A widely conserved protein Rof inhibits transcription termination factor Rho and promotes Salmonella virulence program.

Transcription is crucial for the expression of genetic information and its efficient and accurate termination is required for all living organisms. Rho-dependent termination could rapidly terminate unwanted premature RNAs and play important roles in bacterial adaptation to changing environments. Although Rho has been discovered for about five decades, the regulation mechanisms of Rho-dependent termination are still not fully elucidated. Here we report that Rof is a conserved antiterminator and determine the cryogenic electron microscopy structure of Rho-Rof antitermination complex. Rof binds to the open-ring Rho hexamer and inhibits the initiation of Rho-dependent termination. Rof's N-terminal α-helix undergoes conformational changes upon binding with Rho, and is key in facilitating Rof-Rho interactions. Rof binds to Rho's primary binding site (PBS) and excludes Rho from binding with PBS ligand RNA at the initiation step. Further in vivo analyses in Salmonella Typhimurium show that Rof is required for virulence gene expression and host cell invasion, unveiling a physiological function of Rof and transcription termination in bacterial pathogenesis.

RES-Xre toxin-antitoxin locus knaAT maintains the stability of the virulence plasmid in Klebsiella pneumoniae.

Hypervirulent Klebsiella pneumoniae isolates have been increasingly reported worldwide, especially hypervirulent drug-resistant variants owing to the acquisition of a mobilizable virulence plasmid by a carbapenem-resistant strain. This pLVPK-like mobilizable plasmid encodes various virulence factors; however, information about its genetic stability is lacking. This study aimed to investigate the type II toxin-antitoxin (TA) modules that facilitate the virulence plasmid to remain stable in K. pneumoniae. More than 3,000 TA loci in 2,000 K. pneumoniae plasmids were examined for their relationship with plasmid cargo genes. TA loci from the RES-Xre family were highly correlated with virulence plasmids of hypervirulent K. pneumoniae. Overexpression of the RES toxin KnaT, encoded by the virulence plasmid-carrying RES-Xre locus knaAT, halts the cell growth of K. pneumoniae and E. coli, whereas co-expression of the cognate Xre antitoxin KnaA neutralizes the toxicity of KnaT. knaA and knaT were co-transcribed, representing the characteristics of a type II TA module. The knaAT deletion mutation gradually lost its virulence plasmid in K. pneumoniae, whereas the stability of the plasmid in E. coli was enhanced by adding knaAT, which revealed that the knaAT operon maintained the genetic stability of the large virulence plasmid in K. pneumoniae. String tests and mouse lethality assays subsequently confirmed that a loss of the virulence plasmid resulted in reduced pathogenicity of K. pneumoniae. These findings provide important insights into the role of the RES-Xre TA pair in stabilizing virulence plasmids and disseminating virulence genes in K. pneumoniae.

Microbial and metabolic profiles unveil mutualistic microbe-microbe interaction in obesity-related colorectal cancer.

Obesity is a risk factor for colorectal cancer (CRC), and the involvement of gut microbiota in the pathogenesis of obesity and CRC is widely recognized. However, the landscape of fecal microbiome and metabolome distinguishing patients with obesity-related CRC from obesity remains unknown. Here, we utilize metagenomic sequencing and metabolomics from 522 patients with CRC and healthy controls to identify the characteristics of obese CRC. Our integrated analysis reveals that obesity-related CRC is characterized by elevated Peptostreptococcus stomatis, dysregulated fatty acids and phospholipids, and altered Kyoto Encyclopedia of Genes and Genomes pathways involving glycerophospholipid metabolism and lipopolysaccharide synthesis. Correlation analysis unveils microbial interactions in obesity, where the probiotic Faecalibacterium prausnitzii and the tumor-promoting species P. stomatis may engage in cross-feeding, thereby promoting tumorigenesis. In vitro experiments affirm enhanced growth under cross-feeding conditions. The mutualistic microbe-microbe interaction may contribute to the association between obesity and elevated CRC risk. Additionally, diagnostic models incorporating BMI-specific microbial biomarkers display promise for precise CRC screening.

In vivo RNA interactome profiling reveals 3'UTR-processed small RNA targeting a central regulatory hub.

Small noncoding RNAs (sRNAs) are crucial regulators of gene expression in bacteria. Acting in concert with major RNA chaperones such as Hfq or ProQ, sRNAs base-pair with multiple target mRNAs and form large RNA-RNA interaction networks. To systematically investigate the RNA-RNA interactome in living cells, we have developed a streamlined in vivo approach iRIL-seq (intracellular RIL-seq). This generic approach is highly robust, illustrating the dynamic sRNA interactomes in Salmonella enterica across multiple stages of growth. We have identified the OmpD porin mRNA as a central regulatory hub that is targeted by a dozen sRNAs, including FadZ cleaved from the conserved 3'UTR of fadBA mRNA. Both ompD and FadZ are activated by CRP, constituting a type I incoherent feed-forward loop in the fatty acid metabolism pathway. Altogether, we have established an approach to profile RNA-RNA interactomes in live cells, highlighting the complexity of RNA regulatory hubs and RNA networks.

CpxR promotes the carbapenem antibiotic resistance of Klebsiella pneumoniae by directly regulating the expression and the dissemination of blaKPC on the IncFII conjugative plasmid.

Klebsiella pneumoniae is an important human pathogen known for its resistance to carbapenem antibiotics, especially the increasing carbapenem-resistant hypervirulent variants. The carbapenem resistance is mainly caused by the carbapenemase gene blaKPC which was commonly found on the IncFII transferable plasmids in K. pneumoniae ST11 isolates in regions of China. However, the mechanisms of the plasmid-carrying blaKPC regulation by the host strain are not clear. To investigate the chromosome-encoded two-component system (TCS) that regulates the carbapenem resistance of K. pneumoniae caused by blaKPC, twenty-four TCSs of a carbapenem-resistant classical K. pneumoniae ST11 clinical isolate were knocked out. The deletion mutation of the TCS regulator cpxR exhibited increased sensitivity to carbapenem, which could be restored by complementation with cpxR in trans. Electrophoretic mobility shift, isothermal titration calorimetry and DNase I footprinting results revealed that CpxR directly bound to the promoter DNA of blaKPC and the binding was abolished by disrupting the DNA-binding domain in CpxR. The subsequent in vivo assays using the lacZ reporter system and qPCR showed that CpxR upregulates the transcription of blaKPC. Notably, CpxR was also found to activate the transfer of the blaKPC-carrying IncFII plasmid between the hypervirulent K. pneumoniae and E. coli isolates, in which CpxR promoted the transcription of the tra operon via binding to its promoter region. These results provide an important insight into the regulation of the host factor CpxR in the plasmid-carrying carbapenemase gene in the classical and hypervirulent K. pneumoniae.

Ribose 5-phosphate: the key metabolite bridging the metabolisms of nucleotides and amino acids during stringent response in Escherichia coli?

The bacterial stringent response and its effector alarmone guanosine penta- or tetra - phosphates (p)ppGpp are vital for bacterial tolerance and survival of various stresses in environments (including antibiotics) and host cells (virulence). (p)ppGpp does so by binding to its numerous target proteins and reprograming bacterial transcriptome to tune down the synthesis of nucleotides and rRNA/tRNA, and up-regulate amino acid biosynthesis genes. Recent identification of more novel (p)ppGpp direct binding proteins in Escherichia coli and their deep studies have unveiled unprecedented details of how (p)ppGpp coordinates the nucleotide and amino acid metabolic pathways upon stringent response; however, the mechanistic link between nucleotide and amino acid metabolisms remains still incompletely understood. Here we propose the metabolite ribose 5'-phosphate as the key link between nucleotide and amino acid metabolisms and a working model integrating both the transcriptional and metabolic effects of (p)ppGpp on E. coli physiological adaptation during the stringent response.

Decoding the microbiome: advances in genetic manipulation for gut bacteria.

Studies of the gut microbiota have revealed associations between specific bacterial species or community compositions with health and disease, yet the causal mechanisms underlying microbiota gene-host interactions remain poorly understood. This is partly due to limited genetic manipulation (GM) tools for gut bacteria. Here, we review current advances and challenges in the development of GM approaches, including clustered regularly interspaced short palindromic repeats (CRISPR)-Cas and transposase-based systems in either model or non-model gut bacteria. By overcoming barriers to 'taming' the gut microbiome, GM tools allow molecular understanding of host-microbiome associations and accelerate microbiome engineering for clinical treatment of cancer and metabolic disorders. Finally, we provide perspectives on the future development of GM for gut microbiome species, where more effort should be placed on assembling a generalized GM pipeline to accelerate the application of groundbreaking GM tools in non-model gut bacteria towards both basic understanding and clinical translation.

Salmonella effector SopB reorganizes cytoskeletal vimentin to maintain replication vacuoles for efficient infection.

A variety of intracellular bacteria modulate the host cytoskeleton to establish subcellular niches for replication. However, the role of intermediate filaments, which are crucial for mechanical strength and resilience of the cell, and in bacterial vacuole preservation remains unclear. Here, we show that Salmonella effector SopB reorganizes the vimentin network to form cage-like structures that surround Salmonella-containing vacuoles (SCVs). Genetic removal of vimentin markedly disrupts SCV organization, significantly reduces bacterial replication and cell death. Mechanistically, SopB uses its N-terminal Cdc42-binding domain to interact with and activate Cdc42 GTPase, which in turn recruits vimentin around SCVs. A high-content imaging-based screening identified that MEK1/2 inhibition led to vimentin dispersion. Our work therefore elucidates the signaling axis SopB-Cdc42-MEK1/2 as mobilizing host vimentin to maintain concrete SCVs and identifies a mechanism contributing to Salmonella replication. Importantly, Trametinib, a clinically-approved MEK1/2 inhibitor identified in the screen, displayed significant anti-infection efficacy against Salmonella both in vitro and in vivo, and may provide a therapeutic option for treating drug-tolerant salmonellosis.

RNase III-CLASH brings bacterial RNA networks into focus.

Bacterial small RNAs have emerged as crucial regulators in complex networks controlling diverse phenotypes. Two groups, Mediati et al. and McKellar et al., have leveraged CLASH technology and the global regulatory potential of RNase III to build a rich landscape of RNA interactions in Staphylococcus aureus, revealing post-transcriptional control of virulence and new mechanistic themes for exploration.

Gene sdaB Is Involved in the Nematocidal Activity of Enterobacter ludwigii AA4 Against the Pine Wood Nematode Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus, a plant parasitic nematode, is the causal agent of pine wilt, a devastating forest tree disease. Essentially, no efficient methods for controlling B. xylophilus and pine wilt disease have yet been developed. Enterobacter ludwigii AA4, isolated from the root of maize, has powerful nematocidal activity against B. xylophilus in a new in vitro dye exclusion test. The corrected mortality of the B. xylophilus treated by E. ludwigii AA4 or its cell extract reached 98.3 and 98.6%, respectively. Morphological changes in B. xylophilus treated with a cell extract from strain AA4 suggested that the death of B. xylophilus might be caused by an increased number of vacuoles in non-apoptotic cell death and the damage to tissues of the nematodes. In a greenhouse test, the disease index of the seedlings of Scots pine (Pinus sylvestris) treated with the cells of strain AA4 plus B. xylophilus or those treated by AA4 cell extract plus B. xylophilus was 38.2 and 30.3, respectively, was significantly lower than 92.5 in the control plants treated with distilled water and B. xylophilus. We created a sdaB gene knockout in strain AA4 by deleting the gene that was putatively encoding the beta-subunit of L-serine dehydratase through Red homologous recombination. The nematocidal and disease-suppressing activities of the knockout strain were remarkably impaired. Finally, we revealed a robust colonization of P. sylvestris seedling needles by E. ludwigii AA4, which is supposed to contribute to the disease-controlling efficacy of strain AA4. Therefore, E. ludwigii AA4 has significant potential to serve as an agent for the biological control of pine wilt disease caused by B. xylophilus.

Impact of pseudouridylation, substrate fold, and degradosome organization on the endonuclease activity of RNase E.

The conserved endoribonuclease RNase E dominates the dynamic landscape of RNA metabolism and underpins control mediated by small regulatory RNAs in diverse bacterial species. We explored the enzyme's hydrolytic mechanism, allosteric activation, and interplay with partner proteins in the multicomponent RNA degradosome assembly of Escherichia coli. RNase E cleaves single-stranded RNA with preference to attack the phosphate located at the 5' nucleotide preceding uracil, and we corroborate key interactions that select that base. Unexpectedly, RNase E activity is impeded strongly when the recognized uracil is isomerized to 5-ribosyluracil (pseudouridine), from which we infer the detailed geometry of the hydrolytic attack process. Kinetics analyses support models for recognition of secondary structure in substrates by RNase E and for allosteric autoregulation. The catalytic power of the enzyme is boosted when it is assembled into the multienzyme RNA degradosome, most likely as a consequence of substrate capture and presentation. Our results rationalize the origins of substrate preferences of RNase E and illuminate its catalytic mechanism, supporting the roles of allosteric domain closure and cooperation with other components of the RNA degradosome complex.

The global emergence of a novel Streptococcus suis clade associated with human infections.

Streptococcus suis, a ubiquitous bacterial colonizer in pigs, has recently extended host range to humans, leading to a global surge of deadly human infections and three large outbreaks since 1998. To better understand the mechanisms for the emergence of cross-species transmission and virulence in human, we have sequenced 366 S. suis human and pig isolates from 2005 to 2016 and performed a large-scale phylogenomic analysis on 1,634 isolates from 14 countries over 36 years. We show the formation of a novel human-associated clade (HAC) diversified from swine S. suis isolates. Phylogeographic analysis identified Europe as the origin of HAC, coinciding with the exportation of European swine breeds between 1960s and 1970s. HAC is composed of three sub-lineages and contains several healthy-pig isolates that display high virulence in experimental infections, suggesting healthy-pig carriers as a potential source for human infection. New HAC-specific genes are identified as promising markers for pathogen detection and surveillance. Our discovery of a human-associated S. suis clade provides insights into the evolution of this emerging human pathogen and extend our understanding of S. suis epidemics worldwide.

The conserved 3' UTR-derived small RNA NarS mediates mRNA crossregulation during nitrate respiration.

Small noncoding RNAs (sRNAs) from mRNA 3' UTRs seem to present a previously unrecognized layer of bacterial post-transcriptional control whereby mRNAs influence each other's expression, independently of transcriptional control. Studies in Escherichia coli and Salmonella enterica showed that such sRNAs are natural products of RNase E-mediated mRNA decay and associate with major RNA-binding proteins (RBPs) such as Hfq and ProQ. If so, there must be additional sRNAs from mRNAs that accumulate only under specific physiological conditions. We test this prediction by characterizing candidate NarS that represents the 3' UTR of nitrate transporter NarK whose gene is silent during standard aerobic growth. We find that NarS acts by Hfq-dependent base pairing to repress the synthesis of the nitrite transporter, NirC, resulting in mRNA cross-regulation of nitrate and nitrite transporter genes. Interestingly, the NarS-mediated repression selectively targets the nirC cistron of the long nirBDC-cysG operon, an observation that we rationalize as a mechanism to protect the bacterial cytoplasm from excessive nitrite toxicity during anaerobic respiration with abundant nitrate. Our successful functional assignment of a 3' UTR sRNA from a non-standard growth condition supports the notion that mRNA crossregulation is more pervasive than currently appreciated.

Computational Analysis of RNA-Protein Interactions via Deep Sequencing.

RNA-binding proteins (RBPs) function in all aspects of RNA processes including stability, structure, export, localization and translation, and control gene expression at the posttranscriptional level. To investigate the roles of RBPs and their direct RNA ligands in vivo, recent global approaches combining RNA immunoprecipitation and deep sequencing (RIP-seq) as well as UV-cross-linking (CLIP-seq) have become instrumental in dissecting RNA-protein interactions. However, the computational analysis of these high-throughput sequencing data is still challenging. Here, we provide a computational pipeline to analyze CLIP-seq and RIP-seq datasets. This generic analytic procedure may help accelerate the identification of direct RNA-protein interactions from high-throughput RBP profiling experiments in a variety of bacterial species.

In Vivo Cleavage Map Illuminates the Central Role of RNase E in Coding and Non-coding RNA Pathways.

Understanding RNA processing and turnover requires knowledge of cleavages by major endoribonucleases within a living cell. We have employed TIER-seq (transiently inactivating an endoribonuclease followed by RNA-seq) to profile cleavage products of the essential endoribonuclease RNase E in Salmonella enterica. A dominating cleavage signature is the location of a uridine two nucleotides downstream in a single-stranded segment, which we rationalize structurally as a key recognition determinant that may favor RNase E catalysis. Our results suggest a prominent biogenesis pathway for bacterial regulatory small RNAs whereby RNase E acts together with the RNA chaperone Hfq to liberate stable 3' fragments from various precursor RNAs. Recapitulating this process in vitro, Hfq guides RNase E cleavage of a representative small-RNA precursor for interaction with a mRNA target. In vivo, the processing is required for target regulation. Our findings reveal a general maturation mechanism for a major class of post-transcriptional regulators.

Dual RNA-seq unveils noncoding RNA functions in host-pathogen interactions.

Bacteria express many small RNAs for which the regulatory roles in pathogenesis have remained poorly understood due to a paucity of robust phenotypes in standard virulence assays. Here we use a generic 'dual RNA-seq' approach to profile RNA expression simultaneously in pathogen and host during Salmonella enterica serovar Typhimurium infection and reveal the molecular impact of bacterial riboregulators. We identify a PhoP-activated small RNA, PinT, which upon bacterial internalization temporally controls the expression of both invasion-associated effectors and virulence genes required for intracellular survival. This riboregulatory activity causes pervasive changes in coding and noncoding transcripts of the host. Interspecies correlation analysis links PinT to host cell JAK-STAT signalling, and we identify infection-specific alterations in multiple long noncoding RNAs. Our study provides a paradigm for a sensitive RNA-based analysis of intracellular bacterial pathogens and their hosts without physical separation, as well as a new discovery route for hidden functions of pathogen genes.

A 3' UTR-Derived Small RNA Provides the Regulatory Noncoding Arm of the Inner Membrane Stress Response.

Small RNAs (sRNAs) from conserved noncoding genes are crucial regulators in bacterial signaling pathways but have remained elusive in the Cpx response to inner membrane stress. Here we report that an alternative biogenesis pathway releasing the conserved mRNA 3' UTR of stress chaperone CpxP as an ∼60-nt sRNA provides the noncoding arm of the Cpx response. This so-called CpxQ sRNA, generated by general mRNA decay through RNase E, acts as an Hfq-dependent repressor of multiple mRNAs encoding extracytoplasmic proteins. Both CpxQ and the Cpx pathway are required for cell survival under conditions of dissipation of membrane potential. Our discovery of CpxQ illustrates how the conversion of a transcribed 3' UTR into an sRNA doubles the output of a single mRNA to produce two factors with spatially segregated functions during inner membrane stress: a chaperone that targets problematic proteins in the periplasm and a regulatory RNA that dampens their synthesis in the cytosol.

Transcriptome-Wide Identification of Hfq-Associated RNAs in Brucella suis by Deep Sequencing.

UNLABELLED: Recent breakthroughs in next-generation sequencing technologies have led to the identification of small noncoding RNAs (sRNAs) as a new important class of regulatory molecules. In prokaryotes, sRNAs are often bound to the chaperone protein Hfq, which allows them to interact with their partner mRNA(s). We screened the genome of the zoonotic and human pathogen Brucella suis 1330 for the presence of this class of RNAs. We designed a coimmunoprecipitation strategy that relies on the use of Hfq as a bait to enrich the sample with sRNAs and eventually their target mRNAs. By deep sequencing analysis of the Hfq-bound transcripts, we identified a number of mRNAs and 33 sRNA candidates associated with Hfq. The expression of 10 sRNAs in the early stationary growth phase was experimentally confirmed by Northern blotting and/or reverse transcriptase PCR. IMPORTANCE: Brucella organisms are facultative intracellular pathogens that use stealth strategies to avoid host defenses. Adaptation to the host environment requires tight control of gene expression. Recently, small noncoding RNAs (sRNAs) and the sRNA chaperone Hfq have been shown to play a role in the fine-tuning of gene expression. Here we have used RNA sequencing to identify RNAs associated with the B. suis Hfq protein. We have identified a novel list of 33 sRNAs and 62 Hfq-associated mRNAs for future studies aiming to understand the intracellular lifestyle of this pathogen.

dRNA-Seq Reveals Genomewide TSSs and Noncoding RNAs of Plant Beneficial Rhizobacterium Bacillus amyloliquefaciens FZB42.

Bacillus amyloliquefaciens subsp. plantarum FZB42 is a representative of Gram-positive plant-growth-promoting rhizobacteria (PGPR) that inhabit plant root environments. In order to better understand the molecular mechanisms of bacteria-plant symbiosis, we have systematically analyzed the primary transcriptome of strain FZB42 grown under rhizosphere-mimicking conditions using differential RNA sequencing (dRNA-seq). Our analysis revealed 4,877 transcription start sites for protein-coding genes, identified genes differentially expressed under different growth conditions, and corrected many previously mis-annotated genes. We also identified a large number of riboswitches and cis-encoded antisense RNAs, as well as trans-encoded small noncoding RNAs that may play important roles in the gene regulation of Bacillus. Overall, our analyses provided a landscape of Bacillus primary transcriptome and improved the knowledge of rhizobacteria-host interactions.

Regulatory small RNAs from the 3' regions of bacterial mRNAs.

Most studies of small regulatory RNAs in bacteria have focussed on conserved transcripts in intergenic regions. However, several recent developments including single-nucleotide resolution transcriptome profiling by RNA-seq and increased knowledge of the cellular targets of the RNA chaperone Hfq suggest that the bacterial world of functional small RNAs is more diverse. One emerging class are small RNAs that are identical to the 3' regions of known mRNAs, but are produced either by transcription from internal promoters or by mRNA processing. Using several recently discovered examples of such sRNAs, we discuss their biogenesis and modes of action, and illustrate how they can facilitate mRNA crosstalk in various physiological processes.