A multicopy sRNA of Listeria monocytogenes regulates expression of the virulence adhesin LapB

The multicopy sRNA LhrC of the intracellular pathogen Listeria monocytogenes has been shown to be induced under infection-relevant conditions, but its physiological role and mechanism of action is not understood. In an attempt to pinpoint the exact terms of LhrC expression, cell envelope stress could be defined as a specific inducer of LhrC. In this process, the two-component system LisRK was shown to be indispensable for expression of all five copies of LhrC. lapB mRNA, encoding a cell wall associated protein that was recently identified as an important virulence factor, was disclosed to be directly bound by LhrC leading to an impediment of its translation. Although LhrC binds to Hfq, it does not require the RNA chaperone for stability or lapB mRNA interaction. The mechanism of LhrC-lapB mRNA binding was shown to involve three redundant CU-rich sites and a structural rearrangement in the sRNA. This study represents an extensive depiction of a so far uncharacterized multicopy sRNA and reveals interesting new aspects concerning its regulation, virulence association and mechanism of target binding.     

Small RNAs encoded within genetic islands of Salmonella typhimurium show host-induced expression and role in virulence

The emergence of pathogenic strains of enteric bacteria and their adaptation to unique niches are associated with the acquisition of foreign DNA segments termed ‘genetic islands’. We explored these islands for the occurrence of small RNA (sRNA) encoding genes. Previous systematic screens for enteric bacteria sRNAs were mainly carried out using the laboratory strain Escherichia coli K12, leading to the discovery of ~80 new sRNA genes. These searches were based on conservation within closely related members of enteric bacteria and thus, sRNAs, unique to pathogenic strains were excluded. Here we describe the identification and characterization of 19 novel unique sRNA genes encoded within the ‘genetic islands’ of the virulent strain Salmonella typhimurium. We show that the expression of many of the island-encoded genes is associated with stress conditions and stationary phase. Several of these sRNA genes are induced when Salmonella resides within macrophages. One sRNA, IsrJ, was further examined and found to affect the translocation efficiency of virulence-associated effector proteins into nonphagocytic cells. In addition, we report that unlike the majority of the E. coli sRNAs that are trans regulators, many of the island-encoded sRNAs affect the expression of cis-encoded genes. Our study suggests that the island encoded sRNA genes play an important role within the network that regulates bacterial adaptation to environmental changes and stress conditions and thus controls virulence.     

The ZorO-OrzO type I toxin–antitoxin locus: repression by the OrzO antitoxin

Type I toxin–antitoxin loci consist of two genes: a small, hydrophobic, potentially toxic protein, and a small RNA (sRNA) antitoxin. The sRNA represses toxin gene expression by base pairing to the toxin mRNA. A previous bioinformatics search predicted a duplicated type I locus within Escherichia coli O157:H7 (EHEC), which we have named the gene pairs zorO-orzO and zorP-orzP. We show that overproduction of the zorO gene is toxic to E. coli; co-expression of the sRNA OrzO can neutralize this toxicity, confirming that the zorO-orzO pair is a true type I toxin–antitoxin locus. However, OrzO is unable to repress zorO in a strain deleted for RNase III, indicating that repression requires cleavage of the target mRNA. Sequence analysis and mutagenesis studies have elucidated a nucleotide sequence region (V1) that allows differential recognition of the zorO mRNA by OrzO and not OrzP, and a specific single nucleotide within the V1 of OrzO that is critical for repression of zorO. Although there are 18 nt of complementarity between the OrzO sRNA and the zorO mRNA, not all base pairing interactions are needed for repression; however, the amount needed is dependent on whether there is continuous or discontinuous complementarity to the target mRNA.     

Engineering Halomonas bluephagenesis via small regulatory RNAs

Halomonas bluephagenesis, a robust and contamination-resistant microorganism has been developed as a chassis for “Next Generation Industrial Biotechnology”. The non-model H. bluephagenesis requires efficient tools to fine-tune its metabolic fluxes for enhanced production phenotypes. Here we report a highly efficient gene expression regulation system (PrrF1-2-HfqPa) in H. bluephagenesis, small regulatory RNA (sRNA) PrrF1 scaffold from Pseudomonas aeruginosa and a target-binding sequence that downregulate gene expression, and its cognate P. aeruginosa Hfq (HfqPa), recruited by the scaffold to facilitate the hybridization of sRNA and the target mRNA. The PrrF1-2-HfqPa system targeting prpC in H. bluephagenesis helps increase 3-hydroxyvalerate fraction in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) to 21 mol% compared to 3.1 mol% of the control. This sRNA system repressed phaP1 and minD simultaneously, resulting in large polyhydroxybutyrate granules. Further, an sRNA library targeting 30 genes was employed for large-scale target identification to increase mevalonate production. This work expands the study on using an sRNA system not based on Escherichia coli MicC/SgrS-Hfq to repress gene expression, providing a framework to exploit new powerful genome engineering tools based on other sRNAs.