Study of the kinetics of L-[14C]proline uptake by S. aureus NCTC 8325 revealed high-affinity (Km = 1.7 microM; maximum rate of transport [Vmax] = 1.1 nmol/min/mg [dry weight]) and low-affinity (Km = 132 microM; Vmax = 22 nmol/min/mg [dry weight]) transport systems. Both systems were present in a proline prototrophic variant grown in the absence of proline, although the Vmax of the high-affinity system was three to five times higher than that of the high-affinity system in strain 8325. Both systems were dependent on Na+ for activity, and the high-affinity system was stimulated by lower concentrations of Na+ more than the low-affinity system. The proline transport activity of the low-affinity system was stimulated by increased osmotic strength. The high-affinity system was highly specific for L-proline, whereas the low-affinity system showed a broader substrate specificity. Glycine betaine did not compete with proline for uptake through either system. Inhibitor studies confirmed that proline uptake occurred via Na(+)-dependent systems and suggested the involvement of the proton motive force in creating an Na+ gradient. Hyperosmotic stress (upshock) of growing cultures led to a rapid and large uptake of L-[14C]proline that was not dependent on new protein synthesis. It is suggested that the low-affinity system is involved in adjusting to increased environmental osmolarity and that the high-affinity system may be involved in scavenging low concentrations of proline.     

Isolation and Characterization of a Recombination-Deficient Hfr Strain

The isolation of a rec(-) Hfr strain of Escherichia coli K-12 is described. The method used consisted of mating AB2463 F(-) Rec(-) His(-) Lac(-) with P4X6 Hfr Rec(+) His(+) Lac(+), selecting Rec(-) His(-) Lac(+) recombinants, and searching for Hfr strains. One Hfr rec(-) strain, no. 12, was used as donor in crosses with Rec(+) and Rec(-) recipients. Crosses with Rec(+) recipients are fertile, and those with Rec(-) recipients are almost infertile, the frequency of recombinants being 10(-2) to 10(-3) that found with Rec(+) recipients. The Rec(-) mutant marker is transfered to and integrated into Rec(+) recipients. Zygotic induction of prophage lambda is observed in crosses between two Rec(-) strains. In crosses of F(-) Rec(-) with Hfr Rec(-), the gradient of integration frequencies for markers progressively more distant from the origin is steeper than in the Rec(+) x Rec(-) or the Rec(-) x Rec(+) crosses.     

Mechanism of conjugation and recombination in bacteria. XXI. Role of F factor genes in post-conjugational recombination in Escherichia coli K-12

Temperature sensitive dnaA recipient crossed at the restrictive temperature with HfrH, free from contaminating F+ cells, forms recombinants almost as proficiently as at the permissive temperature. The merozygotes are able to synthesize DNA at 42 degrees C, although the recipient and donor cells do not incorporate 3H-thymine. A substantial fraction of Lac+ recombinants, irrespective of the mating temperature, is temperature resistant (42 C-R); 15% from among those mated at 35 C and 30% from those mated at 42 C. The presence of dnaA mutation in these Lac+ 42 C-R recombinants was ascertained by co-transduction with ilv. Cell division at 42 C is inhibited in the Lac 42 C-R recombinants by acridine orange. The presence of F factor DNA in these recombinants was demonstrated directly by DNA: DNA hybridization. Suppression of dnaA mutation in Lac+ 42 degrees C-R recombinants and their sensitivity to acridine orange at 42 degrees C suggests that at least part of the F factor is integrated into the recombinant chromosome. A large fraction of the Lac+ 42 degrees C-R recombinants (up to 80%) is sensitive to male phage R17 and fertile. In crosses with HfrC there is a marked decrease of recombination frequency at 42 degrees C in the dnaA recipient. The fraction of Lac+ 42 degrees C-R recombinants is low (up to 10%) and the 42 degrees C-R recombinants are neither sensitive to male phage nor fertile. The results are discussed on the basis of the previously proposed model of post-conjugational recombination.     

Identification of a chromosomal determinant of enterotoxin A production in Staphylococcus aureus.

Chromosomal mapping of the determinants for enterotoxin A and enterotoxin B production in three strains of Staphylococcus aureus was attempted by using conventional transformation procedures and a series of multiply marked derivatives of NCTC 8325 as recipients. A gene governing enterotixin A production (entA+) in strain S-6 was located on the chromosome between the pur-110 and ilv-129 markers, very close to a determinant of alpha-hemolysin production, hla+. The entA+ gene of strain FRI-196E was shown not to be located at the same position; its location could not be determined. The entB+ genes of strains S-6 and C243 were not located within the known linkage groups examined. Recombinants were screened for enterotoxin production by a new procedure that combined characteristics of immune serum plate and optimal sensitivity plate procedures. The strains and methods used in this study of enterotoxin determinants should prove useful in genetic studies to locate other chromosomal determinants of S. aureus whose phenotypes are difficult to score or select for.     

Chromosomal mapping of a gene affecting enterotoxin A production in Staphylococcus aureus.

In a previous study, transformation demonstrated that a gene governing enterotoxin A production (entA+) in Staphylococcus aureus strain S-6 was located on the chromosome between the purB110 and ilv-129 markers; in contrast, the entA+ gene of strain FRI-196E was shown not to be located in the same position. In the current study, 54 enterotoxin A-producing strains of S. aureus were examined to locate the entA+ gene. Conventional transformation procedures and a series of multiply marked derivatives of NCTC 8325 were used as recipients for chromosomal mapping. Of the 54 strains tested, 23 were found to contain the entA+ gene at the original locus between the purB110 and ilv-129 markers. Twenty-seven strains could not be analyzed either because their DNA was genetically ineffective in transforming strain 8325 (23 strains), or Pur+ Ilv+ transformants could not be recovered (four strains). Four other strains contained an entA+ gene that could not be located in any of the chromosomal linkage groups. A new insertion site for Tn551 was located within the hla+ gene involved in alpha-toxin production. It eliminated alpha-toxin production and was used to separate the entA+ gene from the hla+ marker in the purB110-ilv-129 region. This segment of the chromosome is shown to consist of the purB110, entA+, hla+, and ilv-129 markers in that order.     

An amber replication mutant of F plasmid mapped in the minimal replication region

We have constructed a mini-F derivative (pKP1013) consisting of a 5.4 kilobase pairs (kb) segment (44.0 to 49.4 kb) of mini-F and fragments carrying the chloramphenicol and spectinomycin resistance genes that originated from the R plasmid NR1. The plasmid pKP1013 replicates autonomously in a manner indistinguishable from that of the parental mini-F. An amber mutant defective in replication has been isolated from pKP1013 by localized mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine. The virtual absence of incorporation of [3H]-thymidine into the plasmid DNA as well as the kinetics of appearance of plasmid-free segregants suggest that plasmid DNA synthesis is primarily affected under nonpermissive conditions. The amber mutation has been mapped within the 530 base pairs (bp) region that extends from 45.25 (XmaI) to 45.78 Kb (PstI) by extensive analysis of in vitro recombinants constructed from rep+ and rep- plasmids.