Clostridioides difficile LuxS mediates inter-bacterial interactions within biofilms.

The anaerobic gut pathogen, Clostridioides difficile, forms adherent biofilms that may play an important role in recurrent C. difficile infections. The mechanisms underlying C. difficile community formation and inter-bacterial interactions are nevertheless poorly understood. C. difficile produces AI-2, a quorum sensing molecule that modulates biofilm formation across many bacterial species. We found that a strain defective in LuxS, the enzyme that mediates AI-2 production, is defective in biofilm development in vitro. Transcriptomic analyses of biofilms formed by wild type (WT) and luxS mutant (luxS) strains revealed a downregulation of prophage loci in the luxS mutant biofilms compared to the WT. Detection of phages and eDNA within biofilms may suggest that DNA release by phage-mediated cell lysis contributes to C. difficile biofilm formation. In order to understand if LuxS mediates C. difficile crosstalk with other gut species, C. difficile interactions with a common gut bacterium, Bacteroides fragilis, were studied. We demonstrate that C. difficile growth is significantly reduced when co-cultured with B. fragilis in mixed biofilms. Interestingly, the absence of C. difficile LuxS alleviates the B. fragilis-mediated growth inhibition. Dual species RNA-sequencing analyses from single and mixed biofilms revealed differential modulation of distinct metabolic pathways for C. difficile WT, luxS and B. fragilis upon co-culture, indicating that AI-2 may be involved in induction of selective metabolic responses in B. fragilis. Overall, our data suggest that C. difficile LuxS/AI-2 utilises different mechanisms to mediate formation of single and mixed species communities.

In planta induced changes in the native plasmid profile of Pseudomonas syringae pathover phaseolicola strain 1302A.

Pseudomonas syringae pv. phaseolicola (Pph) strain 1302A, a causative agent of halo blight in the common bean Phaseolus vulgaris, contains four native plasmids designated pAV505 (150 kb), pAV506 (50 kb), pAV507 (47 kb) and pAV508 (42 kb). Pph 1302A also contains a 106 kb genomic island PPHGI-1 which shares features with integrative and conjugative elements (ICElands) and carries the effector gene avrPphB (hopAR1) which triggers a defensive response in bean cultivars carrying the matching R3 resistance gene. It has been shown that when Pph 1302A is sequentially inoculated (passaged) through resistant bean cultivar Tendergreen (TG) in which the hypersensitive response (HR) is generated, the three largest plasmids are lost and an extra ∼100 kb plasmid is gained, which tests confirmed to be the 106 kb circular form of PPHGI-1. The aim of the current study was to determine if upon further passaging though bean plants, the plasmid profile of Pph 1302A would alter again and if the missing plasmids had been integrated into the chromosome. Pph 1302A-P6, the strain with the altered plasmid profile was passaged twice through TG and of the four re-isolated strains examined all displayed the plasmid profile associated with wildtype Pph 1302A, that is, all four native plasmids had reappeared and the PPHGI-1 plasmid was absent. This demonstrated that the plasmid composition of Pph 1302A-P6 could indeed change on further exposure to the plant environment and also that the seemingly missing native plasmids were still present within the genome, lending evidence to the theory that they had integrated into the chromosome. Furthermore two of these re-isolated strains had lost PPHGI-1 entirely, meaning they no longer triggered a HR on TG and instead generated a disease response. This study clearly demonstrates the plasticity of the bacterial genome and the extent it can be influenced by the plant environment and conditions generated during the HR.