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arfA antisense RNA regulates MscL excretory activity.

ABSTRACT: Excretion of cytoplasmic protein (ECP) is a commonly observed phenomenon in bacteria, and this partial extracellular localisation of the intracellular proteome has been implicated in a variety of stress response mechanisms. In response to hypoosmotic shock and ribosome stalling in Escherichia coli, ECP is dependent upon the presence of the large-conductance mechanosensitive channel and the alternative ribosome-rescue factor A gene products. However, it is not known if a mechanistic link exists between the corresponding genes and the respective stress response pathways. Here, we report that the corresponding mscL and arfA genes are commonly co-located on the genomes of Gammaproteobacteria and display overlap in their respective 3' UTR and 3' CDS. We show this unusual genomic arrangement permits an antisense RNA-mediated regulatory control between mscL and arfA, and this modulates MscL excretory activity in E. coli These findings highlight a mechanistic link between osmotic, translational stress responses and ECP in E. coli, further elucidating the previously unknown regulatory function of arfA sRNA.

SUBMITTER: Morra R

PROVIDER: S-EPMC10070815 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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<i>arfA</i> antisense RNA regulates MscL excretory activity.

Morra Rosa R Pratama Fenryco F Butterfield Thomas T Tomazetto Geizecler G Young Kate K Lopez Ruth R Dixon Neil N

Life science alliance 20230403 6

Excretion of cytoplasmic protein (ECP) is a commonly observed phenomenon in bacteria, and this partial extracellular localisation of the intracellular proteome has been implicated in a variety of stress response mechanisms. In response to hypoosmotic shock and ribosome stalling in <i>Escherichia coli</i>, ECP is dependent upon the presence of the large-conductance mechanosensitive channel and the alternative ribosome-rescue factor A gene products. However, it is not known if a mechanistic link e ...[more]

PMID: 37012050

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Project description:The VicRK 2-component system of Streptococcus mutans regulates genes associated with cell wall biogenesis and biofilm formation. A putative RNase III-encoding gene ( rnc) is located downstream from the vicRKX operon. The goals of this study were to investigate the potential role of VicR in the regulation of adjacent downstream genes and evaluate transcription levels of vicR during planktonic and biofilm growth. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to investigate whether vicRKX and adjacent downstream genes were cotranscribed. Binding of purified recombinant VicR protein to promoter regions of vicR, rnc, and syfA genes was confirmed by electrophoretic mobility shift assay and by chromatin immunoprecipitation analyses. VicR antisense (AS vicR) RNA was detected by Northern blotting and qRT-PCR assays. AS vicR overexpression mutants were constructed, and the biofilm biomass was determined by crystal violet microtiter assay. Adjacent downstream genes rnc, smc, syfA, smu.1511, and syfB were cotranscribed with vicRKX. The predicted promoter regions of vicR, rnc, and syfA genes were directly regulated by VicR. An AS vicR RNA transcript was detected upstream of the rnc gene. Expression of the AS vicR RNA transcript was elevated in planktonic cultures and repressed during biofilm growth. In addition, Western blot data showed that expression of the VicR protein decreased by 35% in planktonic as compared with biofilm cultures. Furthermore, we show that overexpression of AS vicR led to a reduction in biofilm formation. The downstream genes rnc, smc, syfA, smu.1511, and syfB are cotranscribed with vicRKX. VicR is autophosphorylated, and rnc and syfA are directly regulated by VicR. Expression of VicR protein correlated inversely with different levels of AS vicR RNA transcript and growth conditions. The biofilm biomass decreased in the AS vicR overexpression mutant. These data suggest a role for the AS vicR RNA transcript in posttranscriptional regulation of VicR protein production in S. mutans.

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arfA antisense RNA regulates MscL excretory activity.

Publications

<i>arfA</i> antisense RNA regulates MscL excretory activity.

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