Novel Locus Required for Expression of High-Level Macrolide-Lincosamide-Streptogramin B Resistance in Staphylococcus aureus

Abstract

The yycF1(Ts) mutation in Staphylococcus aureus conferred hypersensitivity to macrolide-lincosamide-streptogramin B (MLS_B) antibiotics on strains either containing or lacking ermB. The overexpression of the S. aureus Ssa protein restored the yycF1 mutant to wild-type levels of susceptibility. Inactivation of ssa in an unmutagenized strain dramatically reduced ermB-based resistance. Conditional loss of function or expression of ssa in the yycF1 mutant is proposed to result in the observed hypersensitivity to MLS_B antibiotics.

One of several phenotypic consequences of the yycF1(Ts) mutation in Staphylococcus aureus was a hypersensitivity to macrolide-lincosamide-streptogramin B (MLS_B) antibiotics (9). This hypersensitivity was returned to wild-type levels when the mutant was cultured under anoxic conditions at neutral pH. Accordingly, when the ermB-containing transposon Tn917lac was transduced into the original yycF1(Ts) mutant (strain NT372), erythromycin-resistant (Em^r) transductants could be selected only anaerobically. An isogenic set of strains, SAM1010 [yycF1(Ts) Tn917lac::purA571] and SAM1011 (Tn917lac::purA571), was constructed by using this strategy (Table 1). Even though both strains were marked with ermB, the SAM1010 mutant expressed only an intermediate level of resistance to MLS_B-class antibiotics compared to that expressed by SAM1011 (Table 2). In the absence of resistance genes, the NT372 temperature-sensitive mutant was fourfold more sensitive to erythromycin (ERM) than the unmutagenized parental strain (9). Because the yycF response regulator was reported to effect changes at the level of transcription (3, 4), it is possible that the conditional underexpression of one or more chromosomally carried genes in the yycF1(Ts) mutant of S. aureus could account for this observed hypersensitivity.

TABLE 1.

Strains and plasmids

Strain or plasmid	Relevant genotype or description	Phenotypea	Reference
Strains
SAM23	8325-4		6
NT372	SAM23 yycF1	TS	9
SAM1010	SAM23 yycF1 Tn917lac::purA571	TS, Emr	9
SAM1011	SAM23 Tn917lac::purA571	Emr	9
SAM1011s1	SAM1011 ssa::pMP2376	Tcr	This work
SAM1011s1r1	SAM1011 ssa+	Emr	This work
SAM1287	NT372 pMP532	Emr	This work
Plasmids
pMP16	S. aureus-Escherichia coli shuttle vector	Emr Apr	9
pMP532	pMP16(ssa nhaC)	Emr	This work
pMP1025	pMP16(nhaC)	Emr	This work
pMP1029	pMP16(ssa)	Emr	This work
pMP2376	ssa disruption plasmid	rep(Ts) Tcr Apr	This work

^aTS, temperature sensitive; Tc^r and Ap^r, tetracycline and ampicillin resistance, respectively.

TABLE 2.

Changes in MICs as a result of ssa inactivation

Antibiotic	MIC (μg/ml) for strain:
	SAM1010 (Ts) [yycF1(Ts) ermB]	SAM1011 (ermB)	SAM1011s1 (ssa::tetK ermB)
Macrolides (14 and 16 membered) and lincosamides
Erythromycin	16	>512	8
Roxithromycin	32	>512	4
Josamycin	8	>512	2
Tylosin tartrate	4	>512	2
Lincomycin	1	>512	2
Clindamycin	0.25	>512	0.25
Representative non-MLSB-type antibiotics
Chloramphenicol	16	16	8
Phleomycin	4	4	4
Streptomycin	2	2	2
Vancomycin	1	1	1
Methicillin	1	1	1
Norfloxacin	0.5	0.5	0.5

The NT372 mutant grew very poorly on plating media like Trypticase soy agar (TSA) at 39°C and did not grow at higher temperatures. Thus, by selecting plasmid-based genomic clones that restored high-level Em^r (10 μg/ml) on TSA at this semipermissive growth temperature, it was thought that one or more related genes affecting this partial MLS_B resistance phenotype might be revealed. With a plasmid library (9) constructed from the unmutagenized parental strain (SAM23), a total of 21 Em^r transductants of NT372 were isolated. Under identical selection conditions, over 50,000 Em^r transductants for the isogenic wild type (SAM23) were obtained. Of the 21 Em^r transductants of NT372, 5 could be reselected at 43°C and contained plasmids bearing the original yycFG locus (complementing clones). The remaining 16 clones, represented by strain SAM1287 (Table 1), bore equivalently sized genomic inserts (5.2 kb) that conferred high-level ERM resistance (MIC > 512 μg/ml) but did not fully restore the mutant's ability to grow at the higher temperature (43°C).

The inserts of all 16 clones were sequenced and found to be identical. Subcloning and recomplementation of the NT372 mutant (Fig. 1a) correlated the selection of high-level MLS_B resistance to a single open reading frame (ORF), ssa. The subclone lacking ssa, pMP1025, conferred only very weak growth on solid media at lower ERM concentrations (1 μg/ml), demonstrating that the combination of ssa and ermC was necessary for the expression of high-level MLS_B resistance in the presence of a yycF1 chromosomal mutation. To test this apparent correlation between ssa expression and the observed antibiotic hypersensitivity, inactivation of the ssa locus in an unmutagenized Em^r strain (SAM1011) was attempted.

FIG. 1.

Mutant complementation and gene inactivation. (a) A partial restriction map of the suppressor clone, pMP532, along with subcloning and mutant recomplementation data are shown. Incomplete ORFs are denoted by dashed arrows. Restriction enzyme abbreviations are as follows: C, ClaI; E, EcoRI; and N, NheI. (b) The inactivation of the chromosomal ssa locus resulted in a reduction of the MIC of ERM. The resolution of the plasmid integrant and subsequent restoration of an intact copy of ssa completely restored Em^r.

By using an S. aureus integration plasmid (pMP2376) with a temperature-sensitive origin of replication from pE194ts and the tetK resistance gene from pT181, the chromosomal copy of this ORF (ssa) was inactivated in SAM1011 (Em^r) by Campbell-style integration (Fig. 1b). Briefly, exponentially growing cultures containing the integration plasmid were shifted to a temperature restrictive for plasmid replication (39°C) and allowed to grow to saturation at this temperature. Site-specific integration of the disruption plasmid into the ssa chromosomal locus (SAM1011s1) was confirmed by genomic PCR and Southern blot analysis (data not shown). The MICs of different MLS_B-class antibiotics for the resulting progeny dropped substantially (Table 2), demonstrating that an intact copy of ssa was necessary for the full phenotypic expression of resistance in a strain bearing the ermB gene (SAM1011).

To demonstrate further that the observed change in susceptibility to MLS_B-type antibiotics was a direct result of inactivation of the ssa ORF, the integration plasmid was allowed to resolve from the chromosome and restore a functional copy of the ssa ORF. Single colonies of the integration strain (SAM1011s1) were inoculated into broth (Trypticase soy broth) and cultured in the absence of antibiotic selection at a temperature permissive for plasmid replication (30°C). Serial dilutions of the saturated overnight cultures were plated onto selective medium (TSA with ERM), and numbers of viable cells in these cultures were compared to those observed on nonselective medium (TSA). The number of colonies that had resolved the tandem duplication and had regained phenotypic Em^r were identified at approximately 1:500 CFU/ml, which is in general agreement with our previous observations (9). A subset (eight isolates) of this population was assayed, and all of these isolates had regained full phenotypic Em^r (MIC > 512 μg/ml). Chromosomal DNA was prepared from these eight strains, and a restored copy of the ssa ORF at the predicted size was confirmed by PCR for each (eight out of eight).

The biological function of the ssa gene in S. aureus is not understood. The inactivation of this locus caused no apparent effect upon the viability of the strain in vitro, demonstrating that the expression of the ssa gene was not essential. The ssa ortholog from Staphylococcus epidermidis (8) has been described as encoding a secreted, highly immunogenic protein expressed during the course of infection. The ssa ortholog from S. aureus described here encoded a predicted hydrophilic polypeptide of 267 amino acids, with a predicted transmembrane region at the N terminus (Fig. 2). At the C-terminal end of this ORF was a predicted block of 67 amino acids, which was found to be highly similar (>50% identity) to the equivalent C-terminal residues of six additional hypothetical proteins in the completed genomic sequence of S. aureus strain N315 (7). This apparently conserved motif did not contain the classic gram-positive cell surface anchoring motif, LPXTG (12). The LysM peptidoglycan binding motif (1) was identified in three of these ORFs, suggesting that this group of unassigned proteins may bind to a specific entity on the surface of S. aureus, not unlike the choline binding proteins of Streptococcus pneumoniae (5).

FIG. 2.

Alignment of the C-terminal regions of seven S. aureus hypothetical proteins. The 67 C-terminal amino acids of each hypothetical ORF show significant identity (>50%). The grey box denotes a predicted transmembrane domain, and the striped box indicates similarity to the LysM domain, common among proteins associated with peptidoglycan processing (1). The mottled box indicates the common C-terminal domain corresponding to the alignment above. Biological functions for these ORFs have not been confirmed; therefore, each ORF is named according to the published genomic data (7) for strain N315 (http://www.bio.nite.go.jp/cgi-bin/dogan/genome_top.cgi?′n315′).

Aside from those in coagulase-negative staphylococci (8), there do not appear to be structural orthologs of ssa in the genomes of other gram-positive bacteria that have been sequenced to date. The conditional loss of function of ssa in the yycF1(Ts) mutant of S. aureus (SAM1010) may explain why the SAM1010 strain was hypersensitive to MLS_B-class antibiotics whereas other gram-positive yycF mutants reportedly do not share this defect (3, 4, 10, 11). We have demonstrated that the inactivation of the ssa gene in S. aureus unexpectedly resulted in the loss of full phenotypic expression of ermB-based antibiotic resistance. This is the first report of the expression of a novel uncharacterized cell surface protein as an accessory factor to the manifestation of high-level MLS_B resistance in S. aureus. We propose that this is analogous to the identification of the fem genes in S. aureus, which are chromosomal genes that contribute to the phenotypic expression of methicillin resistance in strains containing the mec determinant (2).