Molecular characterization of the gene encoding the DNA gyrase A subunit of Streptococcus pneumoniae

Abstract

The gene encoding the DNA gyrase A subunit of Streptococcus pneumoniae was cloned and sequenced. The gyrA gene codes for a protein of 822 amino acids homologous to the gyrase A subunit of eubacteria. Translation of the gene in an Escherichia coli expression system revealed a 92-kDa polypeptide. A sequence-directed DNA curvature was identified in the promoter region of gyrA. The bend center was mapped and located between the -35 and -10 regions of the promoter. Primer extension analysis showed that gyrA transcription initiates 6 bp downstream of an extended -10 promoter. The possible implications of the bent DNA region as a regulatory element in the transcription of gyrA are discussed.

FIG. 1

Restriction map of the gyrA region of S. pneumoniae (A) and genetic structure as deduced from the nucleotide sequence (B). P at the left of the gray arrow indicates the promoter. The physical maps of the inserts of pertinent plasmids are also indicated (hatched bars). Bg, BglII; D, DraI; E, EcoRI; EV, EcoRV; H, HindIII; K, KpnI; P, PstI; S, SacI. The DNA probes used in Southern blot experiments are indicated as bars labeled gyrA44-70 (insert of plasmid pQRDR26) and gyrA136-485 (insert of pGYAN31). The oligonucleotides used in PCR experiments are indicated by black arrows (not drawn to scale).

FIG. 2

Nucleotide sequence of a 2,685-bp fragment of S. pneumoniae R6 which contains the gyrA gene. The strand corresponding to the mRNA is shown. Nucleotides and amino acids (italics) are numbered by taking the first gyrA nucleotide as nt 1 and the first GyrA residue as residue 1. The −35 and the extended −10 promoter regions and the putative ribosome-binding site (RBS) are underlined and in boldface. The first nucleotide of the mRNA is indicated as +1. The amino acid residues encoded by the degenerate oligonucleotides used in PCR experiments are also underlined and in boldface. Other pertinent oligonucleotides and restriction endonuclease sites are labeled and underlined.

FIG. 3

Comparison of the amino acid sequences of the DNA gyrase A subunits from S. pneumoniae (Spn), B. subtilis (Bsu) (27), S. aureus (Sau) (21), and E. coli (Eco) (42). Identical amino acids are boxed. Residues in E. coli GyrA that form the active site of the breakage-reunion reaction, including the active Tyr-122 residue that links to DNA, are indicated by arrows. Residues involved in quinolone resistance in S. pneumoniae are indicated by circles (15, 29, 31, 43).

FIG. 4

Protein tree of full-length GyrA and ParC subunits from bacteria. The tree was compiled by using the CLUSTAL multiple sequence alignment program from PCGENE.

FIG. 5

Expression of the GyrA protein. Cultures of E. coli BL21(DE3) containing pGEM3Z (lanes 3 and 5) or pGYAN9 (lanes 4 and 6) were grown in M9 (lanes 5 and 6) or in LB (lanes 3 and 4) medium and induced with IPTG as described in Materials and Methods. Samples containing 15 μg of protein were electrophoresed in an SDS-polyacrylamide gel. Lanes 3 to 6, polypeptides revealed by Coomassie blue staining. The dried gel was exposed for 8 h to Kodak X-Omat for autoradiography. Lane 2 shows the autoradiogram of lane 6. Lane 1, molecular mass protein standards.

FIG. 6

Mapping of the center of curvature by circular permutation analysis. (A) Computer-generated structure of the DNA region from coordinates −128 to −8 of the sequence in Fig. 2. Plot is shown in the YZ plane to show the predicted bend region. (B) Plasmid pBEND-11 was digested separately with the indicated enzymes (E, EcoRI; H, HindIII; EV, EcoRV; N, NheI; B, BamHI) to generate 609-bp fragments containing the insert at different positions relative to their ends. These fragments were isolated from agarose gel slices, and mobility was analyzed by electrophoresis in a 5% polyacrylamide gel run at 4°C. Plasmid pBR322 digested with HpaII was used as the size marker (lane Mw). (C) Physical structure of plasmid pBEND-11. The restriction sites that occur only once within each duplicated region flanking the insert (box) are indicated. The mobility of each permuted fragment was determined and plotted against the distance from the 5′ end (EcoRI site) of the duplicated fragment in the vector to the midpoint of the fragment generated by each of the restriction enzymes used. The line represents the best fit to the experimental data, and the arrow points to their minima, which correspond to the center of curvature (see text for details).

FIG. 7

DNA sequence of the 5′ region of gyrA and localization of the transcription initiation site. Sequenase reactions using plasmid pGYAN6 as the template and gyrA20 as the primer provided a reference sequence ladder. G, A, T, and C indicate the dideoxynucleotides used during the sequencing assay. For primer extension experiments, RNAs obtained from E. coli XL1-Blue containing either pEMBL18⁺ (lane 1, 15 μg of RNA) or pGYAN6 (lane 2, 3 μg of RNA; lane 3, 15 μg of RNA) were used. The arrow indicates the direction of electrophoresis. The extended −10 region, the first nucleotide of the mRNA (+1), and the putative ribosome-binding site (RBS) are framed. The double-strand DNA sequence of the 5′ gyrA region and the deduced amino acid sequence are shown.