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1. Characterization of 475 Novel, Putative Small RNAs (sRNAs) in Carbon-Starved Salmonella enterica Serovar Typhimurium

   https://doi.org/10.3390/antibiotics10030305  

   Houserova, Dominika; Dahmer, Donovan J.; Amin, Shivam V.; King, Valeria M.; Barnhill, Emmaline C.; Zambrano, Mike E.; Dean, Meghan A.; Crucello, Aline; Aria, Kevin M.; Spector, Michael P.; et al (March 2021, Antibiotics)

   Neuwirth, Catherine (Ed.)

   An increasingly apparent role of noncoding RNA (ncRNAs) is to coordinate gene expression during environmental stress. A mounting body of evidence implicates small RNAs (sRNAs) as key drivers of Salmonella stress survival. Generally thought to be 50–500 nucleotides in length and to occur in intergenic regions, sRNAs typically regulate protein expression through base pairing with mRNA targets. In this work, through employing a refined definition of sRNAs allowing for shorter sequences and sRNA loci to overlap with annotated protein-coding gene loci, we have identified 475 previously unannotated sRNAs that are significantly differentially expressed during carbon starvation (C-starvation). Northern blotting and quantitative RT-PCRs confirm the expressions and identities of several of these novel sRNAs, and our computational analyses find the majority to be highly conserved and structurally related to known sRNAs. Importantly, we show that deletion of one of the sRNAs dynamically expressed during C-starvation, sRNA4130247, significantly impairs the Salmonella C-starvation response (CSR), confirming its involvement in the Salmonella CSR. In conclusion, the work presented here provides the first-ever characterization of intragenic sRNAs in Salmonella, experimentally confirms that sRNAs dynamically expressed during the CSR are directly involved in stress survival, and more than doubles the Salmonella enterica sRNAs described to date. 

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2. Genome-Wide Computational Prediction and Analysis of Noncoding RNAs in Oleidesulfovibrio alaskensis G20

   https://doi.org/10.3390/microorganisms12050960  

   Singh, Ram Nageena; Sani, Rajesh K (May 2024, Microorganisms)

   Noncoding RNAs (ncRNAs) play key roles in the regulation of important pathways, including cellular growth, stress management, signaling, and biofilm formation. Sulfate-reducing bacteria (SRB) contribute to huge economic losses causing microbial-induced corrosion through biofilms on metal surfaces. To effectively combat the challenges posed by SRB, it is essential to understand their molecular mechanisms of biofilm formation. This study aimed to identify ncRNAs in the genome of a model SRB, Oleidesulfovibrio alaskensis G20 (OA G20). Three in silico approaches revealed genome-wide distribution of 37 ncRNAs excluding tRNAs in the OA G20. These ncRNAs belonged to 18 different Rfam families. This study identified riboswitches, sRNAs, RNP, and SRP. The analysis revealed that these ncRNAs could play key roles in the regulation of several pathways of biosynthesis and transport involved in biofilm formation by OA G20. Three sRNAs, Pseudomonas P10, Hammerhead type II, and sX4, which were found in OA G20, are rare and their roles have not been determined in SRB. These results suggest that applying various computational methods could enrich the results and lead to the discovery of additional novel ncRNAs, which could lead to understanding the “rules of life of OA G20” during biofilm formation. 

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3. Salmonella Outer Membrane Vesicles contain tRNA Fragments (tRFs) that Inhibit Bacteriophage P22 infection

   https://doi.org/10.1101/2021.11.09.467952  

   Houserova, Dominika; Huang, Yulong; Kasukurthi, Mohan V; Watters, Brianna C; Khan, Fiza F; Mehta, Raj V; Chaudhary, Neil Y; Roberts, Justin T; DeMeis, Jeffrey D; Hobbs, Trevor K; et al (November 2021, bioRxiv)

   ABSTRACT SalmonellaOuter Membrane Vesicles (OMVs) were recently shown to inhibit P22 bacteriophage infection. Furthermore, despite there being several published reports now independently describing (1) the marked prevalence of tRFs within secreted vesicle transcriptomes and (2) roles for specific tRFs in facilitating/inhibiting viral replication, there have been no examinations of the effects of vesicle-secreted tRFs on viral infection reported to date. Notably, while specific tRFs have been reported in a number of bacteria, the tRFs expressed by salmonellae have not been previously characterized. As such, we recently screened small RNA-seq datasets for the presence of recurrent, specifically excised tRFs and identified 31 recurrent, relatively abundant tRFs expressed bySalmonella entericaserovar Typhimurium (SL1344). What’s more, we findS. Typhimurium OMVs contain significant levels of tRFs highly complementary to knownSalmonella enterica-infecting bacteriophage with 17 of 31 tRFs bearing marked complementarity to at least one knownSalmonella enterica-infecting phage (averaging 97.4% complementarity over 22.9 nt). Most notably, tRNA-Thr-CGT-1-1, 44-73, bears 100% sequence complementary over its entire 30 nt length to 29 distinct, annotatedSalmonella enterica-infecting bacteriophage including P22. Importantly, we find inhibiting this tRF in secreted OMVs improves P22 infectivity in a dose dependent manner whereas raising OMV tRF levels conversely inhibits P22 infectivity. Furthermore, we find P22 phage pre-incubation with OMVs isolated from naïve, control SL1344S. Typhimurium, successfully rescues the ability ofS. Typhimurium transformed with a specific tRNA-Thr-CGT-1-1, 44-73 tRF inhibitor to defend against P22. Collectively, these experiments confirm tRFs secreted inS. Typhimurium OMVs are directly involved with and required for the ability of OMVs to defend against bacteriophage predation. As we find the majority of OMV tRFs are highly complementary to an array of knownSalmonella enterica-infecting bacteriophage, we suggest OMV tRFs may primarily function as a broadly acting, previously uncharacterized innate antiviral defense. 

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4. Investigation of the global translational response to oxidative stress in the model archaeon Haloferax volcanii reveals untranslated small RNAs with ribosome occupancy

   https://doi.org/10.1128/msphere.00343-25  

   Dallon, Emma; Moran, Haley M; Chidambaran, Sadhana R; Kian, Arman; Huang, Betty_Y H; Fried, Stephen D; DiRuggiero, Jocelyne (September 2025, mSphere)

   Ellermeier, Craig D (Ed.)

   ABSTRACT Oxidative stress induces a wide range of cellular damage, often causing disease and cell death. While many organisms are susceptible to the effects of oxidative stress, haloarchaea have adapted to be highly resistant. Several aspects of the haloarchaeal oxidative stress response have been characterized; however, little is known about the impacts of oxidative stress at the translation level. Using the model archaeonHaloferax volcanii, we performed RNA-seq and ribosome profiling (Ribo-seq) to characterize the global translation landscape during oxidative stress. We identified 281 genes with differential translation efficiency (TE). Downregulated genes were enriched in ribosomal and translation proteins, in addition to peroxidases and genes involved in the TCA cycle. We also identified 42 small noncoding RNAs (sRNAs) with ribosome occupancy. Size distributions of ribosome footprints revealed distinct patterns for coding and noncoding genes, with 12 sRNAs matching the pattern of coding genes, and mass spectrometry confirming the presence of seven small proteins encoded by these sRNAs. However, the majority of sRNAs with ribosome occupancy had no evidence of coding potential. Of these ribosome-associated sRNAs, 12 had differential ribosome occupancy or TE during oxidative stress, suggesting that they may play a regulatory role during the oxidative stress response. Our findings on ribosomal regulation during oxidative stress, coupled with potential roles for ribosome-associated noncoding sRNAs and sRNA-derived small proteins inH. volcanii, revealed additional regulatory layers and underscored the multifaceted architecture of stress-responsive regulatory networks.IMPORTANCEArchaea are found in diverse environments, including as members of the human microbiome, and are known to play essential ecological roles in major geochemical cycles. The study of archaeal biology has expanded our understanding of the evolution of eukaryotes, uncovered novel biological systems, and revealed new opportunities for applications in biotechnology and bioremediation. Many archaeal systems, however, remain poorly characterized. UsingHaloferax volcaniias a model, we investigated the global translation landscape during oxidative stress. Our findings expand current knowledge of translational regulation in archaea and further illustrate the complexity of stress-responsive gene regulation. 

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5. Small RNAs positively and negatively control transcription elongation through modulation of Rho utilization site accessibility

   https://doi.org/10.1101/2024.02.02.578684  

   Farley, Kristen R; Buechler, Andrew K; Bianco, Colleen M; Vanderpool, Carin K (February 2024, bioRxiv)

   Abstract Bacteria use a multi-layered regulatory strategy to precisely and rapidly tune gene expression in response to environmental cues. Small RNAs (sRNAs) form an important layer of gene expression control and most act post-transcriptionally to control translation and stability of mRNAs. We have shown that at least five different sRNAs inEscherichia coliregulate the cyclopropane fatty acid synthase (cfa) mRNA. These sRNAs bind at different sites in the long 5’ untranslated region (UTR) ofcfamRNA and previous work suggested that they modulate RNase E-dependent mRNA turnover. Recently, thecfa5’ UTR was identified as a site of Rho-dependent transcription termination, leading us to hypothesize that the sRNAs might also regulatecfatranscription elongation. In this study we find that a pyrimidine-rich region flanked by sRNA binding sites in thecfa5’ UTR is required for premature Rho-dependent termination. We discovered that both the activating sRNA RydC and repressing sRNA CpxQ regulatecfaprimarily by modulating Rho-dependent termination ofcfatranscription, with only a minor effect on RNase E-mediated turnover ofcfamRNA. A stem-loop structure in thecfa5’ UTR sequesters the pyrimidine-rich region required for Rho-dependent termination. CpxQ binding to the 5’ portion of the stem increases Rho-dependent termination whereas RydC binding downstream of the stem decreases termination. These results reveal the versatile mechanisms sRNAs use to regulate target gene expression at transcriptional and post-transcriptional levels and demonstrate that regulation by sRNAs in long UTRs can involve modulation of transcription elongation. ImportanceBacteria respond to stress by rapidly regulating gene expression. Regulation can occur through control of messenger RNA (mRNA) production (transcription elongation), stability of mRNAs, or translation of mRNAs. Bacteria can use small RNAs (sRNAs) to regulate gene expression at each of these steps, but we often do not understand how this works at a molecular level. In this study, we find that sRNAs inEscherichia coliregulate gene expression at the level of transcription elongation by promoting or inhibiting transcription termination by a protein called Rho. These results help us understand new molecular mechanisms of gene expression regulation in bacteria. 

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