Insertional inactivation of staphylococcal methicillin resistance by Tn551.

Transposon Tn551 was translocated into the chromosome of a methicillin-resistant (mec) strain of Staphylococcus aureus by heat inactivation of a thermo-sensitive plasmid carrying Tn551 and selection for erythromycin-resistant (Emr) survivors. Two independent chromosomal insertions of Tn551 were obtained which reduced the level of the methicillin resistance by a factor of 50 to 100, making the strains phenotypically methicillin sensitive (Mecs). Each of the Tn551 insertions was on the largest fragment produced by EcoRI digestion of the chromosomal DNA of these strains. The integration sites lie about 1 kilobase apart. These Mecs strains reverted to Mecr at frequencies of 2.4 X 10(-8) and 3.6 X 10(-5), respectively. The majority of Mecr revertants still were Emr; only a few lost the Emr phenotype concomitantly with reversion to the Mecr phenotype. Hybridization data with labeled Tn551 showed complex rearrangements and deletions in the region of the insertion. These two Tn551 insertions do not lie on the same linkage group, II, as the mec determinant. The phenotypic expression of methicillin resistance, therefore, is also dependent upon a chromosomal genetic marker not physically linked to the mec determinant.     

Influence of femB on methicillin resistance and peptidoglycan metabolism in Staphylococcus aureus.

The inactivation of FemB by insertion of Tn551 in the central part of the femB open reading frame was shown to increase susceptibility of methicillin-resistant Staphylococcus aureus strains toward beta-lactam antibiotics to the same extent as did inactivation of femA. Strains carrying the methicillin resistance determinant (mec) and expressing PBP 2' were affected to the same extent as were strains selected for in vitro resistance, which did not express PBP 2'. Both femA and femB, which form an operon, are involved in a yet unknown manner in the glycine interpeptide bridge formation of the S. aureus peptidoglycan. FemB inactivation was shown to reduce the glycine content of peptidoglycan by approximately 40%, depending on the S. aureus strain. The reduction of the interpeptide bridge glycine content led to significant reduction in peptidoglycan cross-linking, as measured by gel permeation high-pressure liquid chromatography of muramidase-digested cell walls. Maximum peptide chain length was reduced by approximately 40%. It is shown that the complete pentaglycine interpeptide bridge is important for the sensitivity against beta-lactam antibiotics and for the undisturbed activity of the staphylococcal cell wall-synthesizing and hydrolyzing enzymes, as was also apparent from electron microscopic examinations, which revealed aberrant placement of cross walls and retarded cell separation, leading to a pseudomulticellular phenotype of the cells for both femA and femB mutants.     

Suppression of autolysis and cell wall turnover in heterogeneous Tn551 mutants of a methicillin-resistant Staphylococcus aureus strain.

Isogenic Tn551 mutants of a highly and uniformly methicillin-resistant strain of Staphylococcus aureus were tested for their rates of autolysis and cell wall degradation in buffer and for cell wall turnover during growth. The normal (relatively fast) autolysis and turnover rates of the parent strain were retained in a Tn551 mutant in which the insert was located within the mec gene and which produced undetectable levels of penicillin-binding protein 2A. On the other hand, autolysis and cell wall turnover rates were greatly reduced in auxiliary mutants, i.e., mutants in which the transposon caused conversion of the high-level and uniform resistance of the parent strain to a variety of distinct heterogeneous expression types and greatly decreased resistance levels. All of these mutants contained an intact mec gene and produced normal amounts of penicillin-binding protein 2A, and one of the mutations was located in the femA region of the staphylococcal chromosome (B. Berger-Bachi, L. Barberis-Maino, A. Strassle, and F. H. Kayser, Mol. Gen. Genet. 219:263-269, 1989). Autolysis rates were related to the degree of residual methicillin resistance and to the sites of Tn551 insertion. Fast cell wall turnover may help expression of high-level methicillin resistance by providing a mechanism for the excision of abnormal (and potentially lethal) structural elements of the cell wall synthesized by the bacteria in the presence of methicillin.     

Peptidoglycan composition of a highly methicillin-resistant Staphylococcus aureus strain. The role of penicillin binding protein 2A.

All clinical isolates of methicillin-resistant Staphylococcus aureus contain an extra penicillin binding protein (PBP) 2A in addition to four PBPs present in all staphylococcal strains. This extra PBP is thought to be a transpeptidase essential for the continued cell wall synthesis and growth in the presence of beta-lactam antibiotics. As an approach of testing this hypothesis we compared the muropeptide composition of cell walls of a highly methicillin-resistant S. aureus strain containing PBP2A and its isogenic Tn551 derivative with reduced methicillin resistance, which contained no PBP2A because of the insertional inactivation of the PBP2A gene. Purified cell walls were hydrolyzed into muropeptides which were subsequently resolved by reversed-phase high-performance liquid chromatography and identified by chemical and mass spectrometric analysis. The peptidoglycan composition of the two strains were identical. Both peptidoglycans were highly cross-linked mainly through pentaglycine cross-bridges, although other, chemically distinct peptide cross-bridges were also present including mono-, tri-, and tetraglycine; alanine; and alanyl-tetraglycine. Our experiments provided no experimental data for a unique transpeptidase activity associated with PBP2A.     

Isolation and characterization of autolysis-defective mutants of Staphylococcus aureus created by Tn917-lacZ mutagenesis.

Two autolysis-defective mutants (Lyt-1 and Lyt-2) of Staphylococcus aureus have been isolated by transposon Tn917-lacZ mutagenesis. The mutants exhibited normal growth rate, cell division, cell size, and adaptive responses to environmental changes. No autolytic activities were detected in a crude autolytic enzyme preparation from the Lyt- mutants. The rate of autolysis of whole cells and cell walls in the mutants were negligible, but mutant cell wall preparations were degraded by crude enzyme preparations from the wild-type strain. Zymographic analyses of enzyme extracts from the mutants showed a single autolytic enzyme band, compared with more than 10 autolytic enzyme bands from the parent strain. Analyses of intracellular and exoprotein fractions gave results similar to those in experiments with total-cell extracts. Southern blot analysis indicated the insertion of a single copy of the transposon into the chromosome of Lyt mutants. Isogenic Lyt mutants constructed by phage phi 11 transduction showed similar phenotypes. Because both Lyt- mutants had Tn917-lacZ inserted in the appropriate orientation, it was possible to determine gene activity under various conditions by measuring beta-galactosidase activity. The gene activity was found to be induced by low pH, low temperature, and high sucrose and high sodium chloride concentrations. From these data, we propose that the mutation lies in either a master regulatory gene or a structural gene which is responsible for the synthesis or processing of a majority of the autolytic enzyme bands.     

Increase in Transduction Efficiency of Tn551 Mediated by the Methicillin Resistance Marker

The transduction efficiency of Tn551, a staphylococcal transposon coding for erythromycin resistance (ermB), was increased by a factor of about five in acceptor strains carrying the gene for methicillin resistance (mec), as compared with methicillin-sensitive strains. This enhancement was independent of the generalized transducing phage used and of the chromosomal insertion site of the Tn551 transposon.     

The sae locus of Staphylococcus aureus encodes a two-component regulatory system

Sae is a regulatory locus that activates the production of several exoproteins in Staphylococcus aureus. A 3.4-kb fragment of a S. aureus genomic library, screened with a probe adjacent to the transposon insertion of a sae::Tn551 mutant, was cloned into a bifunctional vector. This fragment was shown to carry the sae locus by restoration of exoprotein production in sae mutants. The sae locus was mapped to the SmaI-D fragment of the staphylococcal chromosome by pulse-field electrophoresis. Sequence analysis of the cloned fragment revealed the presence of two genes, designated saeR and saeS, encoding a response regulator and a histidine protein kinase, respectively, with high homology to other bacterial two-component regulatory systems.     

Transposon disruption of the complex I NADH oxidoreductase gene (snoD) in Staphylococcus aureus is associated with reduced susceptibility to the microbicidal activity of thrombin-induced platelet microbicidal protein 1

The cationic molecule thrombin-induced platelet microbicidal protein 1 (tPMP-1) exerts potent activity against Staphylococcus aureus. We previously reported that a Tn551 S. aureus transposon mutant, ISP479R, and two bacteriophage back-transductants, TxA and TxB, exhibit reduced in vitro susceptibility to tPMP-1 (tPMP-1(r)) compared to the parental strain, ISP479C (V. Dhawan, M. R. Yeaman, A. L. Cheung, E. Kim, P. M. Sullam, and A. S. Bayer, Infect. Immun. 65:3293-3299, 1997). In the current study, the genetic basis for tPMP-1(r) in these mutants was identified. GenBank homology searches using sequence corresponding to chromosomal DNA flanking Tn551 mutant strains showed that the fourth gene in the staphylococcal mnh operon (mnhABCDEFG) was insertionally inactivated. This operon was previously reported to encode a Na(+)/H(+) antiporter involved in pH tolerance and halotolerance. However, the capacity of ISP479R to grow at pH extremes and in high NaCl concentrations (1 to 3 M), coupled with its loss of transmembrane potential (DeltaPsi) during postexponential growth, suggested that the mnh gene products are not functioning as a secondary (i.e., passive) Na(+)/H(+) antiporter. Moreover, we identified protein homologies between mnhD and the nuo genes of Escherichia coli that encode components of a complex I NADH:ubiquinone oxidoreductase. Consistent with these data, exposures of tPMP-1-susceptible (tPMP-1(s)) parental strains (both clinical and laboratory derived) with either CCCP (a proton ionophore which collapses the proton motive force) or pieracidin A (a specific complex I enzyme inhibitor) significantly reduced tPMP-induced killing to levels seen in the tPMP-1(r) mutants. To reflect the energization of the gene products encoded by the mnh operon, we have renamed the locus sno (S. aureus nuo orthologue). These novel findings indicate that disruption of a complex I enzyme locus can confer reduced in vitro susceptibility to tPMP-1 in S. aureus.     

Localization of a chromosomal mutation affecting expression of extracellular lipase in Staphylococcus aureus.

We describe a Tn551 chromosomal insertion in Staphylococcus aureus S6C that results in sharply reduced expression of extracellular lipase. With Tn917 as a probe, the insertion in the original mutant (KSI905) was localized to a 12.6-kb EcoRI DNA fragment. The 12.6-kb fragment was cloned and used as a probe to identify a 26-kb EcoRI fragment containing the Tn551 insertion site in the S6C parent strain. Restriction endonuclease analysis of the 12.6- and 26-kb EcoRI fragments confirmed that the Tn551 insertion in KSI905 was accompanied by a deletion of 18.7 kb of chromosomal DNA. Tn551 was transduced from KSI905 back into the S6C parent strain. All transductants exhibited the same lipase-negative (Lip-) phenotype and contained the same mutation with respect to both the insertion and the 18.7-kb deletion. The inability to produce lipase was not caused by disruption of the lipase structural gene, since all Lip- mutants carried intact copies of geh. Moreover, the Tn551 insertion was localized to a region of the staphylococcal chromosome at least 650 kb from geh. Taken together, these results suggest that the Tn551 insertion occurred in a region of the chromosome encoding a trans-active element required for the expression of extracellular lipase. A 20-bp oligonucleotide corresponding to a sequence within the region encoding RNA II near the Tn551 insertion site in ISP546 (H.L. Peng, R.P. Novick, B. Kreiswirth, J. Kornblum, and P. Schlievert, J. Bacteriol. 170:4365-4372, 1988) and a 1.75-kb DNA fragment representing the region encoding RNA III were used as gene probes to show that the Tn551 insertion did not occur in the agr locus. We conclude that the genetic element functions independently of agr or as an unrecognized part of that regulatory system.     

Additional DNA in methicillin-resistant Staphylococcus aureus and molecular cloning of mec-specific DNA.

Additional DNA was shown to be present in methicillin-resistant Staphylococcus aureus by one- and two-dimensional restriction endonuclease analyses of the chromosomal DNA. A 3.5-kilobase Bg/II fragment, which was present in methicillin-resistant strains but not in the isogenic methicillin-sensitive parental strain, was cloned into newly constructed plasmid pWDB1 in Escherichia coli. Hybridization of this 3.5-kilobase Bg/II fragment with different methicillin-sensitive and methicillin-resistant S. aureus clinical isolates indicated that the fragment represents part of the methicillin resistance determinant (mec). In addition, the fragment carries a sequence that is present in some large staphylococcal plasmids, as well as in penicillinase plasmid pI524.     

femA, which encodes a factor essential for expression of methicillin resistance, affects glycine content of peptidoglycan in methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains.

femA is a chromosomally encoded factor, occurring naturally in Staphylococcus aureus, which is essential for the expression of high-level methicillin resistance in this organism. The production of a low-affinity penicillin-binding protein, PBP2a or PBP2', which is intimately involved with methicillin resistance in S. aureus, is not influenced by femA. To elucidate a possible physiological function of the 48-kDa protein encoded by femA, several related methicillin-resistant, methicillin-susceptible, and Tn551 insertionally inactivated femA mutants were analyzed for possible changes in cell wall structure and metabolism. Independent of the presence of mec, the methicillin resistance determinant, all femA mutants had a reduced peptidoglycan (PG) glycine content (up to 60% in the molar ratio of glycine/glutamic acid) compared to that of related femA+ parent strains. Additional effects of femA inactivation and the subsequent decrease in PG-associated glycine were (i) reduced digestion of PG by recombinant lysostaphin, (ii) unaltered digestion of PG by Chalaropsis B-muramidase, (iii) reduced cell wall turnover, (iv) reduced whole-cell autolysis, and (v) increased sensitivity towards beta-lactam antibiotics. Also, the PG-associated glycine content of a femA::Tn551 methicillin-susceptible strain was restored concomitantly with the methicillin resistance to a level almost equal to that of its femA+ methicillin-resistant parent strain by introduction of plasmid pBBB31, encoding femA.     

Cloning and characterization of a gene affecting the methicillin resistance level and the autolysis rate in Staphylococcus aureus.

Tn918 mutagenesis of a high-level methicillin-resistant Staphylococcus aureus (methicillin MIC, 800 micrograms/ml) led to the isolation of a low-resistance mutant. The Tn918 insert was transferred back to the parent to produce strain SRM563 (methicillin MIC, 12.5 micrograms/ml), which showed heterogeneous resistance. Twenty-two clinical isolates of methicillin-resistant S. aureus were transformed with DNA of SRM563. In the transformants of most strains, instances of reduced resistance were observed with concomitant increases of autolysis rate induced by Triton X-100 and were generally more profound in high-resistance strains. Two transformants exhibited a decrease of the autolysis rate and little reduction of resistance. In the transformant of methicillin-susceptible strain RN2677, an increase of the autolysis rate and little reduction of resistance were observed. The production of low-affinity penicillin-binding protein (PBP2') did not significantly decrease in the mutants. Insertion of Tn918 occurred within the 3'-terminal region of a novel gene designated llm, which was cloned and sequenced. RNA blot analysis demonstrated that the gene was transcribed. The encoded protein was composed of 351 amino acid residues with a molecular weight of 38,512 and was hydrophobic, suggesting its location on the membrane. The gene was detected by PCR in all S. aureus strains tested but not in the other 26 staphylococcal species. Comparison of the 3'-terminal sequences of the gene among several S. aureus strains showed that, whereas nucleotide substitutions occurred at the third position in seven of eight 3'-terminal codons, only C-terminal amino acid variation of glutamate or aspartate was observed.     

FemA of Staphylococcus aureus: Isolation and immunodetection

Abstract FemA, a cytoplasmic protein necessary for the expression of methicillin resistance in Staphylococcus aureus and also involved in the biosynthesis of staphylococcal cell walls, was detected and quantified in several S. aureus strains under different growth conditions by Western immunoblot. Two types of antigens were used for the production of polyclonal antibodies against FemA: (i) a synthetic peptide comprising 14 amino acids of its C-terminal sequence; and (ii) FemA isolated by preparative gel electrophoresis and electroelution from an overproducing staphylococcal strain. Immunodetection revealed that all investigated strains, either methicillin-resistant or susceptible, expressed FemA during the exponential growth phase in varying amounts. In the stationary phase, the FemA content was diminished. Strains in which femA was inactivated by insertion of Tn557 into the control region of the fem AB operon still expressed about 10% of the protein compared to their parent strains. Tn55 I insertion in the middle of the fem B gene did not affect the FemA expression. In 40 methicillin-susceptible and 6 resistant clinical isolates of S. aureus , the FemA content or its affinity to the antibodies was reduced compared to laboratory parent strains. In susceptible strains, an additional protein of higher molecular weight, present in large quantities, was also able to bind the FemA antibodies. Such a protein was also present in methicillin-resistant isolates, although it was not as pronounced as in the susceptible strains.     

Cloning and sequencing of the gene, fmtC, which affects oxacillin resistance in methicillin-resistant Staphylococcus aureus

Two Tn551 insertional mutants with reduced methicillin resistance were isolated from methicillin-resistant Staphylococcus aureus KSA8. These two mutants showed increased susceptibility to beta-lactam antibiotics and bacitracin, but not to fosfomycin and vancomycin. Tn551 in these mutants was inserted into the same gene, termed fmtC. The fmtC gene has an open reading frame of 840 amino acid residues with an estimated molecular mass of 96.9 kDa. The N-terminal half of the deduced FmtC protein is very hydrophobic, implying that this protein is a membrane-associated protein.     

Sigma-B, a putative operon encoding alternate sigma factor of Staphylococcus aureus RNA polymerase: molecular cloning and DNA sequencing.

We have identified a gene cluster located on the chromosomal SmaI I fragment of a highly methicillin resistant strain of Staphylococcus aureus, consisting of four open reading frames (ORFs), named after the number of deduced amino acid residues, in the sequential order orf333-orf108-orf159-orf256. The gene cluster showed close similarities to the Bacillus subtilis sigB operon both in overall organization and in primary sequences of the gene products. The complete gene cluster (provisionally named sigma-B or sigB) was preceded by an sigmaA-like promoter (PA) and had an internal sigmaB-like promoter sequence (PB) between orf333 and orf108, suggesting a complex regulatory mechanism. The polypeptides encoded by orf333, -108, -159, and -256 showed 62, 67, 71, and 77% homologies, respectively, with the RsbU, RsbV, RsbW, and SigB polypeptides encoded by the B. subtilis sigB operon. A Tn551 insertional mutant, RUSA168 (insert in orf256 of the staphylococcal sigma-B operon), showed drastic reduction in methicillin resistance (decrease in MIC from 1,600 microg ml-1 to 12 to 25 microg ml-1off     

Isolation of transposon Tn551 insertions near chromosomal markers of interest in Staphylococcus aureus.

A procedure was developed to isolate insertions of transposon Tn551 near other markers of interest on the chromosome of Staphylococcus aureus NCTC 8325. When an inoculum of strain 8325-4 carrying a thermosensitive mutant of plasmid pI258 (on which Tn551 resides) was inoculated into brain heart infusion agar plus erythromycin and grown to saturation at 43 degrees C, the transforming DNA extracted from this population of cells contained a random collection of different chromosomal insertions of Tn551; this DNA is referred to as pooled Tn551 DNA. When erythromycin-sensitive recipient strains containing chromosomal markers of interest were transformed with pooled Tn551 DNA, and the resulting Emr transformants were screened for coinheritance of the donor allele of the marker of interest, insertions of Tn551 were isolated near several markers, including fus-149, tet-3490, mec-4916, pig-131, ilv-129, pur-140, and uraA141. Many of the insertions were within the linkage groups that contained these markers, and several insertions occupied different positions between the linkage groups in heretofore undefined regions of the circular chromosomal map of S. aureus. These insertions of transposon Tn551 extend the known limits of the existing linkage groups, provide linkage data and map locations for markers not previously mapped, and provide a means to map markers which cannot be directly selected.     

Involvement of multiple genetic determinants in high-level methicillin resistance in Staphylococcus aureus.

A methicillin-susceptible, novobiocin-resistant strain of Staphylococcus aureus (RN2677; methicillin MIC, 0.8 micrograms/ml) was transformed with DNA prepared from highly and homogeneously methicillin-resistant S. aureus strains (methicillin MIC, greater than or equal to 400 micrograms/ml) or from heterogeneous strains in which the majority of cells had a low level of resistance (methicillin MIC, 6.3 micrograms/ml). All methicillin-resistant transformants showed low and heterogeneous resistance (methicillin MIC, 3.1 micrograms/ml) irrespective of the resistance level of DNA donors. All transformants examined produced normal amounts of the low-affinity penicillin-binding protein (PBP) 2a, and methicillin resistance and the capacity to produce PBP 2a showed the same degree of genetic linkage to the novobiocin resistance marker with both homogeneous and heterogeneous DNA donors. Next, we isolated a methicillin-susceptible mutant from a highly and homogeneously resistant strain which had a Tn551 insertion near or within the PBP 2a gene and thus did not produce PBP 2a. With this mutant used as the recipient, genetic transformation of the methicillin resistance gene was repeated with DNA isolated either from highly and homogeneously resistant strains or from heterogeneous (low-resistance) strains. All transformants obtained expressed high and homogeneous resistance and produced PBP 2a irrespective of the resistance level of the DNA donors. Our findings suggest that (i) the methicillin resistance locus is identical to the structural gene for PBP 2a, (ii) although the ability to produce PBP 2a is essential for resistance, the MICs for the majority of cells are not related to the cellular concentration of PBP 2a, and (iii) high MICs and homogeneous expression of resistance require the products of other distinct genetic elements as well.