Flagellar interference with plasmid uptake in biofilms: a joint experimental and modeling study.

Plasmid conjugation is a key facilitator of horizontal gene transfer (HGT), and plasmids encoding antibiotic resistance drive the increasing prevalence of antibiotic resistance. In natural, engineered, and clinical environments, bacteria often grow in protective biofilms. Therefore, a better understanding of plasmid transfer in biofilms is needed. Our aim was to investigate plasmid transfer in a biofilm-adapted wrinkly colony mutant of Xanthomonas retroflexus (XRw) with enhanced matrix production and reduced motility. We found that XRw biofilms had an increased uptake of the broad host-range IncP-1ϵ plasmid pKJK5 compared to the wild type (WT). Proteomics revealed fewer flagellar-associated proteins in XRw, suggesting that flagella were responsible for reducing plasmid uptake. This was confirmed by the higher plasmid uptake of non-flagellated fliM mutants of the X. retroflexus wrinkly mutant as well as the wild type. Moreover, testing several flagellar mutants of Pseudomonas putida suggested that the flagellar effect was more general. We identified seven mechanisms with the potential to explain the flagellar effect and simulated them in an individual-based model. Two mechanisms could thus be eliminated (increased distances between cells and increased lag times due to flagella). Another mechanism identified as viable in the modeling was eliminated by further experiments. The possibility of steric hindrance of pilus movement and binding by flagella, reducing the frequency of contact and thus plasmid uptake, proved viable, and the three other viable mechanisms had a reduced probability of plasmid transfer in common. Our findings highlight the important yet complex effects of flagella during bacterial conjugation in biofilms.IMPORTANCEBiofilms are the dominant form of microbial life and bacteria living in biofilms are markedly different from their planktonic counterparts, yet the impact of the biofilm lifestyle on horizontal gene transfer (HGT) is still poorly understood. Horizontal gene transfer by conjugative plasmids is a major driver in bacterial evolution and adaptation, as exemplified by the troubling spread of antibiotic resistance. To either limit or promote plasmid prevalence and dissemination, we need a better understanding of plasmid transfer between bacterial cells, especially in biofilms. Here, we identified a new factor impacting the transfer of plasmids, flagella, which are required for many types of bacterial motility. We show that their absence or altered activity can lead to enhanced plasmid uptake in two bacterial species, Xanthomonas retroflexus and Pseudomonas putida. Moreover, we demonstrate the utility of mathematical modeling to eliminate hypothetical mechanisms.

Genotypic variations and interspecific interactions modify gene expression and biofilm formation of Xanthomonas retroflexus.

Multispecies biofilms are important models for studying the evolution of microbial interactions. Co-cultivation of Xanthomonas retroflexus (XR) and Paenibacillus amylolyticus (PA) systemically leads to the appearance of an XR wrinkled mutant (XRW), increasing biofilm production. The nature of this new interaction and the role of each partner remain unclear. We tested the involvement of secreted molecular cues in this interaction by exposing XR and XRW to PA or its supernatant and analysing the response using RNA-seq, colony-forming unit (CFU) estimates, biofilm quantification, and microscopy. Compared to wild type, the mutations in XRW altered its gene expression and increased its CFU number. These changes matched the reported effects for one of the mutated genes: a response regulator part of a two-component system involved in environmental sensing. When XRW was co-cultured with PA or its supernatant, the mutations effects on XRW gene expression were masked, except for genes involved in sedentary lifestyle, being consistent with the higher biofilm production. It appears that the higher biofilm production was the result of the interaction between the genetic context (mutations) and the biotic environment (PA signals). Regulatory genes involved in environmental sensing need to be considered to shed further light on microbial interactions.

Emergent bacterial community properties induce enhanced drought tolerance in Arabidopsis.

Drought severely restricts plant production and global warming is further increasing drought stress for crops. Much information reveals the ability of individual microbes affecting plant stress tolerance. However, the effects of emergent bacterial community properties on plant drought tolerance remain largely unexplored. Here, we inoculated Arabidopsis plants in vivo with a four-species bacterial consortium (Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans, and Paenibacillus amylolyticus, termed as SPMX), which is able to synergistically produce more biofilm biomass together than the sum of the four single-strain cultures, to investigate its effects on plant performance and rhizo-microbiota during drought. We found that SPMX remarkably improved Arabidopsis survival post 21-day drought whereas no drought-tolerant effect was observed when subjected to the individual strains, revealing emergent properties of the SPMX consortium as the underlying cause of the induced drought tolerance. The enhanced drought tolerance was associated with sustained chlorophyll content and endogenous abscisic acid (ABA) signaling. Furthermore, our data showed that the addition of SPMX helped to stabilize the diversity and structure of root-associated microbiomes, which potentially benefits plant health under drought. These SPMX-induced changes jointly confer an increased drought tolerance to plants. Our work may inform future efforts to engineer the emergent bacterial community properties to improve plant tolerance to drought.

High-throughput screening alternative to crystal violet biofilm assay combining fluorescence quantification and imaging.

The crystal violet assay is widely used for biofilm quantitation despite its toxicity and variability. Here, we instead combine fluorescence labelling with the Cytation 5 multi-mode plate reader, to enable simultaneous acquisition of both quantitative and imaging biofilm data. This high-throughput method produces more robust data and provides information about morphology and spatial species organization within the biofilm.

Biofilms can act as plasmid reserves in the absence of plasmid specific selection.

Plasmids facilitate rapid bacterial adaptation by shuttling a wide variety of beneficial traits across microbial communities. However, under non-selective conditions, maintaining a plasmid can be costly to the host cell. Nonetheless, plasmids are ubiquitous in nature where bacteria adopt their dominant mode of life - biofilms. Here, we demonstrate that biofilms can act as spatiotemporal reserves for plasmids, allowing them to persist even under non-selective conditions. However, under these conditions, spatial stratification of plasmid-carrying cells may promote the dispersal of cells without plasmids, and biofilms may thus act as plasmid sinks.

High cell densities favor lysogeny: induction of an H20 prophage is repressed by quorum sensing and enhances biofilm formation in Vibrio anguillarum.

Temperate ϕH20-like phages are repeatedly identified at geographically distinct areas as free phage particles or as prophages of the fish pathogen Vibrio anguillarum. We studied mutants of a lysogenic isolate of V. anguillarum locked in the quorum-sensing regulatory modes of low (ΔvanT) and high (ΔvanO) cell densities by in-frame deletion of key regulators of the quorum-sensing pathway. Remarkably, we find that induction of the H20-like prophage is controlled by the quorum-sensing state of the host, with an eightfold increase in phage particles per cell in high-cell-density cultures of the quorum-sensing-deficient ΔvanT mutant. Comparative studies with prophage-free strains show that biofilm formation is promoted at low cell density and that the H20-like prophage stimulates this behavior. In contrast, the high-cell-density state is associated with reduced prophage induction, increased proteolytic activity, and repression of biofilm. The proteolytic activity may dually function to disperse the biofilm and as a quorum-sensing-mediated antiphage strategy. We demonstrate an intertwined regulation of phage-host interactions and biofilm formation, which is orchestrated by host quorum-sensing signaling, suggesting that increased lysogeny at high cell density is not solely a strategy for phages to piggy-back the successful bacterial hosts but is also a host strategy evolved to take control of the lysis-lysogeny switch to promote host fitness.

Mixed-species biofilms in the food industry: Current knowledge and novel control strategies.

Attachment of microorganisms to food contact surfaces and the subsequent formation of biofilms may cause equipment damage, food spoilage and even diseases. Mixed-species biofilms are ubiquitous in the food industry and they generally exhibit higher resistance to disinfectants and antimicrobials compared to single-species biofilms. The physiology and metabolic activity of microorganisms in mixed-species biofilms are however rather complicated to study, and despite targeted research efforts, the potential role of mixed-species biofilms in food industry is still rather unexplored. In this review, we summarize recent studies in the context of bacterial social interactions in mixed-species biofilms, resistance to disinfectants, detection methods, and potential novel strategies to control the formation of mixed-species biofilms for enhanced food safety and food quality.

Interspecies interactions reduce selection for a biofilm-optimized variant in a four-species biofilm model.

Multispecies biofilms are structured and spatially defined communities, where interspecies interactions impact assembly and functionality. Here, we compared the spatial organization and growth of bacterial cells in differently composed biofilm communities over time to determine links between interspecies interactions and selection for biofilm phenotypes of individual species. An established model community consisting of Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus was used. It was found that interspecies interactions led to varying levels of selection for a new colony phenotype of X. retroflexus, depending on the presence/absence of other species. When M. oxydans was absent, X. retroflexus was not able to establish in the top layers of the biofilm, which led to selection for a hyper-matrix forming phenotype of X. retroflexus that successfully established in the biofilm top layers. No such phenotypic X. retroflexus variants were identified in the presence of M. oxydans. These findings indicate that interspecies interactions may lead to favourable localization of individual species in a multispecies biofilm and thereby reduce selection for competitive phenotypes.

Priority of Early Colonizers but No Effect on Cohabitants in a Synergistic Biofilm Community.

The arrival order of different species to a habitat can strongly impact community assembly and succession dynamics, thus influencing functionality. In this study, we asked how prior colonization of one community member would influence the assembly of a synergistic multispecies biofilm community grown in vitro. We expected that the prior arrival would confer an advantage, in particular for good biofilm formers. Yet, we did not know if the cohabitants would be impaired or benefit from the pre-colonization of one member, depending on its ability to form biofilm. We used a consortium consisting of four soil bacteria; Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus. This consortium has been shown to act synergistically when grown together, thus increasing biofilm production. The results showed that the two good biofilm formers gained a fitness advantage (increase in abundance) when allowed prior colonization on an abiotic surface before the arrival of their cohabitants. Interestingly, the significantly higher number of the pre-colonized biofilm formers did not affect the resulting composition in the subsequent biofilm after 24 h.

Fluidic resistance control enables high-throughput establishment of mixed-species biofilms.

Bacteria often live in communities of mixed species embedded in a self-produced extracellular matrix of polysaccharides, proteins and DNA, termed biofilms. The BioFlux microfluidic flow system is useful for studying biofilm formation in different media under flow. However, analyzing the architecture and maturation of biofilms under flow requires a proper seeding, which can prove difficult when working with bacteria of different sizes, motile bacteria or aiming for a high number of replicates. Here we developed an efficient protocol that exploits viscosity tuning and seeding indicator dyes to improve seeding and allow for high-throughput examination and visualization of consistent mono- and mixed-species biofilm developments under flow.

Big Impact of the Tiny: Bacteriophage-Bacteria Interactions in Biofilms.

Bacteriophages (phages) have been shaping bacterial ecology and evolution for millions of years, for example, by selecting for defence strategies. Evidence supports that bacterial biofilm formation is one such strategy and that biofilm-mediated protection against phage infection depends on maturation and composition of the extracellular matrix. Interestingly, studies have revealed that phages can induce and strengthen biofilms. Here we review interactions between bacteria and phages in biofilms, discuss the underlying mechanisms, the potential of phage therapy for biofilm control, and emphasize the importance of considering biofilms in future phage research. This is especially relevant as biofilms are associated with increased tolerance towards antibiotics and are implicated in the majority of chronic infections.

The impact of the conjugative IncP-1 plasmid pKJK5 on multispecies biofilm formation is dependent on the plasmid host.

Horizontal gene transfer by conjugation has been reported to increase overall biofilm formation. Biofilm is considered a hot spot for plasmid transfer, and it has been found that social interactions during biofilm formation can increase the biomass. In this study, we demonstrate a contrast to previous studies by showing that the conjugative IncP-1 plasmid pKJK5 influences biofilm formation negatively. The results showed that a co-culture (Pseudomonas putida, Kluyvera sp., and Escherichia coli) formed significantly more biofilm than the strains did individually. When pKJK5 was inserted into P. putida, biofilm formation was significantly reduced compared with the co-culture without plasmid. A nonconjugative version of pKJK5 was also used, and the biofilm formation was restored. Visualization with the BioFlux 1000 facility showed that the presence of pKJK5-containing P. putida in the co-culture led to a changed biofilm structure, where the cells showed a higher tendency to attach to other cells rather than surfaces. This study thus indicates that the presence of conjugative plasmids in some species may decrease the surface-associated biofilm formation of a mixed co-culture by facilitating cell-cell attachment with reduced surface attachment as the consequence.