A shift from oral to blood pH is a stimulus for adaptive gene expression of Streptococcus gordonii CH1 and induces protection against oxidative stress and enhanced bacterial growth by expression of msrA

Abstract

Viridans group streptococci (VS) from the oral cavity entering the bloodstream may initiate infective endocarditis (IE). We aimed to identify genes expressed in response to a pH increase from slightly acidic (pH 6.2) to neutral (pH 7.3) as encountered by VS entering the bloodstream from the oral cavity. Using a recently developed promoter-screening vector, we isolated five promoter fragments from the genomic DNA of Streptococcus gordonii CH1 responding to this stimulus. No common regulatory sequences were identified in these promoter fragments that could account for the coordinate expression of the corresponding genes. One of the isolated fragments contained the promoter region and 5' end of a gene highly homologous to the methionine sulfoxide reductase gene (msrA) of various bacterial and eukaryotic species. This gene has been found to be activated in S. gordonii strain V288 in a rabbit model of IE (A. O. Kiliç, M. C. Herzberg, M. W. Meyer, X. Zhao, and L. Tao, Plasmid 42:67-72, 1999). We isolated and characterized the msrA gene of S. gordonii CH1 and constructed a chromosomal insertion mutant. This mutant was more sensitive to hydrogen peroxide, suggesting a role for the streptococcal MsrA in protecting against oxidative stress. Moreover, MsrA appeared to be important for the growth of S. gordonii CH1 under aerobic and anaerobic conditions. Both these properties of MsrA may contribute to the ability of S. gordonii to cause IE.

FIG. 1

Complete nucleotide sequence of the promoter fragment SG_P1224. Putative −35 and −10 promoter regions and Shine-Dalgarno sequences (SD) are underlined. P₁ and P₂ are possible promoter stretches driving expression of the promoterless spectinomycin gene, and P_pbg is a putative promoter driving expression of the inversely oriented pbg-like gene. Translational start sites (ATG) are printed in boldface, and partial open reading frames are shown.

FIG. 2

Complete nucleotide sequence of the msrA gene from S. gordonii CH1 and partial sequences of a pyrD homolog and of a putative open reading frame, located upstream and downstream of the streptococcal msrA, respectively. Putative −35 and −10 promoter hexamers and Shine-Dalgarno sequences (SD) are underlined, and the transcriptional start site of msrA is indicated with an asterisk. Inverted repeats, which might form a transcriptional termination stem-loop, are indicated with arrows. Translational start sites (ATG) are printed in boldface, and the translated amino acid sequences of msrA and of the partial open reading frames upstream and downstream of msrA are shown. Nucleotides 1 to 747 represent the sequence of the isolated promoter fragments SG_P1222 and SG_P1225.

FIG. 3

Determination of the transcription start site of S. gordonii CH1 msrA. The transcription start site is indicated with an arrow, and the putative −10 region in the coding strand is presented in boldface.

FIG. 4

Amino acid sequence alignment of MsrA of S. gordonii (Sg) with MsrA proteins of S. pneumoniae (Sp), Helicobacter pylori (Hp), Haemophilus influenzae (Hi), and E. coli (Ec), and with the homologous PilB of N. gonorrhoeae (Ng). Amino acid sequence alignment was performed with the CLUSTAL program. The shaded boxes enclose residues of the MsrA protein from S. gordonii CH1 that are found at identical positions within one or more of the other MsrA sequences or within N. gonorrhoeae PilB.

FIG. 5

Southern blot of S. gordonii strain CH1 and its msrA insertion mutant MM1. The hybridizing fragment in the wild-type strain is increased in size in the mutant strain by 1.0 kb, due to the inserted erythromycin resistance gene.

FIG. 6

Growth of S. gordonii CH1 (squares), its msrA mutant MM1 (circles), and the complemented mutant (MM1 pMM1229; triangles) under aerobic (top) and anaerobic (bottom) conditions in TH medium. The values are the averages of three experiments, and the standard error of the mean is indicated for each value.