Tn552, one of several closely related beta-lactamase-encoding transposons from Staphylococcus aureus, has a novel set of putative transposition functions. Each is homologous with a well-characterized function from a different type of mobile genetic element. Thus, Tn552 encodes: (i) resL-binL, a co-integrate resolution system homologous with those of Tn3 family elements; (ii) p480, a potential transposase significantly homologous with the DNA integrases of eukaryotic retroviruses and retrotransposons; and (iii) p271, a potential ATP-binding protein that shows homology with the B protein of phage Mu. The 3' terminal nucleotides of Tn552 (CA), adjacent to which p480 might cleave, are the same as those of retroviruses, retrotransposons and phage Mu. The presumptive resolvase (BinL) is very closely related to BinR, which was identified as a DNA invertase and is now shown to resolve an artificial co-integrate in vivo. Furthermore, the structure of the derivative of Tn552 found in the staphylococcal plasmid pI258 can be explained by a BinL (or BinR)-mediated site-specific deletion ('resolution') event. Thus, pI258 contains only the right-hand half of Tn552, which encodes the beta-lactamase and two regulatory proteins. The latter are homologous with the beta-lactamase gene repressor and co-inducer of Bacillus licheniformis. Interestingly, the order of the regulatory genes is reversed in S. aureus compared with Bacillus licheniformis.

Linoleic acid, but not stearic acid, inhibited the growth of Staphylococcus aureus NCTC 8325. Growth inhibition was associated with an increase in the Permeability of the bacterial membrane. The presence of a plasmid conferring resistance to penicillin (PC plasmid, e.g. p1258blaI-) increased the growth inhibitory and membrane permeability effects of linoleic acid. Under growth inhibitory conditions, linoleic acid was incorporated into the lipid of both PC plasmid-containing and PC plasmid-negative bacteria and there was little difference between these cultures in the uptake or fate of linoleic acid. Experiments using a glycerol auxotroph of S. aureus suggested that free linoleic acid, rather than lipid containing this acid, inhibits growth. Linoleic acid probably inhibits growth by increasing the permeability of the bacterial membrane as a result of its surfactant action, and the presence of the PC plasmid increases these effects.

SummaryPurified BlaI, the putative repressor of the ␤-lactamase operon in Staphylococcus aureus, binds specifically to two regions of dyad symmetry (operators) located in the blaZ-blaR1 intergenic region. BlaI binds with similar affinity to the two regions and to the related sequence upstream of the mec gene found in methicillin-resistant strains of S. aureus, providing physical evidence for the cross-talk previously observed between these systems. A change from a lysine in the N-terminus of BlaI to an alanine or deletion of the C-terminal 23 amino acids severely reduces its DNA-binding ability, demonstrating the functional importance of both the N-and C-termini. An operator DNA-protein complex observed with crude cell lysates from repressed cells, indistinguishable from that observed with purified BlaI, was eliminated by induction of the ␤-lactamase operon. Furthermore, BlaI is proteolytically cleaved in response to the addition of inducer in a blaR1-dependent manner, providing primary evidence for the molecular basis of induction. Thus, BlaI is shown to be the repressor of the ␤-lactamase system.

The staphylococcal beta‐lactamase transposon Tn552 is a member of a novel group of transposable elements. The organization of genes in Tn552 resembles that of members of the Tn21 sub‐group of Tn3 family transposons, which transpose replicatively by cointegrate formation and resolution. Thus, a possible resolution site (‘resL’) and a resolvase gene (tnpR or ‘binL’) have been identified. However, consistent with the fact that Tn552 generates 6 bp (rather than 5 bp) flanking direct repeats of target DNA, neither the putative transposase protein, nor the terminal inverted repeats of Tn552 are homologous to those of Tn3 elements. Tn552, like phage Mu and retroelements, is defined by the terminal dinucleotides 5′ TG .. CA 3′. A naturally occurring staphylococcal plasmid, pI9789, contains a Tn552‐derived resolution system (‘resR‐binR’) that acts as a ‘hotspot’ for Tn552 transposition; insertion creates a segment of DNA flanked by inversely repeated resolution sites, one (resR) on pI9789 and the other (resL) on Tn552. The putative Tn552 resolvase, the most closely related of known resolvases to the homologous DNA invertases, initially was identified as a DNA invertase (‘Bin’) as a result of its ability to mediate efficient inversion of this segment in vivo.

The nucleotide sequence of the ileS gene conferring high-level resistance to mupirocin in Staphylococcus aureus J2870 has been determined. The gene sequence is substantially different from that of the native ileS gene of S. aureus, indicating that high-level resistance to mupirocin results from the acquisition of a novel ileS gene.

The atlC gene (1,485 bp), encoding an autolysin which binds fibronectin, and the ica operon, involved in biofilm formation, were isolated from the chromosome of an infectious isolate of Staphylococcus caprae and sequenced. AtlC (155 kDa) is similar to the staphylococcal autolysins Atl, AtlE, Aas (48 to 72% amino acid identity) and contains a putative signal peptide of 29 amino acids and two enzymatic centers (N-acetylmuramoyl-L-alanine amidase and endo-␤-N-acetylglucosaminidase) interconnected by three imperfect fibronectin-binding repeats. The glycine-tryptophan (GW) motif found in the central and end part of each repeat may serve for cell surface anchoring of AtlC as they do in Listeria monocytogenes. The S. caprae ica operon contains four genes closely related to S. epidermidis and S. aureus icaA, icaB, icaC, and icaD genes (> 68% similarity) and is preceded by a gene similar to icaR (>70% similarity). The polypeptides deduced from the S. caprae ica genes exhibit 67 to 88% amino acid identity to those of S. epidermidis and S. aureus ica genes. The ica operon and icaR gene were analyzed in 14 S. caprae strains from human specimens or goats' milk. Some of the strains produced biofilm, and others did not. All strains carry the ica operon and icaR of the same sizes and in the same relative positions, suggesting that the absence of biofilm formation is not related to the insertion of a mobile element such as an insertion sequence or a transposon.Staphylococcus caprae (13) is the predominent species among the staphylococci recovered from mastitis-free goats' milk (5). It is also increasingly recognized as a human pathogen infecting implanted foreign bodies (1,6,14,44,46,52). Despite the amended description of this species (25), its phenotypic identification remains difficult. Therefore, molecular identification methods such as the analysis of ribotypes (1, 5, 12, 52), DNA-DNA hybridization (25), sequencing of the 16S rRNA gene (46), or analysis of the banding patterns on gels of penicillin-binding proteins (24) have been used for ecological studies and investigation of the involvement of S. caprae in infections. Some S. caprae strains from human specimens and goats' milk form biofilms (1, 4). Other strains do not, although the genomes of all strains tested carry nucleotide sequences hybridizing, at low stringency, with the S. epidermidis genes involved in initial adherence (atlE) and biofilm accumulation (the ica operon) (1). S. caprae adherence to fibronectin-and gelatin-coated coverslips is very weak. Nevertheless, surface proteins binding fibronectin have been detected on all S. caprae strains tested (1). The N-terminal part of the 175-kDa fibronectin-binding protein released from the surface of S. caprae clinical isolate 96007 has more than 50% amino acid identity (1) to the N-terminal part of the staphylococcal autolysins Atl (38), AtlE (18), and Aas (20). The aim of this study was to isolate the atlC autolysin gene of isolate 96007 to check whether the purified protein encoded by this gene binds fibronectin. ...

Based on structural and functional properties, three groups of large staphylococcal multiresistance plasmids have been recognized, viz., the pSK1 family, pSK41-like conjugative plasmids, and ␤-lactamase-heavy-metal resistance plasmids. Here we describe an analysis of the replication functions of a representative of each of these plasmid groups. The replication initiation genes from the Staphylococcus aureus plasmids pSK1, pSK41, and pI9789::Tn552 were found to be related to each other and to the Staphylococcus xylosus plasmid pSX267 and are also related to rep genes of several plasmids from other gram-positive genera. Nucleotide sequence similarity between pSK1 and pI9789::Tn552 extended beyond their rep genes, encompassing upstream divergently transcribed genes, orf245 and orf256, respectively. Our analyses revealed that genes encoding proteins related to the deduced orf245 product are variously represented, in several types of organization, on plasmids possessing six seemingly evolutionarily distinct types of replication initiation genes and including both thetamode and rolling-circle replicons. Construction of minireplicons and subsequent functional analysis demonstrated that orf245 is required for the segregational stability of the pSK1 replicon. In contrast, no gene equivalent to orf245 is evident on the conjugative plasmid pSK41, and a minireplicon encoding only the pSK41 rep gene was found to exhibit a segregational stability approaching that of the parent plasmid. Significantly, the results described establish that many of the large multiresistance plasmids that have been identified in clinical staphylococci, which were formerly presumed to be unrelated, actually utilize an evolutionarily related thetamode replication system.Clinical Staphylococcus aureus strains often harbor multiple plasmids, ranging from small rolling-circle (RC) replicating plasmids that are cryptic or encode only a single resistance determinant to larger multiresistance and conjugative plasmids (12,32,36). Three groups of multiresistance plasmids have been recognized in staphylococci. Isolates from the 1960s and 1970s were commonly found to carry multiresistance plasmids conferring resistance to penicillin and heavy metals or other inorganic ions (48). Such ␤-lactamase-heavy-metal resistance plasmids characteristically contain the ␤-lactamase-encoding transposon Tn552 or a derivative and operons mediating resistance to arsenical, cadmium, and/or mercuric ions (36). Some ␤-lactamase-heavy-metal resistance plasmids also contain Tn551, conferring resistance to macrolide-lincosamide-streptogramin type B antibiotics (33); an IS256-bounded composite aminoglycoside resistance transposon, Tn4001 (30); and/or a qacA or qacB antiseptic and disinfectant multidrug resistance determinant (30).pSK41-like conjugative multiresistance plasmids were first detected in strains isolated in the mid 1970s. Such plasmids commonly contain a Tn4001 hybrid structure (6) and IS257-flanked cointegrated copies of small plasmids, such as the aminoglycoside resist...

Ten Staphylococcus caprae strains isolated from four patients and responsible for bone infections following implantation of orthopaedic material were compared to four 5. caprae strains collected from milk samples of healthy goats. The following characteristics were investigated : SmaI patterns, hybridization patterns with pBA2 (ribotypes), slime production, adhesion to matrix proteins (fibrinogen, fibronectin, collagen) and the staphylococcal adhesion genes (fnbA, clfA, cna, at/€, ica, fbe). None of the characteristics enabled us to distinguish the human strains from the goat strains. Slime was occasionally produced by 5. caprae strains but all of them carried nucleotide sequences hybridizing at low stringency with the following genes: at/€ encoding a 5. epidermidis autolysin binding vitronectin and responsible for the primary adhesion to polystyrene, ica operon involved in the biosynthesis of a S. epidermidis extracellular polysaccharide, and the part of c/fA encoding the serine-aspartate repeated region of a 5. aureus cell-wall fibrinogen-binding protein.