Bacterial Domain Research Articles

Bacterial WYL domain transcriptional repressors sense single-stranded DNA to control gene expression.

Bacteria encode a wide array of immune systems to protect themselves against ubiquitous bacteriophages and foreign DNA elements. While these systems' molecular mechanisms are becoming increasingly well known, their regulation remains poorly understood. Here, we show that an immune system-associated transcriptional repressor of the wHTH-WYL-WCX family, CapW, directly binds single-stranded DNA to sense DNA damage and activate expression of its associated immune system. We show that CapW mediates increased expression of a reporter gene in response to DNA damage in a host cell. CapW directly binds single-stranded DNA by-products of DNA repair through its WYL domain, causing a conformational change that releases the protein from double-stranded DNA. In an Escherichiacoli CBASS system with an integrated capW gene, we find that CapW-mediated transcriptional activation is important for this system's ability to prevent induction of a λ prophage. Overall, our data reveal the molecular mechanisms of WYL-domain transcriptional repressors, and provide an example of how bacteria can balance the protective benefits of carrying anti-phage immune systems against the inherent risk of these systems' aberrant activation.

Proposal of Patescibacterium danicum gen. Nov., sp. nov. in the ubiquitous ultrasmall bacterial phylum Patescibacteriota phyl. Nov

Abstract Candidatus Patescibacteria is a diverse bacterial phylum that is notable for members with ultrasmall cell size, reduced genomes, limited metabolic capabilities and dependence on other prokaryotic hosts. Despite the prevalence of the name Ca. Patescibacteria in the scientific literature, it is not officially recognized under the International Code of Nomenclature of Prokaryotes (ICNP) and lacks a nomenclatural type. Here, we rectify this situation by describing two closely related circular metagenome-assembled genomes (MAGs) and by proposing one of them (ABY1TS) to serve as the nomenclatural type for the species Patescibacterium danicumTS gen. Nov., sp. nov. according to the rules of the SeqCode. Rank-normalized phylogenomic inference confirmed the stable placement of P. danicumTS in the class ABY1 within Ca. Patescibacteria. Based on these results, we propose Patescibacterium gen. Nov. to serve as the type genus for associated higher taxa, including the phylum Patescibacteriota phyl. Nov. We complement our proposal with a genomic characterization, metabolic reconstruction, and biogeographical analysis of Patescibacterium. Our results confirm small genome sizes (< 1Mbp), low GC content (>36%), and the occurrence of long gene coding insertions in the 23S rRNA sequences, along with reduced metabolic potential, inferred symbiotic lifestyle, and a global distribution. In summary, this effort, focused on the fourth-largest phylum in the bacterial domain, aims to provide nomenclatural stability in the scientific literature.

The ribosome-associated quality control pathway supports survival in the absence of non-stop ribosome rescue factors.

In bacteria, it is estimated that 2%-4% of all translation reactions terminate with the ribosome stalled on a damaged mRNA lacking a stop codon. Mechanisms that rescue these ribosomes are essential for viability. We determined the functional overlap between the ribosome quality control pathway and the classical non-stop rescue systems [alternative rescue factor (ArfA) and trans-translation] in a representative Firmicute and Proteobacterium, phyla that are evolutionarily distinct. Furthermore, we used a bioinformatics approach to examine the conservation and overlap of various ribosome rescue systems in >15,000 species throughout the bacterial domain. These results provide key insights into ribosome rescue in diverse phyla.

Activation of a helper NLR by plant and bacterial TIR immune signaling.

Plant intracellular nucleotide-binding leucine-rich repeat (NLR) receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain sense pathogen effectors to initiate immune signaling. TIR domains across different kingdoms possess NADase activities and can produce phosphoribosyl adenosine monophosphate/diphosphate (pRib-AMP/ADP) or cyclic ADPR (cADPR) isomers. Lipase-like proteins, EDS1 and PAD4, transduce immune signals from sensor TIR-NLRs to a helper NLR called ADR1, which executes immune function. We report the structure and function of Arabidopsis EDS1-PAD4-ADR1 (EPA) heterotrimer in complex with pRib-AMP/ADP activated by plant or bacterial TIR signaling. 2'cADPR can be hydrolyzed into pRib-AMP and thus activates EPA signaling. Bacterial TIR domains producing 2'cADPR also activate EPA function. Our findings suggest that 2'cADPR may be the storage form of the unstable signaling molecule pRib-AMP.

Stimulation of Batch Mesophilic Anaerobic Digestion by Cellulose- and Polysaccharide-Derived Polymers in Landfill Leachates

The fate of biobased and biodegradable cellulose-derived plastics in landfills represents an important topic from economic and environmental points of view. Anaerobic digestion is a cost-effective waste-to-energy technology. The behaviour of six polymer types—that is, cellulose (C), cellulose acetate (CA), viscose (V), nanocellulose (NC), acetate textile (AT), and heteropolysaccharide pectin (P)—was studied under anaerobic batch mesophilic conditions in a landfill leachate for 147 days. The cumulative biogas production was as follows: C>V=CA>>AT>>NC=P. Metagenomic analysis revealed notable variations in the proportion of bacterial and archaeal domains with the highest archaeal abundance in the presence of CA (80.2%) and C (78.5%). At the end of digestion, cellulolytic, hydrolytic, and dehydrogenase activities were measured in the intact samples, as well as the liquid and solid fractions, under aerobic and anaerobic conditions. Cellulolytic activity in P was detected only in the pellet, while in NC, activity was mostly in the supernatant under both aerobic and anaerobic conditions. Scanning electron microscopy and confocal scanning laser microscopy showed a defragmentation and degradation of polymeric substrates as well as microbial colonisation. Based on the results, landfill leachate is appropriate for the anaerobic biodegradation of cellulose-derived polymers; however, the process is polymer specific.

BEAN and HABAS: Polyphyletic insertions in the DNA-directed RNA polymerase.

The β and β' subunits of the RNA polymerase (RNAP) are large proteins with complex multi-domain architectures that include several insertional domains. Here, we analyze the domain organizations of RNAP-β and RNAP-β' using sequence, experimentally determined structures and AlphaFold structure predictions. We observe that lineage-specific insertional domains in bacterial RNAP-β belong to a group that we call BEAN (broadly embedded annex). We observe that lineage-specific insertional domains in bacterial RNAP-β' belong to a group that we call HABAS (hammerhead/barrel-sandwich hybrid). The BEAN domain has a characteristic three-dimensional structure composed of two square bracket-like elements that are antiparallel relative to each other. The HABAS domain contains a four-stranded open β-sheet with a GD-box-like motif in one of the β-strands and the adjoining loop. The BEAN domain is inserted not only in the bacterial RNAP-β', but also in the archaeal version of universal ribosomal protein L10. The HABAS domain is inserted in several metabolic proteins. The phylogenetic distributions of bacterial lineage-specific insertional domains of β and β' subunits of RNAP follow the Tree of Life. The presence of insertional domains can help establish a relative timeline of events in the evolution of a protein because insertion is inferred to post-date the base domain. We discuss mechanisms that might account for the discovery of homologous insertional domains in non-equivalent locations in bacteria and archaea.

Trans-aconitic acid assimilation system as a widespread bacterial mechanism for environmental adaptation.

The ability of bacteria to use natural carbon sources greatly affects their growth and survival in the environment. Bacteria have evolved versatile abilities to use environmental carbon sources, but their diversity and assimilation pathways remain largely unexplored. Trans-aconitic acid, a geometric isomer of cis-aconitic acid involved in the tricarboxylic acid cycle, has long been considered a natural carbon source metabolizable by bacteria. However, its catabolism and ecological role in linking bacterial interactions with the environments remain unclear. Here, we identify a regulatory system in Bacillus velezensis FZB42 that is capable of sensing and catabolizing trans-aconitic acid. The system consists of a tar operon, an adjacent positive regulatory gene tarR, and a shared promoter. After receiving the trans-aconitic acid signal, the TarR protein interacts directly with the promoter, initiating the expression of the membrane transporter TarB and aconitate isomerase TarA encoded by the operon, which function in importing the trans-aconitic acid and isomerizing it into the central intermediate cis-aconitic acid. Subsequent soil colonization experiments reveal that trans-aconitic acid assimilating ability can give its coding bacteria a growth and competitive advantage. Bioinformatics analyses coupled with bacterial isolation experiments further show that the assimilation system of trans-aconitic acid is widely distributed in the bacterial domain, and its assimilating bacteria also extensively distributed in nature, indicating an important role of trans-aconitic acid metabolism in bacterial carbon acquisition. This work emphasizes the importance of metabolic adaptation to environmental carbon sources for bacterial survival and may provide inspiration for engineering microbes with enhanced environmental competitiveness.

NMR assignment of the conserved bacterial DNA replication protein DnaA domain IV

Chromosomal replication is a ubiquitous and essential cellular process. In bacteria, the master replication initiator DnaA plays a key role in promoting an open complex at the origin (oriC) and recruiting helicase in a tightly regulated process. The C-terminal domain IV specifically recognises consensus sequences of double-stranded DNA in oriC, termed DnaA-boxes, thereby facilitating the initial engagement of DnaA to oriC. Here, we report the 13Cβ and backbone 1H, 15N, and 13C chemical shift assignments of soluble DnaA domain IV from Bacillus subtilis at pH 7.6 and 298 K.

The evolution and functional significance of the programmed ribosomal frameshift in prfB.

Release Factor 2 (RF2) is one of two peptide release factors that terminate translation in bacteria. In Escherichia coli, the gene encoding RF2, prfB, contains an in-frame premature RF2-specific stop codon. Therefore, a programmed ribosomal frameshift is required to translate full-length RF2. Here, we investigate the diversity of prfB frameshifting through bioinformatic analyses of >12,000 genomes. We present evidence that prfB frameshifting autoregulates RF2 levels throughout the bacterial domain since (i) the prfB in-frame stop codon is always TGA or TAA, both of which are recognized by RF2, and never the RF1-specific TAG stop codon, and (ii) species that lack the autoregulatory programmed frameshift likely need higher RF2 levels since, on average, they have significantly higher RF2-specific stop codon usage. Overexpression of prfB without the autoregulatory frameshift motif is toxic to Bacillus subtilis, an organism with intermediate RF2-specific stop codon usage. We did not detect the programmed frameshift in any Actinobacteriota. Consistent with this finding, we observed very low frameshift efficiency at the prfB frameshift motif in the Actinobacterium Mycobacterium smegmatis. Our work provides a more complete picture of the evolution of the RF2 programmed frameshifting motif, and its usage to prevent toxic overexpression of RF2.

Cell envelope diversity and evolution across the bacterial tree of life.

The bacterial cell envelope is a complex multilayered structure conserved across all bacterial phyla. It is categorized into two main types based on the number of membranes surrounding the cell. Monoderm bacteria are enclosed by a single membrane, whereas diderm cells are distinguished by the presence of a second, outer membrane (OM). An ancient divide in the bacterial domain has resulted in two major clades: the Gracilicutes, consisting strictly of diderm phyla; and the Terrabacteria, encompassing monoderm and diderm species with diverse cell envelope architectures. Recent structural and phylogenetic advancements have improved our understanding of the diversity and evolution of the OM across the bacterial tree of life. Here we discuss cell envelope variability within major bacterial phyla and focus on conserved features found in diderm lineages. Characterizing the mechanisms of OM biogenesis and the evolutionary gains and losses of the OM provides insights into the primordial cell and the last universal common ancestor from which all living organisms subsequently evolved.

Protein disulfide isomerase A4 binds to Brucella BtpB and mediates intracellular NAD+/NADH metabolism in RAW264.7 cells

The Toll/interleukin-1 receptor (TIR) signaling domain is distributed widely in mammalian Toll-like receptors and adaptors, plant nucleotide-binding leucine-rich repeat receptors, and specific bacterial virulence proteins. Proteins that possess TIR domain exhibit NADase activity which is distinct from the canonical signaling function of these domains. However, the effects of bacterial TIR domain proteins on host metabolic switches and the underlying mechanism of NADase activity in these proteins remain unclear. Here, we utilized Brucella TIR domain-containing type IV secretion system effector protein, BtpB, to explore the mechanism of NADase activity in host cells. We showed that using ectopic expression BtpB not only generates depletion of NAD+ but also loss of NADH and ATP in RAW264.7 macrophage cells. Moreover, immunoprecipitation-mass spectrometry, co-immunoprecipitation, and confocal microscope assays revealed that BtpB interacted with host protein disulfide isomerase A4 (PDIA4). The Brucella mutant strain deleted the gene for BtpB, significantly decreased PDIA4 expression. Furthermore, our data revealed that PDIA4 played an important role in regulating intracellular NAD+/NADH levels in macrophages, and PDIA4 overexpression restored the decline of intracellular NAD+ and NADH levels induced by Brucella BtpB. The results provide new insights into the metabolic regulatory activity of TIR domain proteins in the critical human and animal pathogen Brucella.

Analysis of changes in intestinal microbiota in female Wistar rats at the stage of breast cancer induction by N-methyl-N-nitrosourea

The intestinal microbiota, having enormous metabolic potential, makes a significant contribution to the physiological and pathological processes of humans and animals and is currently considered as an important factor in the pathogenesis of cancer. The aim of this study is to determine changes in the quantitative and qualitative composition of the intestinal microbiota in Wistar rats during chemical induction of breast cancer (BC). Material and methods. The work was performed on female Wistar rats (n = 40) aged 3 months, weighing 200–210 g, using cultural methods for studying fecal microbiota in intact rats (1 group) on the 1st, 14th, 35th days and in rats with induction of breast cancer and and in rats, whereby N-methyl-N-nitrosourea was administered to induce breast cancer (2 group) on the 1st (before injection of N-methyl-N-nitrosourea), 14th, 35th days after injection of N-methyl-N-nitrosourea. Results and discussion. In all experimental animals, representatives characteristic of the intestinal normobiota of warm-blooded animals predominated, namely: Bifidobacterium spp., Lactobacillus spp., Escherichia coli with pronounced enzymatic properties, Enterococcus spp., Clostridium spp. In addition, Staphylococcus spp., yeast-like fungi of the genus Candida and mold. Escherichia coli with reduced enzymatic activity was also detected. It was established that the isolated bacteria belonged to 3 types, 4 classes, 5 orders, 6 families, 6 genera of the bacterial domain. Also, 2 genera of fungi belonging to the order Saccharomycetales were isolated. The most significant changes in the composition of the intestinal microbiota were noted in rats with chemically induced breast cancer on the 35th day tumor induction: the appearance of pathogenic microflora in the intestine was revealed.

Lipid discovery enabled by sequence statistics and machine learning.

Bacterial membranes are complex and dynamic, arising from an array of evolutionary pressures. One enzyme that alters membrane compositions through covalent lipid modification is MprF. We recently identified that Streptococcus agalactiae MprF synthesizes lysyl-phosphatidylglycerol (Lys-PG) from anionic PG, and a novel cationic lipid, lysyl-glucosyl-diacylglycerol (Lys-Glc-DAG), from neutral glycolipid Glc-DAG. This unexpected result prompted us to investigate whether Lys-Glc-DAG occurs in other MprF-containing bacteria, and whether other novel MprF products exist. Here, we studied protein sequence features determining MprF substrate specificity. First, pairwise analyses identified several streptococ-cal MprFs synthesizing Lys-Glc-DAG. Second, a restricted Boltzmann machine-guided approach led us to discover an entirely new substrate for MprF in Enterococcus , diglucosyl-diacylglycerol (Glc 2 -DAG), and an expanded set of organisms that modify glycolipid substrates using MprF. Overall, we combined the wealth of available sequence data with machine learning to model evolutionary constraints on MprF sequences across the bacterial domain, thereby identifying a novel cationic lipid.

In silico and experimental characterization of a new polyextremophilic subtilisin-like protease from Microbacterium metallidurans and its application as a laundry detergent additive.

Considering the current growing interest in new and improved enzymes for use in a variety of applications, the present study aimed to characterize a novel detergent-stable serine alkaline protease from the extremophilic actinobacterium Microbacterium metallidurans TL13 (MmSP) using a combined in silico and experimental approach. The MmSP showed a close phylogenetic relationship with high molecular weight S8 peptidases of Microbacterium species. Moreover, its physical and chemical parameters computed using Expasy's ProtParam tool revealed that MmSP is hydrophilic, halophilic and thermo-alkali stable. 3D structure modelling and functional prediction of TL13 serine protease resulted in the detection of five characteristic domains: [catalytic subtilase domain, fibronectin (Fn) type-III domain, peptidase inhibitor I9, protease-associated (PA) domain and bacterial Ig-like domain (group 3)], as well as the three amino acid residues [aspartate (D182), histidine (H272) and serine (S604)] in the catalytic subtilase domain. The extremophilic strain TL13 was tested for protease production using agricultural wastes/by-products as carbon substrates. Maximum enzyme activity (390U/gds) was obtained at 8th day fermentation on potato peel medium. Extracellular extract was concentrated and partially purified using ammonium sulfate precipitation methodology (1.58 folds purification fold). The optimal pH, temperature and salinity of MmSP were 9, 60°C and 1M NaCl, respectively. The MmSP protease showed broad pH stability, thermal stability, salt tolerance and detergent compatibility. In order to achieve the maximum stain removal efficacy by the TL 13 serine protease, the operation conditions were optimized using a Box-Behnken Design (BBD) with four variables, namely, time (15-75min), temperature (30-60°C), MmSP enzyme concentration (5-10U/mL) and pH (7-11). The maximum stain removal yield (95 ± 4%) obtained under the optimal enzymatic operation conditions (treatment with 7.5U/mL of MmSP during 30min at 32°C and pH9) was in good agreement with the value predicted by the regression model (98 ± %), which prove the validity of the fitted model. In conclusion, MmSP appears to be a good candidate for industrial applications, particularly in laundry detergent formulations, due to its high hydrophilicity, alkali-halo-stability, detergent compatibility and stain removal efficiency.

Bacterial and Archaeal Communities in Erhai Lake Sediments: Abundance and Metabolic Insight into a Plateau Lake at the Edge of Eutrophication.

The littoral zones of lakes are potential hotspots for local algal blooms and biogeochemical cycles; however, the microbial communities within the littoral sediments of eutrophic plateau lakes remain poorly understood. Here, we investigated the taxonomic composition, co-occurrence networks, and potential functional roles of both abundant and rare taxa within bacterial and archaeal communities, as well as physicochemical parameters, in littoral sediments from Erhai Lake, a mesotrophic lake transitioning towards eutrophy located in the Yunnan-Guizhou Plateau. 16S rRNA gene sequencing revealed that bacterial communities were dominated by Proteobacteria, Bacteroidetes, and Chloroflexi, while Euryarchaeota was the main archaeal phylum. Co-occurrence network analysis revealed that keystone taxa mainly belonged to rare species in the bacterial domain, but in the archaeal domain, over half of keystone taxa were abundant species, demonstrating their fundamental roles in network persistence. The rare bacterial taxa contributed substantially to the overall abundance (81.52%), whereas a smaller subset of abundant archaeal taxa accounted for up to 82.70% of the overall abundance. Functional predictions highlighted a divergence in metabolic potentials, with abundant bacterial sub-communities enriched in pathways for nitrogen cycling, sulfur cycling, and chlorate reduction, while rare bacterial sub-communities were linked to carbon cycling processes such as methanotrophy. Abundant archaeal sub-communities exhibited a high potential for methanogenesis, chemoheterotrophy, and dark hydrogen oxidation. Spearman correlation analysis showed that genera such as Candidatus competibacter, Geobacter, Syntrophobacter, Methanocella, and Methanosarcina may serve as potential indicators of eutrophication. Overall, this study provides insight into the distinct roles that rare and abundant taxa play in the littoral sediments of mesotrophic plateau lakes.

Characterization of the Structural Requirements for the NADase Activity of Bacterial Toll/IL-1R domains in a Course-based Undergraduate Research Experience.

TLRs initiate innate immune signaling pathways via Toll/IL-1R (TIR) domains on their cytoplasmic tails. Various bacterial species also express TIR domain-containing proteins that contribute to bacterial evasion of the innate immune system. Bacterial TIR domains, along with the mammalian sterile α and TIR motif-containing protein 1 and TIRs from plants, also have been found to exhibit NADase activity. Initial X-ray crystallographic studies of the bacterial TIR from Acinetobacter baumannii provided insight into bacterial TIR structure but were unsuccessful in cocrystallization with the NAD+ ligand, leading to further questions about the TIR NAD binding site. In this study, we designed a Course-Based Undergraduate Research Experience (CURE) involving 16-20 students per year to identify amino acids crucial for NADase activity of A. baumannii TIR domain protein and the TIR from Escherichia coli (TIR domain-containing protein C). Students used structural data to identify amino acids that they hypothesized would play a role in TIR NADase activity, and created plasmids to express mutated TIRs through site-directed mutagenesis. Mutant TIRs were expressed, purified, and tested for NADase activity. The results from these studies provide evidence for a conformational change upon NAD binding, as was predicted by recent cryogenic electron microscopy and hydrogen-deuterium exchange mass spectrometry studies. Along with corroborating recent characterization of TIR NADases that could contribute to drug development for diseases associated with dysregulated TIR activity, this work also highlights the value of CURE-based projects for inclusion of a diverse group of students in authentic research experiences.

Microbial carbohydrate active enzyme (CAZyme) genes and diversity from Menagesha Suba natural forest soils of Ethiopia as revealed by shotgun metagenomic sequencing

BackgroundThe global over-reliance on non-renewable fossil fuels has led to the emission of greenhouse gases, creating a critical global environmental challenge. There is an urgent need for alternative solutions like biofuels. Advanced biofuel is a renewable sustainable energy generated from lignocellulosic plant materials, which can significantly contribute to mitigating CO2 emissions. Microbial Carbohydrate Active Enzymes (CAZymes) are the most crucial enzymes for the generation of sustainable biofuel energy. The present study designed shotgun metagenomics approaches to assemble, predict, and annotate, aiming to gain an insight into the taxonomic diversity, annotate CAZymes, and identify carbohydrate hydrolyzing CAZymes from microbiomes in Menagesha suba forest soil for the first time.ResultsThe microbial diversity based on small subunit (SSU) rRNA analysis revealed the dominance of the bacterial domain representing 81.82% and 92.31% in the studied samples. Furthermore, the phylum composition result indicated the dominance of the phyla Proteobacteria (23.08%, 27.27%), Actinobacteria (11.36%, 20.51%), and Acidobacteria (10.26%, 15.91%). The study also identified unassigned bacteria which might have a unique potential for biopolymer hydrolysis. The metagenomic study revealed that 100,244 and 65,356 genes were predicted from the two distinct samples. A total number of 1806 CAZyme genes were identified, among annotated CAZymes, 758 had a known enzyme assigned to CAZymes. Glycoside hydrolases (GHs) CAZyme family contained most of the CAZyme genes with known enzymes such as β-glucosidase, endo-β-1,4-mannanase, exo-β-1,4-glucanase, α-L-arabinofuranosidase and oligoxyloglucan reducing end-specific cellobiohydrolase. On the other hand, 1048 of the identified CAZyme genes were putative CAZyme genes with unknown enzymatical activity and the majority of which belong to the GHs family.ConclusionsIn general, the identified putative CAZymes genes open up an opportunity for the discovery of new enzymes responsible for hydrolyzing biopolymers utilized for biofuel energy generation. This finding is used as a first-hand piece of evidence to serve as a benchmark for further and comprehensive studies to unveil novel classes of bio-economically valuable genes and their encoded products.

Targeting plasmid-encoded proteins that contain immunoglobulin-like domains to combat antimicrobial resistance

Antimicrobial resistance (AMR) poses a significant threat to human health. Although vaccines have been developed to combat AMR, it has proven challenging to associate specific vaccine antigens with AMR. Bacterial plasmids play a crucial role in the transmission of AMR. Our recent research has identified a group of bacterial plasmids (specifically, IncHI plasmids) that encode large molecular mass proteins containing bacterial immunoglobulin-like domains. These proteins are found on the external surface of the bacterial cells, such as in the flagella or conjugative pili. In this study, we show that these proteins are antigenic and can protect mice from infection caused by an AMR Salmonella strain harboring one of these plasmids. Furthermore, we successfully generated nanobodies targeting these proteins, that were shown to interfere with the conjugative transfer of IncHI plasmids. Considering that these proteins are also encoded in other groups of plasmids, such as IncA/C and IncP2, targeting them could be a valuable strategy in combating AMR infections caused by bacteria harboring different groups of AMR plasmids. Since the selected antigens are directly linked to AMR itself, the protective effect extends beyond specific microorganisms to include all those carrying the corresponding resistance plasmids.

Targeting plasmid-encoded proteins that contain immunoglobulin-like domains to combat antimicrobial resistance.

Antimicrobial resistance (AMR) poses a significant threat to human health. Although vaccines have been developed to combat AMR, it has proven challenging to associate specific vaccine antigens with AMR. Bacterial plasmids play a crucial role in the transmission of AMR. Our recent research has identified a group of bacterial plasmids (specifically, IncHI plasmids) that encode large molecular mass proteins containing bacterial immunoglobulin-like domains. These proteins are found on the external surface of the bacterial cells, such as in the flagella or conjugative pili. In this study, we show that these proteins are antigenic and can protect mice from infection caused by an AMR Salmonella strain harboring one of these plasmids. Furthermore, we successfully generated nanobodies targeting these proteins, that were shown to interfere with the conjugative transfer of IncHI plasmids. Considering that these proteins are also encoded in other groups of plasmids, such as IncA/C and IncP2, targeting them could be a valuable strategy in combating AMR infections caused by bacteria harboring different groups of AMR plasmids. Since the selected antigens are directly linked to AMR itself, the protective effect extends beyond specific microorganisms to include all those carrying the corresponding resistance plasmids.

RqcH supports survival in the absence of non-stop ribosome rescue factors.

Ribosomes frequently translate truncated or damaged mRNAs due to the extremely short half-life of mRNAs in bacteria. When ribosomes translate mRNA that lacks a stop codon (non-stop mRNA), specialized pathways are required to rescue the ribosome from the 3' end of the mRNA. The most highly conserved non-stop rescue pathway is trans-translation, which is found in greater than 95% of bacterial genomes. In all Proteobacteria that have been studied, the alternative non-stop ribosome rescue factors, ArfA and ArfB, are essential in the absence of trans-translation. Here, we investigate the interaction between non-stop rescue pathways and RqcH, a ribosome quality control factor that is broadly conserved outside of Proteobacteria. RqcH does not act directly on non-stop ribosomes but adds a degron tag to stalled peptides that obstruct the large ribosomal subunit, which allows the stalled peptide to be cleared from the ribosome by peptidyl-tRNA hydrolase (PTH). We show that Bacillus subtilis can survive without trans-translation and BrfA (Bacillus ArfA homolog), due to the presence of RqcH. We also show that expression of RqcH and its helper protein RqcP rescues the synthetic lethality of ΔssrAΔarfA in Escherichia coli. These results suggest that non-stop ribosome complexes can be disassembled and then cleared because of the tagging activity of RqcH, and that this process is essential in the absence of non-stop ribosome rescue pathways. Moreover, we surveyed the conservation of ribosome rescue pathways in >14,000 bacterial genomes. Our analysis reveals a broad distribution of non-stop rescue pathways, especially trans-translation and RqcH, and a strong co-occurrence between the ribosome splitting factor MutS2 and RqcH. Altogether, our results support a role for RqcH in non-stop ribosome rescue and provide a broad survey of ribosome rescue pathways in diverse bacterial species.