The divergently transcribed Streptococcus parasanguis virulence-associated fimA operon encoding an Mn(2+)-responsive metal transporter and pepO encoding a zinc metallopeptidase are not coordinately regulated

Abstract

The study of how bacteria respond to and obtain divalent metal ions provides insight into the regulation of virulence factors in the host environment. Regulation of metal permease operons in gram-positive bacteria may involve the binding of metal-responsive repressors to palindromic domains in their control regions. The Streptococcus parasanguis fimA operon, which encodes an ATP-binding cassette (ABC) transporter system with sequence homology to the LraI family of metal transporters, possesses a palindromic regulatory region with high homology to that of the Streptococcus gordonii ScaR binding domain. Mapping of the promoter and regulatory regions of fimA and the divergently transcribed pepO gene, which encodes a zinc metalloendopeptidase, indicated that their promoter and regulatory elements overlap. fimA had one transcriptional start site, whereas pepO had three. Analysis of truncated versions of the pepO promoter suggested that all three transcriptional start sites are functional. Analysis of promoter activity under various environmental conditions indicated that the fimA operon promoter and the pepO promoter are not coordinately regulated. The fimA operon is responsive to changes in Mn(2+) concentration, but the pepO promoter is not. A S. parasanguis fimA mutant showed a growth deficiency under conditions of limiting Mn(2+). This deficiency was not alleviated by compensation with either Mg(2+) or Fe(3+). Wild-type S. parasanguis could take up Mn(2+) and Fe(3+), while the fimA mutant showed a marked reduction in this ability. These data suggested that FimA is a component of a metal transporter system capable of transporting both Mn(2+) and Fe(3+). FimA expression itself was shown to be responsive to Mn(2+) concentration, but not to availability of Fe(3+) or Mg(2+).

FIG. 1.

Primer extension (PE) analysis of the fimA (A) and pepO (B and C) transcripts. In lane PE, oligonucleotide was annealed to 10 μg of S. parasanguis FW213 RNA and extended using SuperScript RNase H⁻ reverse transcriptase. The nucleotide sequences of pVT1198 and pVT1327 (lanes G, A, T, and C) were determined using the same oligonucleotide as a primer.

FIG. 2.

Schematic representation of the orientation of the pepO gene and the fimA operon in S. parasanguis. The expanded region shows the nucleotide sequence encompassing the 5′ coding region of pepO, the intergenic region between pepO and the fimA operon, and the 5′ coding region of the fimA operon. The fimA operon sequence (top sequence) and pepO sequence (bottom sequence) are shown. Sequence reported elsewhere (12) (dashed and dotted line) and sequences to which designated primers were designed for PCR and cloning purposes (bold italic type) are indicated. The arrows indicate the location of the start(s) of transcription for either the fimA operon or pepO gene, as determined by primer extension analysis. For the genes of the fimA operon and pepO gene, the start site of translation (bold type) and putative ribosomal binding sites (RBS) (underlined sequence) are shown. The putative −10 and −35 sites of the fimA operon (boxed sequence on grey background) and the pepO promoter (boxed sequence on white background) are indicated.

FIG. 3.

Schematic diagram of the S. parasanguis pepO gene and the fimA operon. The start of transcription of the fimA operon (thick black line) and the three start sites of pepO transcription (thin white boxes) are indicated. The expanded region shows the area of overlap between the fimA operon promoter and the pepO promoter. The fimA operon sequence (top sequence) and pepO sequence (bottom sequence) in the designated 5′ to 3′ orientations are shown. The region that shares high DNA sequence identity with the metallorepressor binding domain in S. gordonii sca operon (20) is boxed on three sides. The putative −10 and −35 sites of the fimA operon (boxed sequence on grey background) and the pepO promoter (boxed sequence on white background) are indicated. The arrows represent transcription products from either the fimA operon promoter or the pepO promoter.

FIG. 4.

Luciferase assay of VT1549 and FW213 strains containing either the fimA operon promoter construct (A) or the pepO promoter construct (B), grown in Chelex-100-treated CDM supplemented with 5.0 μM Mg²⁺ and 0.1 μM Fe³⁺ and various concentrations of Mn²⁺ (0.1 μM [dark grey bars] or 10 μM [light grey bars]). One microgram of total protein of whole-cell extracts in a final volume of 20 μl of RLB was mixed with 100 μl of luciferase assay reagent, and the light produced was measured in a luminometer. These experiments were performed four times; means and standard errors (error bars) are shown and adjusted for controls.

FIG. 5.

Effects of Mg²⁺ and Mn²⁺ concentrations on growth of wild-type FW213 and fimA mutant. Wild-type FW213 (♦) and fimA mutant (▪) were grown in the presence of 1.0 μM Mg²⁺ and various concentrations of Mn²⁺ (A) and 1.0 μM Mn²⁺ and various concentrations of Mg²⁺ (B). Cells were grown for 16 h in Chelex-100-treated CDM supplemented with 1.0 μM Fe³⁺ and metals as indicated, and cell density (OD₆₀₀) was measured.

FIG. 6.

Uptake of ⁵⁴Mn (grey bars) or ⁵⁵Fe (stippled bars) by wild-type FW213 and fimA and fap1 mutant S. parasanguis strains. Cell-associated counts were corrected for cell number as determined by CFU analysis. All assays were done in duplicate; means and standard errors (error bars) are shown. This experiment was performed twice; a representative data set is shown.

FIG. 7.

Competition of manganese (A) and iron (B) uptake. The indicated amount of MnCl₂ or FeCl₃ was added to the uptake assay. All assays were done in duplicate; means and standard errors (error bars) are shown. This experiment was performed twice; a representative data set is shown.

FIG. 8.

Immunoblot detection of FimA expression in wild-type FW213 cells grown in Chelex-100-treated CDM supplemented with metal ions. The cells were grown with the following concentrations of Mn²⁺, Mg²⁺, and Fe³⁺: Mn²⁺ (0.05 to 10 μM), 5.0 μM Mg²⁺, and 1.0 μM Fe³⁺ (A); Mg²⁺ (0.01 to 5.0 μM), 1.0 μM Fe³⁺, and 1.0 μM Mn²⁺ (B); and Fe³⁺ (0.05 to 10 μM), 5.0 μM Mg²⁺, and 1.0 μM Mn²⁺ (C). Proteins were extracted from an equal number of cells as measured by OD₄₉₀, subjected to SDS-polyacrylamide gel electrophoresis, blotted onto nitrocellulose, and probed with polyclonal antiserum directed toward FimA. (D) Immunoblot detection of PepO expression in wild-type FW213 cells grown in Chelex-100-treated CDM supplemented with Mn²⁺ (0.1, 1.0, or 10 μM), 5.0 μM Mg²⁺, and 0.1 μM Fe³. One microgram of total protein per lane was subjected to SDS-polyacrylamide gel electrophoresis, blotted onto nitrocellulose, and probed with polyclonal antiserum directed toward PepO.