Abstract：
Bacterial ribonuclease E (RNase E) is vital for posttranscriptional regulation by degrading and processing RNA. The RraA protein inhibits RNase E activity through protein-protein interactions, exerting a global regulatory effect on gene expression. However, the specific role of RraA remains unclear. In this study, we investigated rraA expression in Vibrio alginolyticus ZJ-T and identified three promoters responsible for its expression, resulting in transcripts with varying 5\'-UTR lengths. During the stationary phase, rraA was significantly posttranscriptionally inhibited. Deletion of rraA had no impact on bacterial growth in rich medium Luria-Bertani broth with salt (LBS) but resulted in decreased biofilm formation and increased resistance to polymyxin B. Transcriptome analysis revealed 350 differentially expressed genes (DEGs) between the wild type and the rraA mutant, while proteome analysis identified 267 differentially expressed proteins (DEPs). Integrative analysis identified 55 genes common to both DEGs and DEPs, suggesting that RraA primarily affects gene expression at the posttranscriptional level. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis demonstrated that RraA facilitates the conversion of fatty acids, propionic acid, and branched-chain amino acids to acetyl-CoA while enhancing amino acid and peptide uptake. Notably, RraA positively regulates the expression of virulence-associated genes, including those involved in biofilm formation and the type VI secretion system. This study expands the understanding of the regulatory network of RraA through transcriptome analysis, emphasizing the importance of proteomic analysis in investigating posttranscriptional regulation.IMPORTANCERraA is an inhibitor protein of ribonuclease E that interacts with and suppresses its endonucleolytic activity, thereby playing a widespread regulatory role in the degradation and maturation of diverse mRNAs and noncoding small RNAs. However, the physiological functions and associated regulon of RraA in Vibrio alginolyticus have not been fully elucidated. Here, we report that RraA impacts virulence-associated physiological processes, namely, antibiotic resistance and biofilm formation, in V. alginolyticus. By conducting an integrative analysis of both the transcriptome and proteome, we revealed the involvement of RraA in carbon metabolism, amino acid catabolism, and transport, as well as in the type VI secretion system. Collectively, these findings elucidate the regulatory influence of RraA on multiple pathways associated with metabolism and pathogenesis in V. alginolyticus.
摘要：
细菌核糖核酸酶E（RNaseE）通过降解和加工RNA对转录后调控至关重要。RraA蛋白通过蛋白质-蛋白质相互作用抑制RNaseE活性，对基因表达发挥整体调节作用。然而,RraA的具体作用尚不清楚.在这项研究中,我们研究了rraA在溶藻弧菌ZJ-T中的表达，并鉴定了三个负责其表达的启动子，产生具有不同5'-UTR长度的转录本。在静止阶段,rraA在转录后被显著抑制。rraA的缺失对含盐的丰富培养基Luria-Bertani肉汤（LBS）中的细菌生长没有影响，但导致生物膜形成减少和对多粘菌素B的抗性增加。转录组分析显示，野生型和rraA突变体之间有350个差异表达基因（DEG），而蛋白质组分析鉴定出267种差异表达蛋白（DEP）。整合分析确定了DEGs和DEP共有的55个基因，这表明RraA主要在转录后水平影响基因表达。KEGG（京都基因和基因组百科全书）分析表明，RraA促进脂肪酸的转化，丙酸,和支链氨基酸乙酰辅酶A，同时增强氨基酸和肽的摄取。值得注意的是,RraA正调控毒力相关基因的表达，包括参与生物膜形成和VI型分泌系统的那些。本研究通过转录组分析扩展了对RraA调控网络的理解,强调蛋白质组学分析在研究转录后调控中的重要性。IMPORTANCERraA是核糖核酸酶E的抑制剂蛋白，与核酸内切酶相互作用并抑制其核酸内切活性，从而在多种mRNA和非编码小RNA的降解和成熟中起着广泛的调节作用。然而,RraA在溶藻弧菌中的生理功能和相关调节子尚未完全阐明。这里,我们报道RraA影响毒力相关的生理过程，即,抗生素耐药性和生物膜形成，在溶藻中。通过对转录组和蛋白质组进行综合分析，我们揭示了RraA参与碳代谢，氨基酸分解代谢，和运输,以及VI型分泌系统。总的来说，这些发现阐明了RraA对与溶藻弧菌代谢和发病机制相关的多种途径的调节作用.
