Genomic and Gene-Expression Comparisons among Phage-Resistant Type-IV Pilus Mutants of Pseudomonas syringae pathovar phaseolicola

Pseudomonas syringae pv. phaseolicola (Pph) is a significant bacterial pathogen of agricultural crops, and phage Φ6 and other members of the dsRNA virus family Cystoviridae undergo lytic (virulent) infection of Pph, using the type IV pilus as the initial site of cellular attachment. Despite the popularity of Pph/phage Φ6 as a model system in evolutionary biology, Pph resistance to phage Φ6 remains poorly characterized. To investigate differences between phage Φ6 resistant Pph strains, we examined genomic and gene expression variation among three bacterial genotypes that differ in the number of type IV pili expressed per cell: ordinary (wild-type), non-piliated, and super-piliated. Genome sequencing of non-piliated and super-piliated Pph identified few mutations that separate these genotypes from wild type Pph–and none present in genes known to be directly involved in type IV pilus expression. Expression analysis revealed that 81.1% of gene ontology (GO) terms up-regulated in the non-piliated strain were down-regulated in the super-piliated strain. This differential expression is particularly prevalent in genes associated with respiration—specifically genes in the tricarboxylic acid cycle (TCA) cycle, aerobic respiration, and acetyl-CoA metabolism. The expression patterns of the TCA pathway appear to be generally up and down-regulated, in non-piliated and super-piliated Pph respectively. As pilus retraction is mediated by an ATP motor, loss of retraction ability might lead to a lower energy draw on the bacterial cell, leading to a different energy balance than wild type. The lower metabolic rate of the super-piliated strain is potentially a result of its loss of ability to retract.

丁香假单胞菌菜豆致病变种（Pseudomonas syringae pv. phaseolicola，Pph）是一类危害农业作物的重要细菌性病原菌。双链RNA病毒科囊泡噬菌体科（Cystoviridae）的噬菌体Φ6及其他成员可通过裂解性（烈性）感染Pph，并以IV型菌毛（type IV pilus）作为细胞附着的初始位点。尽管Pph与噬菌体Φ6的组合作为进化生物学研究的模型系统已被广泛应用，但Pph对噬菌体Φ6的抗性机制仍有待深入解析。为探究抗噬菌体Φ6的Pph菌株间的差异，我们针对三种每细胞IV型菌毛表达量存在差异的细菌基因型开展了基因组与基因表达变异分析，分别为普通野生型、无菌毛型以及超菌毛型。对无菌毛型与超菌毛型Pph的基因组测序结果显示，两类基因型与野生型Pph间的差异突变极少，且未在已知直接参与IV型菌毛表达的基因中检测到突变。表达谱分析表明，无菌毛菌株中上调的81.1%的基因本体（Gene Ontology，GO）术语在超菌毛菌株中呈现下调趋势。这类差异表达在与呼吸作用相关的基因中尤为突出，具体涉及三羧酸循环（tricarboxylic acid cycle，TCA）、有氧呼吸以及乙酰辅酶A代谢相关的基因。三羧酸循环通路的表达模式在无菌毛型与超菌毛型Pph中分别整体上调与整体下调。由于菌毛回缩由ATP马达介导，丧失回缩能力可能会降低细菌细胞的能量消耗，进而形成与野生型不同的能量平衡。超菌毛菌株较低的代谢速率，可能正是其丧失菌毛回缩能力的结果。