BolA is a transcriptional switch that turns off motility and turns on biofilm development

Abstract

Bacteria are extremely versatile organisms that rapidly adapt to changing environments. When bacterial cells switch from planktonic growth to biofilm, flagellum formation is turned off and the production of fimbriae and extracellular polysaccharides is switched on. BolA is present in most Gram-negative bacteria, and homologues can be found from proteobacteria to eukaryotes. Here, we show that BolA is a new bacterial transcription factor that modulates the switch from a planktonic to a sessile lifestyle. It negatively modulates flagellar biosynthesis and swimming capacity in Escherichia coli. Furthermore, BolA overexpression favors biofilm formation, involving the production of fimbria-like adhesins and curli. Our results also demonstrate that BolA is a protein with high affinity to DNA and is able to regulate many genes on a genome-wide scale. Moreover, we show that the most significant targets of this protein involve a complex network of genes encoding proteins related to biofilm development. Herein, we propose that BolA is a motile/adhesive transcriptional switch, specifically involved in the transition between the planktonic and the attachment stage of biofilm formation.

Importance: Escherichia coli cells possess several mechanisms to cope with stresses. BolA has been described as a protein important for survival in late stages of bacterial growth and under harsh environmental conditions. BolA-like proteins are widely conserved from prokaryotes to eukaryotes. Although their exact function is not fully established at the molecular level, they seem to be involved in cell proliferation or cell cycle regulation. Here, we unraveled the role of BolA in biofilm development and bacterial motility. Our work suggests that BolA actively contributes to the decision of bacteria to arrest flagellar production and initiate the attachment to form structured communities, such as biofilms. The molecular studies of different lifestyles coupled with the comprehension of the BolA functions may be an important step for future perspectives, with health care and biotechnology applications.

FIG 1

Classification and graphical representation of the transcriptome assay. (A) Volcano plots representing the transcriptome results corresponding to the time point of 1 h (left side) and 3 h (right side) after bolA induction. Genes associated to an FDR lower than 10% (represented by the dashed horizontal line) were considered significant. The green and red spots represent the downregulated and upregulated genes. The numbers of genes significantly repressed or overexpressed under each condition are indicated by the green and red numbers, respectively. (B) Graphical representation of the gene ontology (GO) biological process enrichments results 1 h after bolA induction. Functional categories that were significantly overrepresented among upregulated or downregulated genes are shown. Bar length corresponds to the average fold change (in absolute value of the bolA++/ΔbolA ratio) of the genes significantly upregulated or downregulated associated with each GO category according to transcriptome results. (C) Graphical representation of GO biological process enrichment results 3 h after bolA induction with the respective fold change (bolA++/ΔbolA). The asterisk represents the statistical significance level of the differential expression of each GO category. Green graphic shows downregulated genes and red the upregulated genes. **, P value associated with the enrichment test was lower than 5 × 10⁻⁴; ***, P value associated with the enrichment test was lower than 5 × 10⁻⁶. No asterisk indicates that the P value associated with the enrichment test was between 5 × 10⁻² and 5 × 10⁻⁴.

FIG 2

Motility assays in the presence of different doses of BolA protein. (A) To measure motility, bacteria were inoculated in swimming agar plates. The plates were incubated at 37°C for 18 h and photographed. (B) Time course analysis of the bacterial swimming. The dispersion radius was quantified between 12 h and 26 h of incubation at 37°C. One-hundred percent represents the detection limit (whole plate covered by the swimming halo). (C) Observation of E. coli flagella using immunofluorescent staining with antiflagellin antibody. Scale bar represents 2 µm.

FIG 3

Influence of the BolA protein in bacterial structures and extracellular components involved in biofilm matrix formation. (A) Effect of BolA on biofilm development in microtiter plates. The thickness of biofilms in cultures of different strains was measured by determining the OD₅₇₀ after staining with crystal violet and normalization by OD₆₀₀ of the planktonic culture. Error bars represent standard deviations. (B) Scanning electron microscopy images of the wt and bolA++ strains. Images were obtained from overpopulated LB plates supplemented with arabinose. The secondary imaging mode of scanning electron microscopy was used. Panels 1 to 3 correspond to wt, while 4 to 6 correspond to the bolA++ strain. The wt displays the regular E. coli bacillus shape, whereas the characteristic BolA overexpression-associated round phenotype can be observed in bolA++ images. The arrows indicate the observed fimbria-like adhesins produced by the bacterial cells in the presence of elevated levels of BolA. (C) A blue color on the toluidine blue O plate indicates the production of extracellular DNA (eDNA). In Congo red plates, the brown, dry, and rough (BDAR) phenotype reveals the production of curli, while the red, dry, and rough (RDAR) phenotype indicates the presence of both curli and cellulose. Regarding the Calcofluor plates, fluorescence detection is indicative of polysaccharide (such as cellulose) production. Finally, a pink color in ruthenium red plates stands for the presence of colanic acid production. (D) Quantification of extracellular sugar, DNA, and protein produced by the different strains expressing BolA after 18 h of growth on an LB plate at 37°C.

FIG 4

Graphical representation of the BolA ChIP-seq results, DNA consensus sequence, and its effect on the transcription of downstream genes. (A) Statistically significant peaks detected for wt 3×Flag and ΔbolA strains. For data processing, the ΔbolA strain was considered the background and subtracted from the wt 3×Flag sample. (B) Graphical representation of the peaks associated with three different targets determined by ChIP-seq. BolA binding regions are spread in the chromosome, not only at the 5′ untranslated region (UTR), but also along the ORFs. (C) The BolA consensus was determined based on the pool of DNA sequences corresponding to the identified peaks. The statistical significance of the consensus obtained is associated with a P value of 6.1 × 10⁻³. (D) Graphic showing the β-galactosidase activity of a construct with the mreBCD promoter region containing the consensus sequence and a second construct without it. The plus sign represents the extracts where the consensus was present, while the minus sign indicates the absence of the consensus. The experiment was performed in the wt and the ΔbolA backgrounds. In agreement with the BolA-dependent mreB downregulation, when the consensus sequence is not present, an increased activity of β-galactosidase can be observed, strengthening the importance of the consensus for the proper BolA-dependent regulation. Additionally, in the ΔbolA background, there are no significant differences between the two extracts, excluding the hypothesis of a construct-dependent effect.

FIG 5

Representative flagellar structure and related proteins coupled with microarrays and ChIP-seq analysis results. ChIP-seq analysis showed the capacity of BolA to directly interact with different genes encoding proteins involved in diverse steps of the flagellar biosynthesis pathway. Of the 33% negatively regulated genes related with the flagellar structure (bolA++/ΔbolA ratios), 45% were identified as direct BolA targets (the proteins in pink boxes are encoded by the target genes). The proteins highlighted in green correspond to the respective downregulated genes. Additionally to the genes indicated in the figure, fliZ, fliA, flgD, and flgJ were also found to be direct targets of BolA.

FIG 6

Model for BolA-mediated regulation of planktonic-to-sessile transition-related mechanisms in Escherichia coli. BolA is a global regulator which has a major direct effect in bacterial motility through the repression of flagellum-associated genes and the induction of tricarboxylic acid (TCA) cycle genes. It modulates the regulation of several carbon metabolism pathways connected to peptidoglycan biosynthesis. Biofilm formation is favored by BolA mainly through the differential regulation of genes involved in lipopolysaccharide (LPS) and cellulose production. The presence of elevated levels of extracellular DNA, proteins, and sugars emphasizes the role of this protein in the production of an extracellular matrix necessary for biofilm development. Concordantly, BolA induction has a positive effect in the expression of several genes encoding fimbria-like adhesins, which are possibly involved in the formation of the three-dimensional structure of biofilms.