A new biofilm-associated colicin with increased efficiency against biofilm bacteria

Abstract

Formation of bacterial biofilm communities leads to profound physiological modifications and increased physical and metabolic exchanges between bacteria. It was previously shown that bioactive molecules produced within the biofilm environment contribute to bacterial interactions. Here we describe new pore-forming colicin R, specifically produced in biofilms formed by the natural isolate Escherichia coli ROAR029 but that cannot be detected under planktonic culture conditions. We demonstrate that an increased SOS stress response within mature biofilms induces SOS-dependent colicin R expression. We provide evidence that colicin R displays increased activity against E. coli strains that have a reduced lipopolysaccharide length, such as the pathogenic enteroaggregative E. coli LF82 clinical isolate, therefore pointing to lipopolysaccharide size as an important determinant for resistance to colicins. We show that colicin R toxicity toward E. coli LF82 is increased under biofilm conditions compared with planktonic susceptibility and that release of colicin R confers a strong competitive advantage in mixed biofilms by rapidly outcompeting sensitive neighboring bacteria. This work identifies the first biofilm-associated colicin that preferentially targets biofilm bacteria. Furthermore, it indicates that the study of antagonistic molecules produced in biofilm and multispecies contexts could reveal unsuspected, ecologically relevant bacterial interactions influencing population dynamics in natural environments.

Figure 1

Bacteriotoxic effect of ROAR029 extracts upon E. coli MG1655F'. (a) Quantification of the effects of ROAR029 planktonic (ROAR029 Pk) and biofilm (ROAR029 Bf) extracts on biofilm formation and growth of MG1655F'. M63B1, control. ***P<0.001. (b) Growth inhibition of ROAR029 planktonic (ROAR029 Pk) and biofilm extracts (ROAR029 Bf) on a lawn of E. coli MG1655. (c) Growth inhibition of ROAR029 colony on a lawn of E. coli MG1655. (d) Effects of ROAR029 biofilm extract (ROAR029 Bf) on a lawn of valine-resistant E. coli MG1655 ilvG+.

Figure 2

ROAR029 bacteriotoxic activity is due to plasmid-encoded colicin regulated by PcnB. (a) Addition of an 8-μl drop of ROAR029 wild type, ROAR029ΔpcnB or ROAR029 bearing a mutagenized plasmid unable to synthesize the bacteriotoxic molecule (ROAR029 pColR−) biofilm extract to a lawn of MG1655. (b) DNA gel of ROAR029 pColR plasmid linearized by the EcoRV enzyme. (c) ROAR029 pColR plasmid map. Arrows indicate transcription polarities of colicin genes. Unique-site restriction enzymes are indicated. (d) Dendrogram of sequences alignment of the 200 last amino acids from colicin-encoding genes of the C-terminal region. Phylogenetic analysis was carried out using the UPGMA method. Numbers represent bootstrap values >75.

Figure 3

ROAR029 colicin is a group A colicin. Effect of addition of an 8-μl drop of ROAR029 (colicin R) or S. boydii M592 (colicin U) biofilm extracts to a lawn of E. coli MG1655 (wild type), MG1655 ΔtolA and MG1655 ΔompA cells.

Figure 4

ROAR029 biofilm-specific production of colicin R is due to an SOS regulatory effect. (a) Sequence analysis revealed two overlapping SOS boxes upstream of the ribosome binding sequence (rbs) and start codon ATG of ROAR029. Insertion site of transposon in pColR+ plasmid is indicated. Transcriptional read-through from kanamycin gene and disruption of LexA box1 binding site may explain constitutive expression of colicin R encoding gene. (b) Effect of LexA induction upon ROAR029 bacteriotoxic activity was assessed using ROAR029 with LexA under the control of an anhydrotetracycline-inducible (aTc) promoter. An 8-μl drop of ROAR029_KmRExTET_LexA treated with increasing concentrations of aTc ranging from 0 to 1000 ng ml⁻¹ was added to a lawn of MG1655. (c) Inhibition halo of E. coli MG1655 when treated with an 8-μl drop of ROAR029 biofilm extract (ROAR029 Bf) with and without induction of the SOS system by 0.1 μg ml⁻¹ of mitomycin C added after inoculation. (d) Growth inhibition of E. coli MG1655 by biofilm extracts produced by E. coli TG1 (black) and TG1 lexAind3 (gray) transformed by different ROAR029 plasmids; active kanamycin-resistant pColR_Km, defective colicin mutant (pColR-, dashed) and constitutively expressed colicin (pColR+, shaded).

Figure 5

ROAR029 colicin activity is dependent on LPS length. (a) SDS-polyacrylamide (14%) gel showing LPS reconstruction of rough E. coli MG1655 and E. coli TG1 strains. (b) Effect of ROAR029 biofilm extract on bacterial lawns of rough (MG1655 and TG1) and smooth (MG1655 wbbL+ and TG1 wbbL+) strains. (c) SDS-polyacrylamide gel (14%) of E. coli 536 wild type and LPS (rfaH, manB, waaG and waaC), capsule (kps15) and colanic acid (cpsG) mutants. (d) Effect of ROAR029 biofilm extract on E. coli 536 wild type and LPS mutants lawns. (e) Growth curves showing the effect of ROAR029 biofilm extract addition to cultures of E. coli 536 and mutants with different LPS lengths. The graph shows means of eight independent wells from a representative experiment. ΔrfaHc, complemented rfaH mutant.

Figure 6

ROAR029 excludes MG1655F' from mixed biofilms. Percentage of ROAR029 pColR_Km, pColR– or pColR+ and MG1655F' after 24 h of mixed biofilms inoculated at different initial ratios (1:1, 1:10, 1:100 and 1:10 000).

Figure 7

Colicin R has enhanced activity towards biofilms of LF82. Differential effect of ROAR029 biofilm extract on planktonic (exponential and stationary phase) and biofilm cells (resuspended and non-resuspended) of two pathogenic E. coli strains, 55989 with full LPS and LF82 with no LPS. Survival was determined by CFU counts after 2-h exposure to equal quantities of ROAR029 biofilm extract containing colicin R. ***P<0.001.