Proteomics profiling of xanthomonas retroflexus colony variants

Plasmid conjugation is a key facilitator of horizontal gene transfer. Since plasmids often carry antibiotic resistance genes, they are crucial drivers of the world-wide rise of antibiotic resistance among pathogens. 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. Proteomics revealed fewer flagellum 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 X. retroflexus wild type and wrinkly mutant. Moreover, testing several flagella mutants of Pseudomonas putida suggested that the flagella effect was more general. We identified seven mechanisms with the potential to explain the flagella 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 modelling was eliminated by further experiments. Four additional proposed mechanisms have a reduced probability of plasmid transfer in common. Our findings highlight the important yet complex effects of flagella during bacterial conjugation in biofilms.

质粒接合（plasmid conjugation）是水平基因转移（horizontal gene transfer）的关键介导因子。由于质粒通常携带抗生素抗性基因，它们是全球范围内病原菌抗生素抗性攀升的核心驱动因素。在自然、工程化及临床环境中，细菌往往定植于具有保护作用的生物被膜（biofilms）中。因此，亟需更深入地解析生物被膜内的质粒转移过程。本研究旨在探究基质产生能力增强、运动性降低的反折黄单胞菌（Xanthomonas retroflexus）生物被膜适应性皱落突变株（XRw）中的质粒转移情况。实验结果显示，相较于野生型菌株，XRw生物被膜对广谱宿主范围的IncP-1ϵ质粒pKJK5的摄取量显著升高。蛋白质组学（Proteomics）分析发现，XRw中鞭毛相关蛋白（flagellum associated proteins）的表达量更低，提示鞭毛可能是导致质粒摄取量降低的关键因素。这一推论得到了实验验证：反折黄单胞菌野生型及皱落突变株的非鞭毛ΔfliM突变体的质粒摄取量均显著升高。此外，针对恶臭假单胞菌（Pseudomonas putida）的多株鞭毛突变体的测试结果表明，鞭毛对质粒转移的调控效应具有一定普遍性。我们共提出七种可解释鞭毛调控效应的潜在机制，并通过基于个体的模型（individual-based model）进行了模拟验证。其中两种机制可被排除：一是鞭毛导致细胞间距增大，二是鞭毛引发的延迟时间延长。另有一种在建模阶段被认为可行的机制，通过后续实验被进一步排除。其余四种已提出的机制均会降低质粒转移的普遍发生概率。本研究结果凸显了生物被膜内细菌接合过程中，鞭毛所发挥的重要且复杂的调控作用。