质粒接合是水平基因转移(HGT)的关键促进因素,和编码抗生素抗性的质粒推动了抗生素抗性的日益普遍。在自然,工程,和临床环境,细菌通常生长在保护性生物膜中。因此,需要更好地了解生物膜中的质粒转移。我们的目的是研究适应生物膜的皱纹菌落突变体中反弯曲黄单胞菌(XRw)的质粒转移,该突变体具有增强的基质产量和降低的运动性。我们发现与野生型(WT)相比,XRw生物膜对宽宿主范围的IncP-1ε质粒pKJK5的摄取增加。蛋白质组学显示XRw中鞭毛相关蛋白较少,表明鞭毛是减少质粒摄取的原因。X.后屈肌皱纹突变体以及野生型的非鞭毛fliM突变体的较高质粒摄取证实了这一点。此外,测试了恶臭假单胞菌的几种鞭毛突变体,表明鞭毛作用更为普遍。我们确定了七种可能解释鞭毛效应的机制,并在基于个体的模型中进行了模拟。因此可以消除两种机制(由于鞭毛,细胞之间的距离增加和滞后时间增加)。通过进一步的实验消除了在建模中被鉴定为可行的另一种机制。菌毛运动和鞭毛结合的空间位阻的可能性,减少接触频率,从而减少质粒摄取,证明是可行的,和其他三种可行的机制降低了共同的质粒转移的可能性。我们的发现强调了鞭毛在生物膜中细菌结合过程中的重要而复杂的作用。重要生物膜是微生物生命的主要形式,生活在生物膜中的细菌与浮游细菌明显不同。然而,生物膜生活方式对水平基因转移(HGT)的影响仍然知之甚少。通过接合质粒的水平基因转移是细菌进化和适应的主要驱动因素,例如抗生素耐药性的令人不安的传播。为了限制或促进质粒的流行和传播,我们需要更好地了解细菌细胞之间的质粒转移,尤其是在生物膜中。这里,我们发现了一个影响质粒转移的新因素,鞭毛,这是许多类型的细菌运动所必需的。我们表明,它们的缺失或活性改变可以导致两种细菌的质粒摄取增强,后弯曲黄单胞菌和恶臭假单胞菌。此外,我们证明了数学建模消除假设机制的实用性。
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.