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. 2017 Sep 19;8(5):e00443-17.
doi: 10.1128/mBio.00443-17.

BolA Is Required for the Accurate Regulation of c-di-GMP, a Central Player in Biofilm Formation

Ricardo N Moreira  1 , Clémentine Dressaire  2 , Susana Barahona  2 , Lisete Galego  2 , Volkhard Kaever  3 , Urs Jenal  4 , Cecília M Arraiano  1
Affiliations

BolA Is Required for the Accurate Regulation of c-di-GMP, a Central Player in Biofilm Formation

Ricardo N Moreira et al. mBio. .

Abstract

The bacterial second messenger cyclic dimeric GMP (c-di-GMP) is a nearly ubiquitous intracellular signaling molecule involved in the transition from the motile to the sessile/biofilm state in bacteria. C-di-GMP regulates various cellular processes, including biofilm formation, motility, and virulence. BolA is a transcription factor that promotes survival in different stresses and is also involved in biofilm formation. Both BolA and c-di-GMP participate in the regulation of motility mechanisms leading to similar phenotypes. Here, we establish the importance of the balance between these two factors for accurate regulation of the transition between the planktonic and sessile lifestyles. This balance is achieved by negative-feedback regulation of BolA and c-di-GMP. BolA not only contributes directly to the motility of bacteria but also regulates the expression of diguanylate cyclases and phosphodiesterases. This expression modulation influences the synthesis and degradation of c-di-GMP, while this signaling metabolite has a negative influence in bolA mRNA transcription. Finally, we present evidence of the dominant role of BolA in biofilm, showing that, even in the presence of elevated c-di-GMP levels, biofilm formation is reduced in the absence of BolA. C-di-GMP is one of the most important bacterial second messengers involved in several cellular processes, including virulence, cell cycle regulation, biofilm formation, and flagellar synthesis. In this study, we unravelled a direct connection between the bolA morphogene and the c-di-GMP signaling molecule. We show the important cross-talk that occurs between these two molecular regulators during the transition between the motile/planktonic and adhesive/sessile lifestyles in Escherichia coli This work provides important clues that can be helpful in the development of new strategies, and the results can be applied to other organisms with relevance for human health.IMPORTANCE Bacterial cells have evolved several mechanisms to cope with environmental stresses. BolA-like proteins are widely conserved from prokaryotes to eukaryotes, and in Escherichia coli, in addition to its pleiotropic effects, this protein plays a determinant role in bacterial motility and biofilm formation regulation. Similarly, the bacterial second messenger c-di-GMP is a molecule with high importance in coordinating the switch between planktonic and sessile life in bacteria. Here we have unravelled the importance of accurate regulation of cross-talk between BolA and c-di-GMP for a proper response in the regulation of these bacterial lifestyles. This finding underlines the complexity of bacterial cell regulation, revealing the existence of one additional tool for fine-tuning such important cellular molecular mechanisms. The relationship between BolA and c-di-GMP gives new perspectives regarding biofilm formation and opens the possibility to extend our studies to other organisms with relevance for human health.

Keywords: BolA; biofilm; c-di-GMP; diguanylate cyclase; motility; phosphodiesterase.

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Figures

FIG 1
FIG 1
Influence of BolA in cell motility and c-di-GMP regulation. (A) To measure motility, bacteria were inoculated in swimming agar plates. The plates were incubated at 37°C for 17 h and pictures were taken using ImageScanner III (GE Healthcare Life Sciences). Significant differences, relative to the wt strain, were determined by measuring the diameter of the swimming halo. ****, P < 0,0001 (analysis of variance [ANOVA] test). (B) Quantification of c-di-GMP metabolite in bacteria expressing different amounts of BolA. In the absence of BolA, the c-di-GMP concentration in the cells was increased about 1.7-fold. In the presence of high levels of this protein, the c-di-GMP concentration was reduced. ****, P ≤ 0.0001 (Student’s t test).
FIG 2
FIG 2
Validation of microarrays results by RT-PCR and analysis of BolA effect on the intracellular c-di-GMP levels in ydaM and yhjH knockout mutants. (A) The levels of several DGCs and PDEs differentially regulated by BolA were determined to validate the data from the microarrays. In all reactions, 50 ng of total RNA that had been extracted 3 h after the addition of arabinose to the media was used. The results of parallel RT-PCR procedures run in the absence of reverse transcriptase yielded no product. The results of RT-PCR performed with primers specific for 16S rRNA showed that there were no significant variations in the amount of RNA used in each sample. The images are representative of results from at least three independent RT-PCR experiments performed with RNAs from two different extractions. (B and C) A bolA ydaM double mutant showed a reversion effect on the elevated levels of c-di-GMP caused by a single bolA mutation (B). In the bolA++ strain lacking the yhjH gene, inhibition of c-di-GMP production was retained (C). Both observations indicate a modulation of c-di-GMP levels dependent on BolA through the regulation of ydaM and yhjH gene expression. ****, P ≤ 0.0001 (one-way ANOVA test).
FIG 3
FIG 3
Real-time biolayer interferometry measurement of 0.5, 1.0, and 1.5 M BolA in immobilized substrate. BolA association and dissociation data are labeled and represented by the dashed red line. BSA protein and flhDC promoter sequence were used as negative controls.
FIG 4
FIG 4
Motility assay of a wt strain growing in LB and in LB supplemented with 10 µM c-di-GMP, cGMP, GTP, pGpG, or guanosine. When growth medium was supplemented with c-di-GMP, similar effects of elevated intracellular levels of this metabolite were observed, representing inhibition of bacterial motility. The same effect was observed when the medium was supplemented with cGMP, GTP, and guanosine but not when it was supplemented with pGpG. cAMP, a metabolite that is not related to c-di-GMP, was used as negative control under the same conditions. Significant differences were determined by measuring the swimming halo diameter after 10 h of growth. ****, P < 0.0001 (one-way ANOVA test).
FIG 5
FIG 5
Influence of c-di-GMP in the transcription of bolA. (A and B) bolA mRNA relative expression levels determined by qRT-PCR. Samples in the exponential (A) and stationary (B) phases of growth were analyzed. The levels of expression of bolA were compared in wt and bolA+ strains in the presence or absence of c-di-GMP. All values are relative to expression of the wt strain in the exponential phase. (C) Graph showing the β-galactosidase activity of a construct with the bolA promoter region. The expression of β-galactosidase was analyzed in the wt strain cultured in LB media or in LB media supplemented with 1 µM c-di-GMP. *, P ≤ 0.05; **, P ≤ 0.01 (Student’s t test).
FIG 6
FIG 6
Influence of YdaM and YhjH in the transcription of bolA. (Left) In a mutant strain with a deletion of yhjH (ΔyhjH mutant) and consequently with higher intracellular levels of c-di-GMP, bolA expression was reduced. (Right) When ydaM was deleted, with the concomitant decrease of c-di-GMP levels, expression of bolA mRNA was slightly increased in comparison to the wt strain levels. ***, P ≤ 0.001 (Student’s t test).
FIG 7
FIG 7
Cross-talk between the transcription factor BolA and the second messenger c-di-GMP in bacterial biofilm formation. BolA and c-di-GMP are known to be important players in biofilm development. (A) Our model suggests that a negative-feedback modulation, which leads to a balance between these two factors, is needed for a proper physiological response. (B) Additionally, the absence of BolA leads to less-robust biofilm formation, even in the presence of high levels of c-di-GMP, which evidences the determinant function of this protein in the regulation of biofilm formation.
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