Fig 1.
Graphic representation of annotated plasmids pIS15_43 and pIS04_68.
%GC content is illustrated in the center of the plasmid diagrams. Regions of pIS04_68 (IncR) that are typically associated with the IncX1-type plasmids (such as pIS15_43) have been highlighted in both sequence maps.
Fig 2.
Nucleotide sequence alignment of mrkA promoters of plasmids from non-Klebsiella hosts (PmrkA[P]) and K. pneumoniae (PmrkA[Kp]).
Residues that do not align with PmrkA[Kp] are indicated in red, which are all associated with IS1 elements (red lining). Blue background highlights conserved PmrkA[Kp] components and green background denotes the unique -35 box found in PmrkA[P]. TSS: transcriptional start site. PmrkA[P] is predicted to be a stronger promoter than PmrkA[Kp] due to an optimized -35 box and a 2 bp longer spacer region between the -10 box and -35 box.
Fig 3.
Transcriptional activity of promoters PmrkA[Kp] and PmrkA[P] in E. coli.
β-galactosidase assays were performed after growth in LB at 37°C and samples were taken over a period of 8.5h. Growth (black) and β-galactosidase activity (green) of PmrkA[P]-lacZ fusion. Growth (gray) and β-galactosidase activity (blue) of PmrkA[Kp]-lacZ fusion (Error bars denote ±SEM, n = 3).
Fig 4.
Genetic map of the mrkABCDF operon and flanking putative regulatory elements encoded on all fully sequenced representative replicons available in GenBank.
Red: mrkABCDF genes. Orange: mrkH and mrkHL genes. Purple: mrkI and mrkIL genes. Green: mrkJ and mrkJL genes. Blue: putative genes encoding incomplete EAL domains. Dark gray: IS/transposon elements. Light gray: phage associated genes. (”) Plasmids pKPN_CZ and pKPN_262 have been isolated from Klebsiella hosts while all other plasmids originate from non-Klebsiella hosts.
Fig 5.
Maximum likelihood phylogeny based on the nucleotide sequence of PapD_N and PapD_C of mrkB.
(*) Indicates that mrkB is encoded on a plasmid. (#) Indicates that mrkB may be part of a putative genomic island. Percentages of bootstrap support (1000 replicates) are shown.
Fig 6.
Influence of plasmid encoded mrkABCDF expression on biofilm-formation and motility of E. coli.
(a) Biofilm formation by CV staining of attached cells after 24h growth at 37°C in microtitre plate wells. PmrkA[P]-mrkABCDF made more biofilm than both PmrkA[Kp]-mrkABCDF and the control: pLOW2 without mrkABCDF. (Error bars denote ±SEM, n = 3). (b) Swimming motility assay performed in 1% tryptone medium with 0.3% agar at 37°C. Negative controls (MG1655 ΔflhD) confirm the applicability of the assay.
Fig 7.
Examination of putative genes, all encoded on pOLA52, pIS15_43 and pIS04_68 that might influence the biofilm or motility behavior of the bacterial host.
Bottom: CV biofilm assay. E. coli harboring constructs with the mrkABCDF cassette (mrk) formed more biofilm than those without but no difference was found among the biofilm formers (Error bars denote ±SEM, n = 3). Middle: swimming motility assay. Pictures were taken after 8h or 16h at 37°C. No differences were found (all swam equal distances, ++) with the exception of the negative control MG1655 ΔflhD (did not swim, −). MG1655 was used a positive control for swimming (not shown). Top: CR assay. Pictures were taken after 2 days at 30°C. No differences were found (equal amounts of CR was bound, ++) with the exception of pRham-Prhamxeal for which less CR was bound by the colony (+). Pseudomonas aeruginosa PA14 and P. aeruginosa PA14 Δpel were used as positive and negative controls, respectively (not shown).
Fig 8.
Conjugative transfer frequencies of pOLA52 with and without an intact mrkABCDF operon from motile and non-motile donors.
Transfer frequencies were calculated as the number of transconjugants (T) per donor (D). ΔflhD: non-motile E. coli MG1655 flhD deletion mutant unable to synthesize flagella. MG1655: motile E. coli MG1655. pOLA52-oqxB::KANR: type 3 fimbriae positive plasmid. pOLA52-mrkC::KANR: type 3 fimbriae negative plasmid. Each transfer frequency was different from the other (Error bars denote ±SEM, n = 3).