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. 2009 Nov;75(21):6783-91.
doi: 10.1128/AEM.00974-09. Epub 2009 Aug 28.

Conjugative plasmid transfer and adhesion dynamics in an Escherichia coli biofilm

Cheryl-Lynn Y Ong  1 , Scott A Beatson, Alastair G McEwan, Mark A Schembri
Affiliations

Conjugative plasmid transfer and adhesion dynamics in an Escherichia coli biofilm

Cheryl-Lynn Y Ong et al. Appl Environ Microbiol. 2009 Nov.

Abstract

A conjugative plasmid from the catheter-associated urinary tract infection strain Escherichia coli MS2027 was sequenced and annotated. This 42,644-bp plasmid, designated pMAS2027, contains 58 putative genes and is most closely related to plasmids belonging to incompatibility group X (IncX1). Plasmid pMAS2027 encodes two important virulence factors: type 3 fimbriae and a type IV secretion (T4S) system. Type 3 fimbriae, recently found to be functionally expressed in E. coli, played an important role in biofilm formation. Biofilm formation by E. coli MS2027 was specifically due to expression of type 3 fimbriae and not the T4S system. The T4S system, however, accounted for the conjugative ability of pMAS2027 and enabled a non-biofilm-forming strain to grow as part of a mixed biofilm following acquisition of this plasmid. Thus, the importance of conjugation as a mechanism to spread biofilm determinants was demonstrated. Conjugation may represent an important mechanism by which type 3 fimbria genes are transferred among the Enterobacteriaceae that cause device-related infections in nosocomial settings.

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Figures

FIG. 1.
FIG. 1.
Circular diagram of plasmid pMAS2027. The outer circle indicates (to scale) the genetic organization of ORFs within the plasmid. The direction of transcription of each ORF is indicated. The middle circle indicates the G+C content, and the inner circle indicates the GC skew. Genes are color coded as indicated. The image was constructed using cgview (37). CDS, coding sequences.
FIG. 2.
FIG. 2.
Physical map comparing the genes encoding the T4S system (and adjacent genes) in plasmid pMAS2027 to the corresponding regions in plasmids pSE34 (Salmonella serovar Enteritidis; EU219533), pOU1115 (Salmonella serovar Dublin; DQ115388), p2ESCUM (E. coli; CU928149), pOLA52 (E. coli; EU370913), pOU1114 (Salmonella serovar Dublin; DQ115387), R6K (E. coli; AJ006342), and pHI4320 (P. mirabilis; AM942760) and the pathogenicity island (PAI) in E. coli ECOR31 (AY233333). The genes encoding the T4S system are blue and include ddp1 (taxA), taxC, actX, pilX1 to pilX11, and taxB. Adjacent genes (gray) encode hypothetical proteins with unknown functions. No sequence information is available for genes outside the T4S system gene cluster in E. coli R6K. The direction of transcription of each gene is indicated.
FIG. 3.
FIG. 3.
Biofilm formation by E. coli MS2027 and derivatives. Strains were grown in urine for 16 h in PVC microtiter plates under various conditions, as indicated. Biofilm formation was quantified by staining adhered cells with 0.1% crystal violet, resuspending them in ethanol-acetate (80:20), and measuring the absorbance at 595 nm. The results are the averages and standard deviations of three independent experiments. The data are the results for MS2027, MS2362 [MS2027(pMAS2027orf27::cam)], MS2181 [MS2027(pMAS2027mrk::cam)], MS2103 [MS2027(pMAS2027pilX::kan)], and MS2183 [MS2027(pMAS2027mrk::cam, pilX::kan)].
FIG. 4.
FIG. 4.
Flow chamber biofilm formation for (A) E. coli MS2091 (MS2027 attB::bla-PA1/04/03-gfpmut3b*-T0;Gfp), (B) MS2519 [MS2027(pMAS2027orf27::gfp-kan)], (C) MS2517 [MS2027(pMAS2027mrk::gfp-kan)], and (D) MS2518 [MS2027(pMAS2027pilX::gfp-kan)]. Biofilm development was monitored by confocal scanning laser microscopy 24 h after inoculation. The large micrographs show horizontal sections. To the right of and above each large image are images of the yz plane and the xz plane, respectively, obtained at the positions indicated by the lines.
FIG. 5.
FIG. 5.
(A) Flow chamber biofilm formation for E. coli MS2199 (Rfp+) and mixed cultures of E. coli MS2199 (Rfp+) and MS2517 [MS2027(pMAS2027mrk::gfp-kan)] (Gfp+), MS2199 (Rfp+) and MS2518 [MS2027(pMAS2027pilX::gfp-kan)] (Gfp+), and MS2199 (Rfp+) and MS2519 [MS2027(pMAS2027orf27::gfp-kan)] (Gfp+). Magnification, ×40. Biofilm development was monitored by confocal scanning laser microscopy at 16 h, 30 h, and 40 h after inoculation. The micrographs show horizontal sections. To the right of and above each large panel are images of the yz plane and the xz plane, respectively, obtained at the positions indicated by the lines. The largest micrograph shows a higher magnification (×100) of a defined region of the MS2199-MS2519 mixed biofilm. (B) SEM micrograph of a mixed MS2199-MS2519 microtiter plate biofilm. Putative T4S pilus structures are indicated by arrows.
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