Biofilms are a major form of microbial life in which cells form dense surface associated communities that can persist for many generations. The long-life of biofilm communities means that they can be strongly shaped by evolutionary processes. Here, we review the experimental study of evolution in biofilm communities. We first provide an overview of the different experimental models used to study biofilm evolution and their associated advantages and disadvantages. We then illustrate the vast amount of diversification observed during biofilm evolution, and we discuss (i) potential ecological and evolutionary processes behind the observed diversification, (ii) recent insights into the genetics of adaptive diversification, (iii) the striking degree of parallelism between evolution experiments and real-life biofilms and (iv) potential consequences of diversification. In the second part, we discuss the insights provided by evolution experiments in how biofilm growth and structure can promote cooperative phenotypes. Overall, our analysis points to an important role of biofilm diversification and cooperation in bacterial survival and productivity. Deeper understanding of both processes is of key importance to design improved antimicrobial strategies and diagnostic techniques.

Post-transcriptional regulation is a very important mechanism to control gene expression in changing environments. In the past decade, a lot of interest has been directed towards the role of small RNAs in bacterial post-transcriptional regulation. However, small RNAs are not the only molecules controlling gene expression at this level, RNA-binding proteins play an important role as well. CsrA and Hfq are the two best studied bacterial proteins of this type, but recently, additional proteins involved in post-transcriptional control have been identified. This review focuses on the general working mechanisms of post-transcriptionally active RNA-binding proteins, which include (i) adaptation of the susceptibility of mRNAs and sRNAs to RNases, (ii) modulating the accessibility of the ribosome binding site of mRNAs, (iii) recruiting and assisting in the interaction of mRNAs with other molecules and (iv) regulating transcription terminator / antiterminator formation, and gives an overview of both the well-studied and the newly identified proteins that are involved in post-transcriptional regulatory processes. Additionally, the post-transcriptional mechanisms by which the expression or the activity of these proteins is regulated, are described. For many of the newly identified proteins, however, mechanistic questions remain. Most likely, more post-transcriptionally active proteins will be identified in the future.

Salmonella, an important foodborne pathogen, forms biofilms in many different environments. The composition of these biofilms differs depending on the growth conditions, and their development is highly coordinated in time. To develop efficient treatments, it is therefore essential that biofilm formation and its inhibition be understood in different environments and in a time-dependent manner. Many currently used techniques, such as transcriptomics or proteomics, are still expensive and thus limited in their application. Therefore, a GFP-promoter fusion library with 79 important Salmonella biofilm genes was developed (covering among other things matrix production, fimbriae and flagella synthesis, and c-di-GMP regulation). This library is a fast, inexpensive, and easy-to-use tool, and can therefore be conducted in different experimental setups in a time-dependent manner. In this paper, four possible applications are highlighted to illustrate and validate the use of this reporter fusion library.

We previously synthesized several series of compounds, based on the 5-aryl-2-aminoimidazole scaffold, that showed activity preventing the formation of Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa biofilms. Here, we further studied the activity spectrum of a number of the most active N 1- and 2 N -substituted 5-aryl-2-aminoimidazoles against a broad panel of biofilms formed by monospecies and mixed species of bacteria and fungi. An N 1-substituted compound showed very strong activity against the biofilms formed by Gram-negative and Gram-positive bacteria and the fungus Candida albicans but was previously shown to be toxic against various eukaryotic cell lines. In contrast, 2 N -substituted compounds were nontoxic and active against biofilms formed by Gram-negative bacteria and C. albicans but had reduced activity against biofilms formed by Gram-positive bacteria. In an attempt to develop nontoxic compounds with potent activity against biofilms formed by Gram-positive bacteria for application in antibiofilm coatings for medical implants, we synthesized novel compounds with substituents at both the N 1 and 2 N positions and tested these compounds for antibiofilm activity and toxicity. Interestingly, most of these N 1-,2 N -disubstituted 5-aryl-2-aminoimidazoles showed very strong activity against biofilms formed by Gram-positive bacteria and C. albicans in various setups with biofilms formed by monospecies and mixed species but lost activity against biofilms formed by Gram-negative bacteria. In light of application of these compounds as anti-infective coatings on orthopedic implants, toxicity against two bone cell lines and the functionality of these cells were tested. The N 1-,2 N -disubstituted 5-aryl-2-aminoimidazoles in general did not affect the viability of bone cells and even induced calcium deposition. This indicates that modulating the substitution pattern on positions N 1 and 2 N of the 5-aryl-2-aminoimidazole scaffold allows fine-tuning of both the antibiofilm activity spectrum and toxicity.

Nowadays, the identification of small non-coding RNAs takes a prominent role in deciphering complex bacterial phenotypes. Evidences are given that the post-transcriptional layer of regulation mediated by sRNAs plays an important role in the formation of bacterial biofilms. These sRNAs exert their activity on various targets, be it directly or indirectly linked to biofilm formation. First, and best described, are the sRNAs that act in core regulatory pathways of biofilm formation, such as those regulating motility and matrix production. Second, overlaps between the regulation of biofilm formation and the outer membrane (OM) are becoming obvious. Additionally, different studies indicate that defects in the OM itself affect biofilm formation through this shared cascade, thereby forming a feedback mechanism. Interestingly, it is known that the OM itself is extensively regulated by different sRNAs. Third, biofilms are also linked to global metabolic changes. There is also evidence that metabolic pathways and the process of biofilm formation share sRNAs.

Biofilm formation is an important survival strategy of Salmonella in all environments. By mutant screening, we showed a knock-out mutant of fabR, encoding a repressor of unsaturated fatty acid biosynthesis (UFA), to have impaired biofilm formation. In order to unravel how this regulator impinges on Salmonella biofilm formation, we aimed at elucidating the S. Typhimurium FabR regulon. Hereto, we applied a combinatorial high-throughput approach, combining ChIP-chip with transcriptomics. All the previously identified E. coli FabR transcriptional target genes (fabA, fabB and yqfA) were shown to be direct S. Typhimurium FabR targets as well. As we found a fabB overexpressing strain to partly mimic the biofilm defect of the fabR mutant, the effect of FabR on biofilms can be attributed at least partly to FabB, which plays a key role in UFA biosynthesis. Additionally, ChIP-chip identified a number of novel direct FabR targets (the intergenic regions between hpaR/hpaG and ddg/ydfZ) and yet putative direct targets (i.a. genes involved in tRNA metabolism, ribosome synthesis and translation). Next to UFA biosynthesis, a number of these direct targets and other indirect targets identified by transcriptomics (e.g. ribosomal genes, ompA, ompC, ompX, osmB, osmC, sseI), could possibly contribute to the effect of FabR on biofilm formation. Overall, our results point at the importance of FabR and UFA biosynthesis in Salmonella biofilm formation and their role as potential targets for biofilm inhibitory strategies.