The exploration of antibiotic resistance genes (ARGs) in drinking water reservoirs is an emerging field. Using a curated database, we enhanced the ARG detection and conducted a comprehensive analysis using 2.2 Tb of deep metagenomic sequencing data to determine the distribution of ARGs across 16 drinking water reservoirs and associated environments. Our findings reveal a greater diversity of ARGs in sediments than in water, underscoring the importance of extensive background surveys. Crucial ARG carriers-specifically Acinetobacter, Pseudomonas, and Mycobacterium were identified in drinking water reservoirs. Extensive analysis of the data uncovered a considerable concern for drinking water safety, particularly in regions reliant on river sources. Mobile genetic elements have been found to contribute markedly to the propagation of ARGs. The results of this research suggest that the establishment of drinking water reservoirs for supplying raw water may be an effective strategy for alleviating the spread of water-mediated ARGs.

Adenosine-to-inosine (A-to-I) RNA editing is an important posttranscriptional event in eukaryotes; however, many features remain largely unexplored in prokaryotes. This study focuses on a serine-to-proline recoding event (S128P) that originated in the mRNA of fliC, which encodes a flagellar filament protein; the editing event was observed in RNA-seq samples exposed to oxidative stress. Using Sanger sequencing, we show that the S128P editing event is induced by H₂O₂. To investigate the in vivo interaction between RNAs and TadA, which is the principal enzyme for A-to-I editing, genome-wide RNA immunoprecipitation–coupled high-throughput sequencing (iRIP-Seq) analysis was performed using HA-tagged TadA from Xanthomonas oryzae pv. oryzicola. We found that TadA can bind to the mRNA of fliC and the binding motif is identical to that previously reported by Bar-Yaacov and colleagues. This editing event increased motility and enhanced tolerance to oxidative stress due to changes in flagellar filament structure, which was modelled in 3D and measured by TEM. The change in filament structure due to the S128P mutant increased biofilm formation, which was measured by the 3D laser scanning confocal microscopy. RNA-seq revealed that a gene cluster that contributes to siderophore biosynthesis and Fe³⁺ uptake was upregulated in S128P compared with WT. Based on intracellular levels of reactive oxygen species and an oxidative stress survival assay, we found that this gene cluster can contribute to the reduction of the Fenton reaction and increases biofilm formation and bacterial virulence. This oxidative stress response was also confirmed in Pseudomonas putida. Overall, our work demonstrates that A-to-I RNA editing plays a role in bacterial pathogenicity and adaptation to oxidative stress. Adenosine-to-inosine (A-to-I) RNA editing is an important posttranscriptional event in eukaryotes that has only been recently documented in bacteria. In this study, we use multiple ‘omic’ approaches to show that A-to-I RNA editing can occur in fliC, a flagellar filament protein. We show that TadA, which encodes adenosine deaminase, can directly bind to mRNA of target genes through recognition of a GACG motif. This editing event changes a single amino acid residue from serine to proline in FliC, resulting in a structural change in the flagellar filament. This posttranscriptional editing event contributes to virulence and increases tolerance to oxidative stress by enhancing biofilm formation. Our results provide insight into a new mechanism that bacterial pathogens use to adapt to oxidative stress, which can also increase virulence.

Enterobacter cloacaecomplex (Ecc) species are widely distributed opportunistic pathogens mainly associated with humans and plants. In this study, the genomes of clinical isolates includingE. hormaechei, E. kobei, andE. ludwigiiand non-clinical isolate includingE. nimipressuraliswere analysed in combination with the genome ofE. asburiaeby using the reference strainE. cloacaesubsp.cloacaeATCC 13047; the Ecc strains were tested on artificial sputum media (ASM), which mimics the host, to evaluate T6SS genes as a case study. All five Ecc strains were sequenced in our lab. Comparative genome analysis of the Ecc strains revealed that genes associated with the survival of Ecc strains, including genes of metal-requiring proteins, defence-associated genes and genes associated with general physiology, were highly conserved in the genomes. However, the genes involved in virulence and drug resistance, specifically those involved in bacterial secretion, host determination and colonization of different strains, were present in different genomic regions. For example, T6SS accessory and core components, T4SS, and multidrug resistance genes/efflux system genes seemed vital for the survival of Ecc strains in various environmental niches, such as humans and plants. Moreover, the ASM host-mimicking growth medium revealed significantly high expression of T6SS genes, including PrpC, which is a regulatory gene of the T6SS, in all tested Ecc strains compared to the control medium. The variations in T6SS gene expression in ASM vs. control showed that the ASM system represents a simple, reproducible and economical alternative to animal models for studies such as those aimed at understanding the divergence of Ecc populations. In summary, genome sequencing of clinical and environmental Ecc genomes will assist in understanding the epidemiology of Ecc strains, including the isolation, virulence characteristics, prevention and treatment of infectious disease caused by these broad-host-range niche-associated species.

Circular RNAs (circRNAs) are prevalent in eukaryotic cells and have been linked to disease progressions. Their unique circular structure and stability make them potential biomarkers and therapeutic targets. Compared with animal models, plant circRNA research is still in its infancy. The lack of effective tools to specifically knock down circRNAs without affecting host gene expression has slowed the progress of plant circRNA research. Here, we have developed a CRISPR–Cas13d tool that can specifically knock down circRNAs in plant systems, successfully achieving the targeted knockdown of circRNAs in rice (Oryza sativa). We further focused on Os-circANK (a circRNA derived from Ankyrin repeat-containing protein), a circRNA differentially expressed in rice upon pathogen infection. Physiological and biochemical experiments revealed that Os-circANK functions as a sponge for miR398b, suppressing the cleavage of Cu/Zn–superoxidase dismutase (CSD)1/CSD2/copper chaperone for superoxide dismutase/superoxidase dismutaseX through competing endogenous RNA, leading to reduced reactive oxygen species levels following Xanthomonas oryzae pv. oryzae (Xoo) infection and a negative regulation of rice resistance to bacterial blight. Our findings indicate Os-circANK inhibits rice resistance to bacterial blight via the microRNA398b(miR398b)/CSD/SOD pathway.

Noncoding small RNAs (sRNAs) packaged in bacterial outer membrane vesicles (OMVs) function as novel mediators of interspecies communication. While the role of bacterial sRNAs in enhancing virulence is well established, the role of sRNAs in the interaction between OMVs from phytopathogenic bacteria and their host plants remains unclear. In this study, we employ RNA sequencing to characterize differentially packaged sRNAs in OMVs of the phytopathogen Xanthomonas oryzae pv. oryzicola (Xoc). Our candidate sRNA (Xosr001) was abundant in OMVs and involved in the regulation of OsJMT1 to impair host stomatal immunity. Xoc loads Xosr001 into OMVs, which are specifically ttransferred into the mechanical tissues of rice leaves. Xosr001 suppresses OsJMT1 transcript accumulation in vivo, leading to a reduction in MeJA accumulation in rice leaves. Furthermore, the application of synthesized Xosr001 sRNA to the leaves of OsJMT1-HA-OE transgenic line results in the suppression of OsJMT1 expression by Xosr001. Notably, the OsJMT1-HA-OE transgenic line exhibited attenuated stomatal immunity and disease susceptibility upon infection with ΔXosr001 compared to Xoc. These results suggest that Xosr001 packaged in Xoc OMVs functions to suppress stomatal immunity in rice.

Ilyonectria mors-panacis is the cause of a serious disease hampering the production of Panax notoginseng, an important Chinese medicinal herb, widely used for its anti-inflammatory, antifatigue, hepato-protective, and coronary heart disease prevention effects. Here, we report the first Illumina-Pacbio hybrid sequenced draft genome assembly of I. mors-panacis strain G3B and its annotation. The availability of this genome sequence not only represents an important tool toward understanding the genetics behind the infection mechanism of I. mors-panacis strain G3B but also will help illuminate the complexities of the taxonomy of this species.