Clostridium difficile is an opportunistic entero-pathogen causing post-antibiotic and nosocomial diarrhea upon microbiota dysbiosis. Although biofilms could contribute to colonization, little is known about their development and physiology. Strain 630Δerm is able to form, in continuous-flow micro-fermentors, macro-colonies and submersed biofilms loosely adhesive to glass. According to gene expression data, in biofilm/planktonic cells, central metabolism is active and fuels fatty acid biosynthesis rather than fermentations. Consistently, succinate is consumed and butyrate production is reduced. Toxin A expression, which is coordinated to metabolism, is down-regulated, while surface proteins, like adhesins and the primary Type IV pili subunits, are over-expressed. C-di-GMP level is probably tightly controlled through the expression of both diguanylate cyclase-encoding genes, like dccA, and phosphodiesterase-encoding genes. The coordinated expression of genes controlled by c-di-GMP and encoding the putative surface adhesin CD2831 and the major Type IV pilin PilA1, suggests that c-di-GMP could be high in biofilm cells. A Bacillus subtilis SinR-like regulator, CD2214, and/or CD2215, another regulator co-encoded in the same operon as CD2214, control many genes differentially expressed in biofilm, and in particular dccA, CD2831 and pilA1 in a positive way. After growth in micro-titer plates and disruption, the biofilm is composed of robust aggregated structures where cells are embedded into a polymorphic material. The intact biofilm observed in situ displays a sparse, heterogeneous and high 3D architecture made of rods and micro-aggregates. The biofilm is denser in a mutant of both CD2214 and CD2215 genes, but it is not affected by the inactivation of neither CD2831 nor pilA1. dccA, when over-expressed, not only increases the biofilm but also triggers its architecture to become homogeneous and highly aggregated, in a way independent of CD2831 and barely dependent of pilA1. Cell micro-aggregation is shown to play a major role in biofilm formation and architecture. This thorough analysis of gene expression reprogramming and architecture remodeling in biofilm lays the foundation for a deeper understanding of this lifestyle and could lead to novel strategies to limit C. difficile spread.

SummaryAlmost one‐third of crop yields are lost every year due to microbial alterations and diseases. The main control strategy to limit these losses is the use of an array of chemicals active against spoilage and unwanted pathogenic microorganisms. Their massive use has led to extensive environmental pollution, human poisoning and a variety of diseases. An emerging alternative to this chemical approach is the use of microbial biocontrol agents. Biopesticides have been used with success in several fields, but a better understanding of their mode of action is necessary to better control their activity and increase their use. Very few studies have considered that biofilms are the preferred mode of life of microorganisms in the target agricultural biotopes. Increasing evidence shows that the spatial organization of microbial communities on crop surfaces may drive important bioprotection mechanisms. The aim of this review is to summarize the evidence of biofilm formation by biocontrol agents on crops and discuss how this surface‐associated mode of life may influence their biology and interactions with other microorganisms and the host and, finally, their overall beneficial activity.