Identification of novel stage-specific genetic requirements through whole genome transcription profiling of Vibrio cholerae biofilm development

Abstract

Bacterial biofilm formation has been described as a developmental process. This process may be divided into three stages: the planktonic stage, the monolayer stage and the biofilm stage. Bacteria in the planktonic stage are not attached to each other or to a surface; bacteria in the monolayer stage are attached to surfaces as single cells; and bacteria in the biofilm stage are attached to surfaces as cellular aggregates. In a study limited to the Vibrio cholerae flaA, mshA and vps genes, we previously demonstrated that transcription in monolayer cells is distinct from that in biofilm cells and that the genetic requirements of monolayer formation are distinct from those of biofilm formation. In this work, we sought to identify additional stage-specific genetic requirements through microarray analysis of the V. cholerae transcriptome during biofilm development. These studies demonstrated unique patterns of transcription in the planktonic, monolayer and biofilm stages of biofilm development. Based on our microarray results, we selected cheY-3 as well as two previously uncharacterized genes, bap1 and leuO, for targeted mutation. The DeltacheY-3 mutant displayed a defect in monolayer but not biofilm formation, suggesting that chemotaxis plays a stage-specific role in formation of the V. cholerae monolayer. Mutants carrying deletions in bap1 and leuO formed monolayers that were indistinguishable from those formed by wild-type V. cholerae. In contrast, these mutants displayed greatly decreased biofilm accumulation. Our microarray analyses document modulation of the transcriptome of V. cholerae as it progresses through the stages in biofilm development. These studies demonstrate that microarray analysis of the transcriptome of biofilm development may greatly accelerate the discovery of novel targets for stage-specific inhibition of biofilm development.

Fig. 1. Schematic representation of the state of V. cholerae under each of the conditions in this work

A. Monolayer experiment. Test: Monolayer (−M)/Reference: Planktonic (−M). B. Planktonic experiment. Test: Planktonic (+M)/Reference: Planktonic (−M). C. Biofilm experiment. Test: Biofilm (+M)/Reference: Planktonic (+M). D. vpsA monolayer experiment. Test: ΔvpsA mutant monolayer (+M)/Reference: ΔvpsA planktonic (+M). +M and −M refer to the presence and absence of mannose in the growth medium respectively.

Fig. 2

Cluster analysis of the monolayer, planktonic, biofilm and vpsA monolayer experiments described in this work. The intensity of red or green represents the extent of gene induction or repression respectively. Yellow represents no change in gene transcription.

Fig. 3

Modulation of transcription of vps genes and similarly regulated genes in the planktonic and vpsA experiments. The intensity of red or blue represents the extent of gene induction or repression respectively. Yellow represents no change in gene transcription. Putative gene products and functions are listed in a table below.

Fig. 4. Monolayer formation, biofilm formation and vpsL transcription by wild-type V. cholerae, a Δbap1 mutant, a ΔleuO mutant and a ΔvpsA mutant

A. Total surface area covered by wild-type and mutant monolayers formed over 3 h. B. Quantification of wild-type and mutant biofilm accumulation in LB. C. Quantification of vpsL transcription in wild-type and mutant V. cholerae harbouring a chromosomal fusion of the vpsL promoter to the lacZ gene.

Fig. 5. Effect of AMM treatment on wild-type V. cholerae and ΔcheY-3 mutant monolayers formed over 24 h

A. Phase contrast micrographs of monolayers before (Monolayer) and after (Monolayer + 0.1% AMM) treatment with AMM. B. Total surface area covered by monolayer cells before and after treatment with AMM. C. Quantification of wild-type V. cholerae and ΔcheY-3 mutant biofilm accumulation in LB.