Putative surface proteins encoded within a novel transferable locus confer a high-biofilm phenotype to Enterococcus faecalis - PubMed (original) (raw)
Putative surface proteins encoded within a novel transferable locus confer a high-biofilm phenotype to Enterococcus faecalis
Preeti M Tendolkar et al. J Bacteriol. 2006 Mar.
Abstract
Enterococci are opportunistic pathogens and among the leading causes of nosocomial infections. Enterococcus faecalis, the dominant species among infection-derived isolates, has recently been recognized as capable of forming biofilms on abiotic surfaces in vitro as well as on indwelling medical devices. A few bacterial factors known to contribute to biofilm formation in E. faecalis have been characterized. To identify additional factors which may be important to this process, we utilized a Tn917-based insertional mutagenesis strategy to generate a mutant bank in a high-biofilm-forming E. faecalis strain, E99. The resulting mutant bank was screened for mutants exhibiting a significantly reduced ability to form biofilms. One mutant, P101D12, which showed greater than 70% reduction in its ability to form biofilms compared to the wild-type parent, was further characterized. The single Tn917 insertion in P101D12 was mapped to a gene, bee-2, encoding a probable cell wall-anchored protein. Sequence information for the region flanking bee-2 revealed that this gene was a member of a locus (termed the bee locus for biofilm enhancer in enterococcus) comprised of five genes encoding three putative cell wall-anchored proteins and two probable sortases. Contour-clamped homogeneous electric field gel and Southern hybridization analyses suggested that the bee locus is likely harbored on a large conjugative plasmid. Filter mating assays using wild-type E99 or mutant P101D12 as a donor confirmed that the bee locus could transfer conjugally at high frequency to recipient E. faecalis strains. This represents the first instance of the identification of a mobile genetic element conferring biofilm-forming property in E. faecalis.
Figures
FIG. 1.
The presence of a single Tn_917_ insertion within the bee locus in mutant strain P101D12 was confirmed by Southern analysis using a Tn_917_-specific probe (a 1.9-kb internal region of Tn_917_ containing two HindIII restriction sites) as described previously (10). (A) Genomic DNA obtained from wild-type strain E99 and mutant P101D12 was restricted with HindIII (lanes 1 and 2) and EcoRI (lanes 4 and 5). (B) Southern hybridization was performed subsequently with the Tn_917_ probe obtained by using primers Tn_917_-1 and Tn_917_-2. The HindIII (lane 3)- and EcoRI (lane 6)-restricted plasmid pTV1-OK was used as the positive control. For size reference, a 1-kb DNA ladder (New England Biolabs, Beverly, MA) was used (lane M).
FIG. 2.
RT-PCR analyses to assess the expression and transcriptional linkage of genes within the bee locus in the wild-type strain E99 and mutant P101D12. (A) Schematic of the bee locus. The bee locus is comprised of three genes, bee-1, bee-2, and bee-3, encoding putative cell wall-anchored proteins, downstream of which are two ORFs, srt-1 and srt-2, encoding putative sortase enzymes. The locations of various primers used for the RT-PCR analysis are indicated. The position of the Tn_917_ insertion (▾) in mutant P101D12 is also indicated. Line arrows indicate putative transcriptional units. (B) Total RNA extracted from E99 was reverse transcribed using random hexamers and reverse transcriptase. An aliquot of the cDNA was then amplified by PCR with gene-specific primers. Primers P101D12-2/P101D12-4 (lane 2), P101D12-3/P101D12-1 (lane 3), P101D12-2/E99-2 (lane 4), Bee-12/P101D12-1 (lane 5), Sor-1L/Sor-1R (lane 6), P101D12-13/Sor-2R (lane 7), and P101D12-11/Bee-13 (lane 8) yielded 259-, 203-, 954-, 532-, 650-, 485-, and 581-bp amplification products, respectively. This implies that the genes bee-1, bee-2, and bee-3 may be cotranscribed as one transcriptional unit, with srt-1 and srt-2 cotranscribed independently. (C) Total RNA extracted from mutant strain P101D12 was used for RT-PCR. Primers P101D12-2/P101D12-4 (lane 2) yielded an expected 259-bp amplification product, indicating that bee-1 was being transcribed. However, no amplification products were obtained when primer pairs P101D12-3/P101D12-1 (lane 3), P101D12-2/E99-2 (lane 4), or Bee-12/P101D12-1 (lane 5) were used for amplification, suggesting that Tn_917_ insertion had abrogated not only the expression of bee-2 but also that of the downstream gene bee-3. Sor-1L/Sor-1R (lane 6), P101D12-13/Sor-2R (lane 7), and P101D12-11/Bee-13 (lane 8) yielded 650-, 485-, and 581-bp amplification products, respectively, implying that the Tn_917_ insertion in bee-2 did not abrogate expression of srt-1 and srt-2. For size reference, a 100-bp DNA ladder (Invitrogen, Carlsbad, CA) was used (lane 1).
FIG. 3.
Biofilm formation by wild-type strain E99, mutant P101D12(pAT28), and complemented strains BLG1 (P101D12 transformed with pAT28 harboring bee-2 downstream of the aphA-3 promoter), BLG2 (P101D12 transformed with pAT28 harboring bee-3 downstream of the aphA-3 promoter), and BLG12 (P101D12 transformed with pAT28 harboring bee-2 and bee-3 downstream of the aphA-3 promoter). The y axis represents the optical density (OD) of dissolved crystal violet measured at 595 nm. The error bars represent the mean ± standard error.
FIG. 4.
CHEF gel and Southern hybridization analysis of transconjugants obtained from filter mating experiments employing mutant P101D12 as the donor and OG1RF as the recipient. (A) CHEF gel of I-ceuI-restricted DNA from three primary transconjugants, POTC-1, POTC-2, and POTC-3 (lanes 1, 2, and 3, respectively); donor strain P101D12 (lane 4); and recipient strain OG1RF (lane 5). (B and C) Also shown are Southern hybridization analyses of I-ceuI-restricted DNA from the transconjugants (lanes 1 to 3), donor strain P101D12 (lane 4), and recipient strain OG1RF (lane 5) using a 23S rRNA gene probe (B) and a Tn_917_-specific probe (C). Bacteriophage lambda concatemers were used as molecular size markers (lane M; New England Biolabs, Beverly, MA).
FIG. 5.
CHEF gel and Southern hybridization analysis of transconjugants obtained from filter mating experiments employing primary transconjugant POTC-2 as the donor and JH2SS as the recipient. (A) I-ceuI-restricted DNA from three secondary transconjugants, POJTC-1, POJTC-2, and POJTC-3 (lanes 1, 2, and 3, respectively); donor POTC-2 (lane 4); and recipient JH2SS (lane 5). (B and C) Also shown are Southern hybridization analyses of I-ceuI-restricted DNA from the transconjugants (lanes 1 to 3), donor strain POTC-2 (lane 4), and recipient strain JH2SS (lane 5) using a 23S rRNA gene probe (B) and a Tn_917_-specific probe (C). Bacteriophage lambda concatemers were used as molecular size markers (lane M; New England Biolabs, Beverly, MA).
FIG. 6.
Biofilm assay on three transconjugants, IG9, IIB3, and IIE7, obtained by filter mating wild-type E99 with E. faecalis strain OG1RF. Crystal violet binding was used to measure the 24-h biofilm densities of IG9, IIB3, IIE7, and parent strain OG1RF grown in TSB supplemented with 0.5% glucose. The error bars represent the mean ± standard error.
FIG. 7.
CHEF gel and Southern hybridization analysis of transconjugants obtained from filter mating experiments employing wild-type E99 as the donor and OG1RF as the recipient. (A) I-ceuI-restricted DNA from donor E99 (lane 1); three transconjugants, IG9, IIB3, and IIE7 (lanes 2, 3, and 4, respectively); and recipient strain OG1RF (lane 5). (B and C) Also shown are Southern hybridization analyses of I-ceuI-restricted DNA from donor strain E99 (lane 1), the transconjugants (lanes 2 to 4), and recipient strain OG1RF (lane 5) using a 23S rRNA gene probe (B) and a bee locus-specific probe (C). Bacteriophage lambda concatemers were used as molecular size markers (lane M; New England Biolabs, Beverly, MA).
References
- Depardieu, F., M. G. Bonora, P. E. Reynolds, and P. Courvalin. 2003. The vanG glycopeptide resistance operon from Enterococcus faecalis revisited. Mol. Microbiol. 50**:**931-948. -PubMed
- Donlan, R. M. 2001. Biofilm formation: a clinically relevant microbiological process. Clin. Infect. Dis. 33**:**1387-1392. -PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources