Biofilm formation by uropathogenic E. coli is mediated by a range of cell surface factors, including fimbriae, flagella, and adhesins. Often, the genes encoding these factors are located on mobile genetic elements, such as plasmids, transposons, and pathogenicity islands. Here we determined the complete nucleotide sequence of a conjugative plasmid isolated from a strain of uropathogenic E. coli that caused CAUTI and defined the properties of this plasmid that are associated with biofilm growth.
Plasmid pMAS2027 is most closely related to conjugative plasmids belonging to the IncX group (36
), and its nucleotide sequence strongly suggested that it belonged to IncX1, a subset of this group (20
). The genetic organization of pMAS2027 is similar to that previously described for several characterized virulence plasmids from S. enterica
). Indeed, of the 58 ORFs identified on pMAS2027, 40 (69%) exhibit the strongest nucleotide sequence similarity to S. enterica
genes. The major genetic load region of pMAS2027 comprised the mrk
genes (encoding type 3 fimbriae), which appear to be located on a mobile genetic element. Recently, a large conjugative plasmid (pOLA52) isolated from swine manure that also contains both the mrkABCDF
genes was described (27
). The mrkABCDF
genes of pOLA52 are flanked by transposon-like sequences, and the nucleotide sequences of these genes are >94% identical to the nucleotide sequences of the mrkABCDF
genes of pMAS2027. A comparison of the pilX1
genes of the two plasmids also revealed a high degree of nucleotide sequence conservation, except for the eex
genes. Although pOLA52 is approximately 10 kb larger than pMAS2027 and contains genes that impart multidrug resistance, the similarity between the backbone sequences of the two plasmids is striking considering that the plasmids were identified in strains isolated from two very different environments (i.e., swine manure and the urine of a patient with CAUTI).
The ability to produce type 3 fimbriae was an absolute requirement for biofilm growth of E. coli
MS2027. No biofilm formation was observed with mutants that lacked the mrkABCDF
genes but retained the ability to produce conjugative T4S pili. Thus, unlike the F pilus (16
), T4S pili do not mediate binding to abiotic surfaces and do not promote biofilm formation. Although the role of type 3 fimbriae in biofilm formation was consistent in previous studies performed with plasmid pOLA52 (4
), we found that deletion of mrkABCDF
resulted in a fivefold increase in the conjugation efficiency of pMAS2027. This finding is in contrast to the results of studies performed with pOLA52, where mutation of the mrkC
gene (which abrogates production of type 3 fimbriae) caused a dramatic reduction in conjugation efficiency (4
). It is possible that this discrepancy is due to differences in the makeup of other cell surface components that might interfere with this process between the E. coli
strains harboring pMAS2027 and pOLA52.
Mixed-culture flow chamber assays were employed to examine the contribution of conjugative plasmid transfer to biofilm development. Maintenance of transconjugant MS2199(pMAS2027orf27::cam) cells within a biofilm was dependent on the production of type 3 fimbriae. Thus, the genetic load of pMAS2027 (mrkABCDF) defined its ability to spread laterally within the biofilm. Genes encoding type 3 fimbriae have been identified in many gram-negative uropathogens, including Klebsiella spp., E. coli, Enterobacter spp., P. mirabilis, Serratia spp., Yersinia spp., and Providentia spp. It is likely that the widespread occurrence of type 3 fimbria-encoding genes in these pathogens is associated with plasmid transfer within biofilms in the hospital setting.