Table_3_The Impact of Type VI Secretion System, Bacteriocins and Antibiotics on Bacterial Competition of Pectobacterium carotovorum subsp. brasiliense and the Regulation of Carbapenem Biosynthesis by Iron and the Ferric-Uptake Regulator.doc

The complexity of plant microbial communities provides a rich model for investigating biochemical and regulatory strategies involved in interbacterial competition. Within these niches, the soft rot Enterobacteriaceae (SRE) represents an emerging group of plant–pathogens causing soft rot/blackleg diseases resulting in economic losses worldwide in a variety of crops. A preliminary screening using next-generation sequencing of 16S rRNA comparatively analyzing healthy and diseased potato tubers, identified several taxa from Proteobacteria to Firmicutes as potential potato endophytes/plant pathogens. Subsequent to this, a range of molecular and computational techniques were used to determine the contribution of antimicrobial factors such as bacteriocins, carbapenem and type VI secretion system (T6SS), found in an aggressive SRE (Pectobacterium carotovorum subsp. brasiliense strain PBR1692 – Pcb1692) against these endophytes/plant pathogens. The results showed growth inhibition of several Proteobacteria by Pcb1692 depends either on carbapenem or pyocin production. Whereas for targeted Firmicutes, only the Pcb1692 pyocin seems to play a role in growth inhibition. Furthermore, production of carbapenem by Pcb1692 was observably dependent on the presence of environmental iron and oxygen. Additionally, upon deletion of fur, slyA and expI regulators, carbapenem production ceased, implying a complex regulatory mechanism involving these three genes. Finally, the results demonstrated that although T6SS confers no relevant advantage during in vitro competition, a significant attenuation in competition by the mutant strain lacking a functional T6SS was observed in planta.
IMPORTANCESoft rot Enterobacteriaceae (SRE) represents important phytopathogens causing soft rot/blackleg diseases in a variety of crops leading to huge economic losses worldwide. These pathogens have been isolated alongside other bacteria from different environments such as potato tubers, stems, roots and from the soil. In these environments, SREs coexist with other bacteria where they have to compete for scarce nutrients and other resources. In this report, we show that Pectobacterium carotovorum subsp. brasiliense strain PBR1692 – Pcb1692, which represents one of the SREs, inhibits growth of several different bacteria by producing different antimicrobial compounds. These antimicrobial compounds can be secreted inside or outside the plant host, allowing Pcb1692 to effectively colonize different types of ecological niches. By analyzing the genome sequences of several SREs, we show that other SREs likely deploy similar antimicrobials to target other bacteria.

植物微生物群落的复杂性为探究细菌间竞争相关的生化与调控策略提供了优质研究模型。在这些生态位中，软腐肠杆菌科（Soft rot Enterobacteriaceae, SRE）是一类新兴植物病原菌，可引发软腐病/黑胫病，在全球范围内对多种作物造成经济损失。本研究通过对健康与患病马铃薯块茎开展基于16S rRNA的高通量测序比较分析，完成初步筛选，鉴定出从变形菌门（Proteobacteria）到厚壁菌门（Firmicutes）的多个类群，均为潜在的马铃薯内生菌/植物病原菌。在此基础上，本研究采用一系列分子与计算生物学技术，探究高致病力软腐肠杆菌科菌株——巴西亚种马铃薯软腐病菌*Pectobacterium carotovorum subsp. brasiliense* PBR1692（简称Pcb1692）所产生的抗菌因子（如细菌素（bacteriocins）、碳青霉烯（carbapenem）与VI型分泌系统（type VI secretion system, T6SS））对上述内生菌/植物病原菌的拮抗作用。实验结果显示，Pcb1692对部分变形菌门细菌的生长抑制作用依赖于碳青霉烯或绿脓菌素（pyocin）的合成；而针对靶标厚壁菌门细菌时，仅Pcb1692产生的绿脓菌素似乎参与了生长抑制过程。此外，Pcb1692合成碳青霉烯的过程显著依赖环境中铁离子与氧气的存在。敲除fur、slyA与expI这三个调控基因后，Pcb1692便停止合成碳青霉烯，这暗示该抗菌因子的合成存在由这三个基因参与的复杂调控机制。最后，研究结果表明：尽管VI型分泌系统在体外竞争实验中未体现出显著竞争优势，但在植物体内实验中，缺失功能性T6SS的突变株的竞争能力出现了明显衰减。

【研究意义】软腐肠杆菌科（SRE）是一类重要的植物病原菌，可引发多种作物的软腐病与黑胫病，在全球范围内造成巨额经济损失。该类病原菌可与其他细菌一同从马铃薯块茎、茎秆、根系以及土壤等多种环境中分离得到。在这些生境中，软腐肠杆菌科菌株会与其他细菌共存，并为获取有限的营养与其他资源展开竞争。本研究表明，作为软腐肠杆菌科代表菌株之一的巴西亚种马铃薯软腐病菌PBR1692（简称Pcb1692），可通过合成多种抗菌物质抑制多种不同细菌的生长。这些抗菌物质可在植物宿主内部或外部分泌，帮助Pcb1692有效定殖于不同类型的生态位中。通过对多株软腐肠杆菌科菌株的基因组序列进行分析，我们发现其他软腐肠杆菌科菌株大概率也会通过类似的抗菌物质实现对其他细菌的靶向拮抗。