Construction and analysis of deletion and complementation strains of the extMNOPQS gene cluster in Geobacter sulfurreducens PCA

抄録

Elemental sulfur respiration plays a pivotal role in the microbial sulfur cycle; however, the molecular mechanisms underlying this process remain poorly understood in many microorganisms, including Geobacter sulfurreducens PCA. A previous RNA-seq analysis showed that the upstream region of the ext gene cluster (extHIJKL) is transcriptionally upregulated in response to sulfur, whereas the downstream region (extMNOPQS) did not exhibit clear induction. However, the specific role of extMNOPQS in elemental sulfur reduction remains unclear. In this study, we investigated the function of extMNOPQS using transcriptional analysis and mutational approaches. RT-qPCR revealed that the expression of extM was not significantly induced by elemental sulfur, in contrast to the sulfur-responsive extHIJKL gene cluster, suggesting distinct transcriptional regulation. To examine its physiological role, we constructed a deletion mutant (ΔextMNOPQS) and a plasmid-based complementation strain. Western blotting confirmed the absence of ExtM in the deletion mutant and its elevated expression in the complemented strain. Growth assays showed that deletion of extMNOPQS did not severely impair cell growth, although the complemented strain exhibited slightly reduced growth when compared with the wild-type. Functional analysis using halo assays demonstrated that the ΔextMNOPQS mutant was markedly impaired in sulfur reduction, and this phenotype was almost completely restored by complementation. These findings indicate that extMNOPQS is essential for elemental sulfur respiration in G. sulfurreducens and may serve as a critical electron transfer module within the ext gene cluster.