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ABSTRACT 8-Oxo-7,8-dihydroguanine (8-oxoG) is a major RNA modification caused by oxidative stresses and has been implicated in carcinogenesis, neurodegeneration, and aging. Several RNA-binding proteins have been shown to have a binding preference for 8-oxoG-modified RNA in eukaryotes and protect cells from oxidative stress. To date, polynucleotide phosphorylase (PNPase) is one of the most well-characterized proteins in bacteria that recognize 8-oxoG-modified RNA, but how PNPase cooperates with other proteins to process oxidized RNA is still unclear. Here, we use RNA affinity chromatography and mass spectrometry to search for proteins that preferably bind 8-oxoG-modified RNA in Deinococcus radiodurans, an extremophilic bacterium with extraordinary resistance to oxidative stresses. We identified four proteins that preferably bind to oxidized RNA: PNPase (DR_2063), DEAD box RNA helicase (DR_0335/RhlB), ribosomal protein S1 (DR_1983/RpsA), and transcriptional termination factor (DR_1338/Rho). Among these proteins, PNPase and RhlB exhibit high-affinity binding to 8-oxoG-modified RNA in a dose-independent manner. Deletions of PNPase and RhlB caused increased sensitivity of D. radiodurans to oxidative stress. We further showed that PNPase and RhlB specifically reduce the cellular availability of 8-oxoG-modified RNA but have no effect on oxidized DNA. Importantly, PNPase directly interacts with RhlB in D. radiodurans; however, no additional phenotypic effect was observed for the double deletion of pnp and rhlB compared to the single deletions. Overall, our findings suggest the roles of PNPase and RhlB in targeting 8-oxoG-modified RNAs and thereby constitute an important component of D. radiodurans resistance to oxidative stress. IMPORTANCE Oxidative RNA damage can be caused by oxidative stress, such as hydrogen peroxide, ionizing radiation, and antibiotic treatment. 8-oxo-7,8-dihydroguanine (8-oxoG), a major type of oxidized RNA, is highly mutagenic and participates in a variety of disease occurrences and development. Although several proteins have been identified to recognize 8-oxoG-modified RNA, the knowledge of how RNA oxidative damage is controlled largely remains unclear, especially in nonmodel organisms. In this study, we identified four RNA binding proteins that show higher binding affinity to 8-oxoG-modified RNA compared to unmodified RNA in the extremophilic bacterium Deinococcus radiodurans, which can endure high levels of oxidative stress. Two of the proteins, polynucleotide phosphorylase (PNPase) and DEAD-box RNA helicase (RhlB), interact with each other and reduce the cellular availability of 8-oxoG-modified RNA under oxidative stress. As such, this work contributes to our understanding of how RNA oxidation is influenced by RNA binding proteins in bacteria.

Abstract Networks of transcriptional and post-transcriptional regulators are critical for bacterial survival and adaptation to environmental stressors. While transcriptional regulators provide rapid activation and/or repression of a wide-network of genes, post-transcriptional regulators, such as small RNAs (sRNAs), are also important to fine-tune gene expression. However, the mechanisms of sRNAs remain poorly understood, especially in less-studied bacteria. Deinococcus radiodurans is a gram-positive bacterium resistant to extreme levels of ionizing radiation (IR). Although multiple unique regulatory systems (e.g., the Radiation and Desiccation Response (RDR)) have been identified in this organism, the role of post-transcriptional regulators has not been characterized within the IR response. In this study, we have characterized an sRNA, PprS (formerly Dsr2), as a post-transcriptional coordinator of IR recovery in D. radiodurans. PprS showed differential expression specifically under IR and knockdown of PprS resulted in reduced survival and growth under IR, suggesting its importance in regulating post-radiation recovery. We determined a number of potential RNA targets involved in several pathways including translation and DNA repair. Specifically, we confirmed that PprS binds within the coding region to stabilize the pprM (DR_0907) transcript, a RDR modulator. Overall, these results are the first to present an additional layer of sRNA-based control in DNA repair pathways associated with bacterial radioresistance.

Plagiodera versicolora (Coleoptera: Chrysomelidae) is one of the most destructive pests of the Salicaceae worldwide, which has established complex interactions with surrounding organisms. Uncovering the molecular mechanisms of some antagonistic interactions would facilitate the development of environmentally friendly pest insect management strategies. Suitable reference genes are essential for reliable qPCR and gene expression analysis in molecular studies; however, a comprehensive assessment of reference genes in P. versicolora is still lacking. In this study, the stability of seven housekeeping genes (including Actin, EF1A, α-tubulin, RPL13a, RPS18, RPL8 and UBC) was investigated under both biotic (developmental stages, tissues, sex and pathogen treatment) and abiotic (RNA interference treatment, temperature treatment) conditions. The geNorm, NormFinder, BestKeeper, and ΔCt programs were used to analyze gene expression data. The RefFinder synthesis analysis was applied to suggest a handful of appropriate reference genes for each experimental condition. RPS18 and EF1A were the most reliable reference genes in different development stages; RPS18 and RPL8 were most stable in female and male adults, different tissues, different temperatures, and pathogen treatment; α-tubulin and RPL13a were most stable after dietary RNAi treatment. The research provides a strong basis for future research into the molecular biology of P. versicolora.

The proper functioning of many proteins requires their transport to the correct cellular compartment or their secretion. Signal recognition particle (SRP) is a major protein transport pathway responsible for the co-translational movement of integral membrane proteins as well as periplasmic proteins. Deinococcus radiodurans is a ubiquitous bacterium that expresses a complex phenotype of extreme oxidative stress resistance, which depends on proteins involved in DNA repair, metabolism, gene regulation, and antioxidant defense. These proteins are located extracellularly or subcellularly, but the molecular mechanism of protein localization in D. radiodurans to manage oxidative stress response remains unexplored. In this study, we characterized the SRP complex in D. radiodurans R1 and showed that the knockdown (KD) of the SRP RNA (Qpr6) reduced bacterial survival under hydrogen peroxide and growth under chronic ionizing radiation. Through LC-mass spectrometry (MS/MS) analysis, we detected 162 proteins in the periplasm of wild-type D. radiodurans, of which the transport of 65 of these proteins to the periplasm was significantly reduced in the Qpr6 KD strain. Through Western blotting, we further demonstrated the localization of the catalases in D. radiodurans, DR_1998 (KatE1) and DR_A0259 (KatE2), in both the cytoplasm and periplasm, respectively, and showed that the accumulation of KatE1 and KatE2 in the periplasm was reduced in the SRP-defective strains. Collectively, this study establishes the importance of the SRP pathway in the survival and the transport of antioxidant proteins in D. radiodurans under oxidative stress.

ABSTRACT The large intestinal pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 detects host cues to regulate virulence gene expression during colonization and infection. However, virulence regulatory mechanisms of EHEC O157:H7 in the human large intestine are not fully understood. Herein, we identified a virulence-regulating pathway where the PhoQ/PhoP two-component regulatory system senses low magnesium levels and signals to the O island 119-encoded Z4267 (LmiA; low magnesium-induced regulator A), directly activating loci of enterocyte effacement genes to promote EHEC O157:H7 adherence in the large intestine. Disruption of this pathway significantly decreased EHEC O157:H7 adherence in the mouse intestinal tract. Moreover, feeding mice a magnesium-rich diet significantly reduced EHEC O157:H7 adherence in vivo. This LmiA-mediated virulence regulatory pathway is also conserved among several EHEC and enteropathogenic E. coli serotypes; therefore, our findings support the use of magnesium as a dietary supplement and provide greater insights into the dietary cues that can prevent enteric infections. IMPORTANCE Sensing specific gut metabolites is an important strategy for inducing crucial virulence programs by enterohemorrhagic Escherichia coli (EHEC) O157:H7 during colonization and infection. Here, we identified a virulence-regulating pathway wherein the PhoQ/PhoP two-component regulatory system signals to the O island 119-encoded low magnesium-induced regulator A (LmiA), which, in turn, activates locus of enterocyte effacement (LEE) genes to promote EHEC O157:H7 adherence in the low-magnesium conditions of the large intestine. This regulatory pathway is widely present in a range of EHEC and enteropathogenic E. coli (EPEC) serotypes. Disruption of this pathway significantly decreased EHEC O157:H7 adherence in the mouse intestinal tract. Moreover, mice fed a magnesium-rich diet showed significantly reduced EHEC O157:H7 adherence in vivo, indicating that magnesium may help in preventing EHEC and EPEC infection in humans.

sRNAs represent a powerful class of regulators that influences multiple mRNA targets in response to environmental changes. However, very few direct sRNA–sRNA interactions have been deeply studied in any organism. Zymomonas mobilis is a bacterium with unique ethanol-producing metabolic pathways in which multiple small RNAs (sRNAs) have recently been identified, some of which show differential expression in ethanol stress. In this study, we show that two sRNAs (Zms4 and Zms6) are upregulated under ethanol stress and have significant impacts on ethanol tolerance and production in Z. mobilis. We conducted multi-omics analysis (combining transcriptomics and sRNA-immunoprecipitation) to map gene networks under the influence of their regulation. We confirmed that Zms4 and Zms6 bind multiple RNA targets and regulate their expressions, influencing many downstream pathways important to ethanol tolerance and production. In particular, Zms4 and Zms6 interact with each other as well as many other sRNAs, forming a novel sRNA–sRNA direct interaction network. This study thus uncovers a sRNA network that co-orchestrates multiple ethanol related pathways through a diverse set of mRNA targets and a large number of sRNAs. To our knowledge, this study represents one of the largest sRNA–sRNA direct interactions uncovered so far.

Spiders are a key predator of insects across ecosystems and possess great potential as pest control agents. Unfortunately, it is difficult to artificially cultivate multiple generations of most spider species. Since gut bacterial flora has been shown to significantly alter nutrient availability, it is plausible that the spiders’ microbial community plays a key role in their unsuccessful breeding. However, both the gut microbial composition and its influencing factors in many spiders remain a mystery. In this study, the gut microbiota of Campanicola campanulata, specialists who prey on ants and are widely distributed across China, was characterized. After, the impact of diet and diet-associated bacteria on gut bacterial composition was evaluated. First, two species of prey ants (Lasius niger and Tetramorium caespitum) were collected from different locations and fed to C. campanulata. For each diet, we then profiled the nutritional content of the ants, as well as the bacterial communities of both the ants and spiders. Results showed that the protein and carbohydrate content varied between the two prey ant species. We isolated 682 genera from 356 families in the ants (dominant genera including Pseudomonas, Acinetobacter, Paraburkholderia, Staphylococcus, and Novosphingobium), and 456 genera from 258 families in the spiders (dominated by Pseudomonas). However, no significant differences were found in the gut microbiota of spiders that were fed the differing ants. Together, these results indicate that nutritional variation and diet-associated bacterial differences have a limited impact on the microbial composition of spider guts, highlighting that spiders may have a potentially stable internal environment and lay the foundation for future investigations into gut microbiota.

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 (O157) is one of the most notorious human pathogens, causing severe disease in humans worldwide. O157 specifically colonizes the large intestine of mammals after passing through the small intestine, and this process is influenced by differential signals between the two regions. Small regulatory RNAs (sRNAs) are able to sense and respond to environmental changes and regulate diverse physiological processes in pathogenic bacteria. Although some sRNAs of O157 have been extensively investigated, whether these molecules can sense differences between the small and large intestine and influence the preferential colonization in the large intestine by O157 remains unknown. In this study, we identified a new sRNA, Esr055, in O157 which senses the low DNA concentration in the large intestine and contributes to the preferential colonization of the bacteria in this region. The number of O157 wild-type that adhered to the colon is 30.18 times higher than the number that adhered to the ileum of mice, while the number of the 1Esr055 mutant that adhered to the colon decreased to 13.27 times higher than the number adhered to the ileum. Furthermore, we found that the expression of Esr055 is directly activated by the regulator, DeoR, and its expression is positively affected by DNA, which is significantly more abundant in the ileum than in the colon of mice. Additionally, combining the results of informatics predictions and transcriptomic analysis, we found that several virulence genes are up-regulated in the ΔEsr055 mutant and five candidate genes (z0568, z0974, z1356, z1926, and z5187) may be its direct targets. [ABSTRACT FROM AUTHOR]