Cross-talk mechanisms in biofilm formation and responses to environmental and physiological stress in Escherichia coli

Abstract

Switching from single-cell (planktonic) to biofilm growth (and vice versa) is regulated by a variety of environmental and physiological cues. Signals leading to activation of stress responses often lead to biofilm formation which, in turn, can trigger induction of stress response mechanisms, suggesting direct cross-talk between the two cellular processes. Regulatory mechanisms of this process include two-component regulatory systems, master regulators such as the rpoS gene and signal molecules such as cyclic-di-GMP, in a tight and complex interplay.

Introduction

It is a widely accepted notion that, in natural habitats, microorganisms are predominantly associated with solid surfaces and organized in communities known as biofilms. Although sometimes found as single-species communities, microbial biofilms typically comprise a large number of different microbial species, both prokaryotic (bacteria) and eukaryotic (unicellular algae or fungi) [13]. Bacteria living in a biofilm differ from their “planktonic” counterparts, living as single cells, not only at the morphological level, but also in their physiological and metabolic state. These differences in the cell's physiological state are reflected by substantial changes in their gene expression pattern, as highlighted by functional genomic studies comparing biofilms to planktonic cells [2], [61], [62], [77]. A defining feature of bacterial biofilms, in striking contrast to planktonic bacteria, lies in extensive production of extracellular polysaccharides (EPSs) which, together with cell surface-associated proteins and nucleic acids, constitutes the so-called “biofilm matrix” [7], [13], [63], [75]. EPS biosynthesis and matrix production by biofilm cells are a major example of reprogramming of gene expression and protein biosynthesis taking place in biofilm cells and contributing to its further development [17], [52], [61], [70]. Gene expression regulation for biofilm determinants requires a combination of different environmental signals which can modulate the activity of complex regulatory networks involving both specific and global regulators. Interestingly, while very few regulatory networks presiding in biofilm formation and maintenance appear to be exclusively dedicated to this cellular process [26], many global regulators, including regulators responding to cellular and environmental stresses, two-component regulatory systems and quorum sensing-related regulators, can directly affect transition from single cells to biofilm, biofilm maintenance and even biofilm dispersal, both in Gram-positive and Gram-negative bacteria [18], [19], [38], [51], [68].

Interestingly, in many bacterial species, biofilm formation seems to be promoted by non-optimal growth conditions or even by cellular stresses. Growth in defined medium results in increased biofilm formation compared to LB (rich) medium in environmental isolates of Escherichia coli [8], while growth at temperatures much lower than the optimum stimulates production of adhesion factors such as curli fibers in Enterobacteria [58] and the EPS poly-N-acetyl-glucosamine (PNAG) in Yersinia pestis [54]. Environmental stresses such as high metal concentrations also stimulate biofilm formation [3], [31]. Finally, antibiotics at subinhibitory concentrations are able to elicit biofilm formation in Pseudomonas aeruginosa via the cyclic-di-GMP signaling system [34] (for a description of cyclic-di-GMP signaling system, see Section 4). In turn, biofilm formation seems to trigger several stress responses in the bacterial cell: for instance, the CpxA/CpxR stress response system, activated by accumulation of misfolded proteins in the periplasm and typically induced by high temperature or high osmolarity, is also turned on, even in the absence of such environmental stresses, in biofilm cells. Likewise, transcription of the recA gene, which responds to DNA damage, is also turned on in E. coli biofilms and, remarkably, the presence of a functional recA allele is necessary for efficient biofilm formation [2]. Another example of a stress-related factor the expression of which is increased upon biofilm formation is the Dps protein, which protects DNA from iron-mediated oxidative damage in the stationary phase of growth [47]; Dps is overproduced in bacteriophage-resistant E. coli MG1655 mutants highly proficient in biofilm formation [43]. However, the most dramatic, albeit indirect, evidence for activation of stress responses in biofilm cells comes from the observation that prophages are induced in mature P. aeruginosa biofilms, leading to sloughing and dispersal of the biofilm itself [72]: indeed, prophage induction is typically activated by stress conditions and, in particular, by DNA damage. Thus, there seems to be a fairly tight connection between biofilm formation and cellular response to various stresses, including DNA damage. In this communication I will address two examples of the interaction between stress responses and biofilm formation in the enterobacterium E. coli. In addition to the already mentioned CpxA/CpxR system, I will focus on the role of the general stress response protein RpoS in production of curli fibers, an important biofilm determinant, and its interplay with GGDEF proteins, responsible for the biosynthesis of cyclic-di-GMP (c-di-GMP), a signal molecule pivotal for production of adhesion factors and biofilm formation. However, it must be stressed that, besides the CpxA/CpxR system and the cyclic-di-GMP signal molecule, several more examples of biofilm formation/cellular stress relay mechanisms can be found in the literature. A list of stress genes either involved in cell adhesion and establishment of biofilm or induced by biofilm formation and maturation in E. coli is shown in Table 1.