Cloning and nucleotide sequence of the DNA gyrase (gyrA) gene from Mycoplasma hominis and characterization of quinolone-resistant mutants selected in vitro with trovafloxacin

Abstract

We report the cloning and characterization of the gyrA gene of the Mycoplasma hominis DNA gyrase, which was previously shown to be associated with quinolone resistance in this organism. The 2,733-bp gyrA gene encodes a protein of 911 amino acids with a calculated molecular mass of 102.5 kDa. As expected, M. hominis GyrA exhibits higher homology with the GyrA subunits of the gram-positive bacteria Clostridium acetobutylicum, Bacillus subtilis, Streptococcus pneumoniae, and Staphylococcus aureus than with its Escherichia coli counterpart. Knowing the entire sequence of the gyrA gene of M. hominis could be very useful for confirming the role of the GyrA subunit in fluoroquinolone resistance. Twenty-nine mutants of M. hominis were selected stepwise for resistance to trovafloxacin, a new potent fluoroquinolone, and their gyrA, gyrB, parC, and parE quinolone resistance-determining regions were characterized. Three rounds of selection yielded 3 first-step, 12 second-step, and 14 third-step mutants. The first-step mutants harbored a single substitution, Glu460-->Lys (E. coli coordinates), in ParE. GyrA changes, Ser83-->Leu, Glu87-->Lys, and Ala119-->Glu or Val, were found only in the second round of selection. At the third step, additional substitutions, at ParC Ser80, Ser81, and Glu84 and ParE Leu440, associated with high-level resistance to fluoroquinolones, appeared. Thus, high-level resistance to trovafloxacin required three steps and was associated with alterations in both fluoroquinolone targets. According to these genetic data, in M. hominis, as in Staphylococcus aureus and Streptococcus pneumoniae, topoisomerase IV seems to be the primary target of trovafloxacin.

FIG. 1

Restriction map and organization of the M. hominis PG21 gyrA locus. E, EcoRI; H, HindIII; B, BglII; N, NsiI. ?, hypothetical gene. ORFs are numbered 1 to 5. Arrowheads indicate the transcription sense of the ORFs. Sizes of DNA inserts are indicated in parentheses.

FIG. 2

Protein tree for full-length GyrA and ParC subunits from nine bacteria: M. hominis (; this study), M. genitalium (16), M. pneumoniae (22), U. urealyticum, B. subtilis (26), S. aureus (13, 30), S. pneumoniae (1, 34), C. acetobulylicum (44), and E. coli (38, 42). The tree was compiled by using the CLUSTAL W multiple-alignment program.

FIG. 3

Alignment of the M. hominis (Mh) GyrA amino acid sequence with those of its counterparts in M. genitalium (Mg) (16), M. pneumoniae (Mp) (22), B. subtilis (Bs) (26), S. aureus (Sa) (30), S. pneumoniae (Sp) (1), C. acetobutylicum (Ca) (44), and E. coli (Ec) (42). An asterisk indicates a residue identical in all eight proteins. Residues involved in quinolone resistance in M. hominis are indicated by diamonds. Dashes indicate gaps.

FIG. 3

Alignment of the M. hominis (Mh) GyrA amino acid sequence with those of its counterparts in M. genitalium (Mg) (16), M. pneumoniae (Mp) (22), B. subtilis (Bs) (26), S. aureus (Sa) (30), S. pneumoniae (Sp) (1), C. acetobutylicum (Ca) (44), and E. coli (Ec) (42). An asterisk indicates a residue identical in all eight proteins. Residues involved in quinolone resistance in M. hominis are indicated by diamonds. Dashes indicate gaps.

FIG. 4

Relationships between M. hominis PG21 and fluoroquinolone-resistant mutants IT1 to IIIT2F11 selected by stepwise exposure to trovafloxacin. The numbers outside the boxes indicate the trovafloxacin concentrations (in micrograms per milliliter) used in the selection steps. The superscripts +, °, and ∗ indicate the presence of mutations in GyrA, ParC, and ParE, respectively (Table 1).