A small RNA controlled AND-gate regulates extrachromosomal DNA transfer in Salmonella

Horizontal gene transfer via plasmid conjugation is a major driving force in microbial evolution. Transfer of conjugative plasmids is a complex process that needs to be synchronized with the physiological state of the bacterial host. While several host transcription factors are known to control the plasmid-borne transfer control genes, RNA-based regulatory circuits for host-plasmid communication remain unknown. Here, we describe a post-transcriptional mechanism whereby the Hfq-dependent small RNA, RprA, inhibits transfer of pSLT, the virulence plasmid of Salmonella enterica. RprA employs two different seed pairing domains to recognize and activate the mRNAs of both the sigma-factor S and RicI, a cytoplasmic membrane protein. The latter is a hitherto unknown conjugation inhibitor whose transcription requires S. Together, RprA and S constitute a feed-forward loop with AND-gate logic which tightly controls RicI synthesis for selective suppression of plasmid conjugation under membrane stress. This study reports the first sRNA-controlled feed-forward loop based on double target activation and an unexpected function for a core-genome encoded small RNA in controlling extrachromosomal DNA transfer. To determine the targets of the small regulatory RNAs RprA/RprA-proc. in S. Typhimurium, we looked at the effect of a short pulse of RprA/RprA-proc. over-expression on the Salmonella transcriptome. To achieve over-expression, the rprA/rprA proc. genes were cloned in the pBAD plasmid and induced with 0.2% L-arabinose for 10 min. We then extracted the total RNA for transcriptional profiling. A strain carrying the pBAD plasmid w/o insert was used as negative control (also induced by L-arabinose). 3 biological replicates were performed. This sRNA target identification strategy has been described in Papenfort et al; Molecular Microbiology (2006) 62(6), 1674–1688.

通过质粒接合实现的水平基因转移是微生物演化的核心驱动力之一。接合型质粒的转移是一个复杂过程，需与细菌宿主的生理状态同步协调。尽管目前已知若干宿主转录因子可调控质粒携带的转移控制基因，但用于宿主与质粒间通信的RNA调控通路仍未被揭示。本研究揭示了一种转录后调控机制：依赖Hfq的小RNA（small RNA, sRNA）RprA可抑制肠炎沙门氏菌毒性质粒pSLT的接合转移。RprA通过两个不同的种子配对结构域，分别识别并激活σ因子σ^S以及胞质膜蛋白RicI的mRNA。后者是迄今为止尚未被报道的接合转移抑制因子，其转录依赖σ^S。综上，RprA与σ^S共同构成了一个带有“与门”逻辑的前馈环路，可精准调控RicI的合成，从而在膜应激条件下选择性抑制质粒接合转移。本研究首次报道了基于双靶标激活的sRNA调控前馈环路，同时揭示了核心基因组编码的小RNA在调控染色体外DNA转移中的意外功能。为鉴定鼠伤寒沙门氏菌中RprA/RprA-proc.这一调控小RNA的靶基因，我们分析了短时脉冲式过表达RprA/RprA-proc.对沙门氏菌转录组的影响。为实现过表达，我们将rprA/rprA-proc.基因克隆至pBAD质粒中，并以0.2% L-阿拉伯糖诱导10分钟。随后我们提取总RNA用于转录谱分析。我们以仅携带空pBAD质粒的菌株作为阴性对照（同样经L-阿拉伯糖诱导）。本实验设置3次生物学重复。该sRNA靶基因鉴定策略已在Papenfort等人发表于《Molecular Microbiology》（2006年，第62卷第6期，1674–1688页）的研究中进行过阐述。