In enterobacteria, the CsgD protein activates production of two extracellular structures: thin aggregative fimbriae (curli) and cellulose. While curli fibres promote biofilm formation and cell aggregation, the evidence for a direct role of cellulose as an additional determinant for biofilm formation is not as straightforward. The MG1655 laboratory strain of Escherichia coli only produces limited amounts of curli and cellulose; however, ectopic csgD expression results in strong stimulation of curli and cellulose production. We show that, in a csgD-overexpressing derivative of MG1655, cellulose production negatively affects curli-mediated surface adhesion and cell aggregation, thus acting as a negative determinant for biofilm formation. Consistent with this observation, deletion of the bcsA gene, necessary for cellulose production, resulted in a significant increase in curli-dependent adhesion. We found that cellulose production increased tolerance to desiccation, suggesting that the function of cellulose might be related to resistance to environmental stresses rather than to biofilm formation. Production of the curli/cellulose network in enterobacteria typically takes place at low growth temperature (,32 6C), but not at 37 6C. We show that CsgD overexpression can overcome temperature-dependent control of the curliencoding csgBA operon, but not of the cellulose-related adrA gene, suggesting very tight temperature control of cellulose production in E. coli MG1655.

Bacteria can switch from a single-cell (planktonic) mode to a multicellular community (biofilm) mode via production of cell-cell aggregation and surface adhesion factors. In this report, we present evidence that the CsgD protein, a transcription regulator involved in biofilm formation in Escherichia coli, modulates the expression of the rpoS ( S ) regulon. Protein pattern analysis of E. coli cells in stationary phase shows that CsgD affects the expression of several proteins encoded by S -dependent genes. CsgD regulation of Sdependent genes takes place at gene transcription level, does not bypass the need for rpoS, and is abolished in an rpoS-null mutant. Consistent with these results, we find that CsgD expression leads to an increase in S intracellular concentration. Increase in S cellular amount is mediated by CsgD-dependent transcription activation of iraP, encoding a factor involved in S protein stabilization. Our results strongly suggest that the CsgD regulatory protein plays a major role as a relay between adhesion factors production and S -dependent gene expression via S protein stabilization. Direct coordination between biofilm formation and expression of the rpoS regulon could positively impact important biological processes, such as host colonization or response to environmental stresses.

BackgroundNext generation sequencing (NGS) technologies have revolutionized gene expression studies and functional genomics analysis. However, further improvement of RNA sequencing protocols is still desirable, in order to reduce NGS costs and to increase its accuracy. In bacteria, a major problem in RNA sequencing is the abundance of ribosomal RNA (rRNA), which accounts for 95-98% of total RNA and can therefore hinder sufficient coverage of mRNA, the main focus of transcriptomic studies. Thus, efficient removal of rRNA is necessary to achieve optimal coverage, good detection sensitivity and reliable results. An additional challenge is presented by microorganisms with GC-rich genomes, in which rRNA removal is less efficient.ResultsIn this work, we tested two commercial kits for rRNA removal, either alone or in combination, on Burkholderia thailandensis. This bacterium, chosen as representative of the important Burkholderia genus, which includes both pathogenic and environmental bacteria, has a rather large (6.72 Mb) and GC-rich (67.7%) genome. Each enriched mRNA sample was sequenced through paired-end Illumina GAIIx run in duplicate, yielding between 10 and 40 million reads. We show that combined treatment with both kits allows an mRNA enrichment of more than 238-fold, enabling the sequencing of almost all (more than 90%) B. thailandensis transcripts from less than 10 million reads, without introducing any bias in mRNA relative abundance, thus preserving differential expression profile.ConclusionsThe mRNA enrichment protocol presented in this work leads to an increase in detection sensitivity up to 770% compared to total RNA; such increased sensitivity allows for a corresponding reduction in the number of sequencing reads necessary for the complete analysis of whole transcriptome expression profiling. Thus we can conclude that the MICROBExpress/Ovation combined rRNA removal method could be suitable for RNA sequencing of whole transcriptomes of microorganisms with high GC content and complex genomes enabling at the same time an important scaling down of sequencing costs.

In Gram-negative bacteria, production of adhesion factors and extracellular polysaccharides (EPS) is promoted by the activity of diguanylate cyclases (DGCs), a class of enzymes able to catalyse the synthesis of the signal molecule bis-(39,59)-cyclic di-guanylic acid (c-di-GMP). In this report we show that in Escherichia coli, overexpression of the YddV protein, but not of other DGCs such as AdrA and YcdT, induces the production of the EPS poly-N-acetylglucosamine (PNAG) by stimulating expression of pgaABCD, the PNAG-biosynthetic operon. Stimulation of PNAG production and activation of pgaABCD expression by the YddV protein are abolished by inactivation of its GGDEF motif, responsible for DGC activity. Consistent with the effects of YddV overexpression, inactivation of the yddV gene negatively affects pgaABCD transcription and PNAG-mediated biofilm formation. pgaABCD regulation by the yddV gene also takes place in a mutant carrying a partial deletion of the csrA gene, which encodes the main regulator of pgaABCD expression, suggesting that YddV does not regulate pgaABCD through modulation of CsrA activity. Our results demonstrate that PNAG production does not simply respond to intracellular c-di-GMP concentration, but specifically requires the DGC activity of the YddV protein, thus supporting the notion that in E. coli, c-di-GMP biosynthesis by a given DGC protein triggers regulatory events that lead to activation of specific sets of EPS biosynthetic genes or proteins. INTRODUCTIONMost bacteria are able to switch between two different 'lifestyles': single cells (planktonic mode) and biofilm, i.e. a sessile microbial community. Biofilm and planktonic cells differ significantly in their physiology, in their gene expression pattern and even in their morphology. In particular, biofilm cells are characterized by production of adhesion factors and extracellular polysaccharides (EPS), resistance to environmental stresses, and lower sensitivity to antibiotics compared with planktonic cells (Costerton et al., 1995;Anderl et al., 2000;Harrison et al., 2007Harrison et al., , 2009).Transition from planktonic cells to biofilm is regulated by environmental and physiological cues, relayed to the bacterial cell by signal molecules or 'second messengers'. A second messenger, bis-(39,59)-cyclic diguanylic acid, better known as cyclic-di-GMP (c-di-GMP), plays a pivotal role in biofilm formation and maintenance by stimulating production of EPS and adhesion factors (Ross et al., 1991;Simm et al., 2004;Kader et al., 2006;Weber et al., 2006). In addition, c-di-GMP biosynthesis affects important cellular processes, such as morphological differentiation and cell replication in Caulobacter crescentus (Paul et al., 2004), cell motility (Méndez-Ortiz et al., 2006; Jonas et al., 2008) and virulence factor production (Kulasakara et al., 2006;Hammer & Bassler, 2009). In Enterobacteria, c-di-GMP seems to be involved in regulation of adhesion factors, such as curli and cellulose, important for adaptation and survival outside the warm-blooded hos...

In bacteria, intracellular amounts of the signal molecule cyclic di-GMP (c-di-GMP) are determined by biosynthetic enzymes, or diguanylate cyclases (DGCs), and degradative enzymes, or c-di-GMP phosphodiesterases (c-PDEs). In Escherichia coli, the production of curli fibers, an important adhesion factor, responds to c-di-GMP. The yddV-dos operon, which encodes a DGC and a c-PDE acting as a protein complex, is highly expressed at a low growth temperature and in the stationary phase, i.e. conditions that also stimulate curli production. We show that perturbations in the balance between YddV and Dos, obtained either through inactivation of the yddV gene or through overproduction of either YddV or Dos, strongly affect curli production. Both YddV and Dos proteins regulate the transcription of the csgBAC operon, which encodes curli structural subunits, while not affecting the expression of the regulatory operon csgDEFG. Consistent with the role of both YddV and Dos proteins as oxygen sensors, their effects on csgBAC gene expression were dramatically reduced in cells grown under anoxic conditions. Our results show that the yddV-dos operon plays an important role in the expression of curli-encoding genes in aerobically growing E. coli, and suggest that YddV and Dos, through their opposite activities, might finely tune curli production in response to oxygen availability.