Cloning and nucleotide sequences of the topoisomerase IV parC and parE genes of Mycoplasma hominis

Abstract

The topoisomerase IV parC and parE genes from the wall-less organism Mycoplasma hominis PG21 were cloned and sequenced. The coupled genes are located far from the DNA gyrase genes gyrA and gyrB. They encode proteins of 639 and 866 amino acids, respectively. As expected, the encoded ParE and ParC proteins exhibit higher homologies with the topoisomerase IV subunits of the gram-positive bacteria Staphylococcus aureus and Streptococcus pneumoniae than with their Escherichia coli counterparts. The conserved regions include the Tyr residue of the active site and the region involved in quinolone resistance (quinolone resistance-determining region [QRDR]) in ParC and the ATP-binding site and the QRDR in ParE.

FIG. 1

Restriction map of DNA insert of plasmid pMHE6. Positions of the parE and parC probes are indicated by the short, solid rectangles. E, EcoRI; N, NsiI; Hp, HpaI; B, BglII; P, PstI; H, HindIII; e, EcoRV. ORFs are numbered 1 to 6. T, putative transcription terminator.

FIG. 2

DNA sequence of the M. hominis parE-parC locus and deduced amino acid sequences of the ParC and ParE subunits of topoisomerase IV. Nucleotides are numbered according to the 7,603-bp sequence submitted to GenBank (accession no. AF036961). Primers MH11 (11), MH13 (13), MH28 (28), and MH29 (29), RBS, and restriction sites NsiI, HindIII, and BglII are indicated.

FIG. 2

DNA sequence of the M. hominis parE-parC locus and deduced amino acid sequences of the ParC and ParE subunits of topoisomerase IV. Nucleotides are numbered according to the 7,603-bp sequence submitted to GenBank (accession no. AF036961). Primers MH11 (11), MH13 (13), MH28 (28), and MH29 (29), RBS, and restriction sites NsiI, HindIII, and BglII are indicated.

FIG. 3

Alignment of M. hominis (Mh) ParE sequence with its counterparts from S. aureus (Sa), M. genitalium (Mg), and E. coli (Ec). An asterisk indicates an identical residue for all four protein sequences. The ParE (GrlB) sequences of S. aureus, M. genitalium, and E. coli are from references , , and and , respectively.

FIG. 4

Alignment of M. hominis (Mh) ParC sequence with its counterparts from S. aureus (Sa), M. genitalium (Mg), and E. coli (Ec). An asterisk indicates an identical residue for all four protein sequences. The ParC (GrlA) sequences of S. aureus, M. genitalium, and E. coli are from references , , and and , respectively.