Methyl-accepting Chemotaxis Proteins Research Articles

Chemoreceptors in Sinorhizobium meliloti require minimal pentapeptide tethers to provide adaptational assistance.

Sensory adaptation in bacterial chemotaxis is mediated by posttranslational modifications of methyl-accepting chemotaxis proteins (MCPs). In Escherichia coli, the adaptation proteins CheR and CheB tether to a conserved C-terminal receptor pentapeptide. Here,we investigated the function of the pentapeptide motif (N/D)WE(E/N)F in Sinorhizobium meliloti chemotaxis. Isothermal titration calorimetry revealed stronger affinity of the pentapeptides to CheR and activated CheB relative to unmodified CheB. Strains with mutations of the conserved tryptophan in one or all four MCP pentapeptides resulted in a significant decrease or loss of chemotaxis to glycine betaine, lysine, and acetate, chemoattractants sensed by pentapeptide-bearing McpX and pentapeptide-lacking McpU and McpV, respectively. Importantly, we discovered that the pentapeptide mediates chemotaxis when fused to the C-terminus of pentapeptide-lacking chemoreceptors via a flexible linker. We propose that adaptational assistance and a threshold number of available sites enable the efficient docking of adaptation proteins to the chemosensory array. Altogether, these results demonstrate that S. meliloti effectively utilizes a pentapeptide-dependent adaptation system with a minimal number of tethering units to assist pentapeptide-lacking chemoreceptors and hypothesize that the higher abundance of CheR and CheB in S. meliloti compared to E. coli allows for ample recruitment of adaptation proteins to the chemosensory array.

Nematicidal activity and action mode of a methyl-accepting chemotaxis protein from Pseudomonas syringae against Caenorhabditis elegans

The conventional phytopathogen Pseudomonas syringae reportedly possesses several virulence determinants against Caenorhabditis elegans; however, their action mechanisms remain elusive. This study reports the nematicidal activity and action receptor of a methyl-accepting chemotaxis protein (MCP03) of a wild-type P. syringae MB03 against C. elegans. Purified MCP03 exhibited nematicidal toxicity against C. elegans at a half-lethal concentration of 124.4 μg mL−1, alongside detrimental effects on the growth and brood size of C. elegans. Additionally, MCP03-treated worms exhibited severe pathological destruction of the intestine and depressed wrinkles of the cuticle. Yeast two-hybrid assays identified a subunit of COP9 signalosome, namely CSN-5, which functioned as an MCP03 action receptor. In vitro pull-down verified the binding interaction between MCP03 and CSN-5. RNA interference assays confirmed that MCP03 antagonizes CSN-5, thereby adversely affecting the brood size and cuticle integrity of C. elegans. Following MCP03 infection, the expression of genes related to reproduction, growth, and cuticle formation, such as kgb-1, unc-98, and col-117, was considerably downregulated, indicating pathological changes in MCP03-treated nematodes. Therefore, we proposed that MCP03 antagonizes CSN-5, causing lethality as well as detrimental effects on the fertility, growth, and morphogenesis of C. elegans, which can provide new insights into the signaling pathways and mechanisms underlying the nematicidal action of MCP03 toward C. elegans.

Chemotaxis-mediated degradation of PAHs and heterocyclic PAHs under low-temperature stress by Pseudomonas fluorescens S01: Insights into the mechanisms of biodegradation and cold adaptation

As wellknown persistent contaminants, polycyclic aromatic hydrocarbons (PAHs) and heterocyclic polyaromatic hydrocarbons (Heterocyclic PAHs)'s fates in cryogenic environments are remains uncertain. Herein, strain S01 was identified as Pseudomonas fluorescens, a novel bacterium tolerant to low temperature and capable of degrading PAHs and heterocyclic PAHs. Strain S01 exhibited growth at 5–40 ℃ and degradation rate of mixed PAHs and heterocyclic PAHs reached 52% under low-temperature. Through comprehensive metabolomic, genomic, and transcriptomic analyses, we reconstructed the biodegradation pathway for PAHs and heterocyclic PAHs in S01 while investigating its response to low temperature. Further experiments involving deletion and replacement of methyl-accepting chemotaxis protein (MCP) confirmed its crucial role in enabling strain S01's adaptation to dual stress of low temperature and pollutants. Additionally, our analysis revealed that MCP was upregulated under cold stress which enhanced strain S01's motility capabilities leading to increased biofilm formation. The establishment of biofilm promoted preservation of distinct cellular membrane stability, thereby enhancing energy metabolism. Consequently, this led to heightened efficiency in pollutant degradation and improved cold resistance capabilities. Our findings provide a comprehensive understanding of the environmental fate of both PAHs and heterocyclic PAHs under low-temperature conditions while also shedding light on cold adaptation mechanism employed by strain S01.

Structural and bioinformatics analysis of single-domain substrate-binding protein from Rhodothermus marinus

Substrate-binding proteins (SBPs) are key elements in determining the substrate specificity and high affinity of the ATP-binding cassette uptake system. A typical SBP has two domains that recognize substrates and are responsible for the specific substrate delivery. Conversely, in GenBank, genes for SBPs constituting a single domain SBP are often found in vicinity of a methyl-accepting chemotaxis protein gene. However, the molecular function and mechanism of single domain SBPs are not fully elucidated. To understand their molecular functions, we performed a crystallographic study of single domain SBP from Rhodothermus marinus (RmSBP). RmSBP crystals were soaked in solution containing NaBr or HgCl2 and their structures determined at 1.75 and 2.3 Å resolution, respectively. RmSBP soaked in NaBr exhibited disorder of the α2-helix, β5-to β6-strand loop, and C-terminus region, showing the structural dynamic region of RmSBP. RmSBP soaked in HgCl2 showed that Hg2+ bound to Cys145 located between the α5-and α6-helices. The structural properties of RmSBP were compared with those of single domain SBP homologs. These results will contribute to continued identification of the molecular function and mechanism of single domain SBPs.

Effects of uranium mining on soil bacterial communities and functions in the Qinghai-Tibet plateau

The microecological effects of plateau uranium mining are still unknown. In this study, we used 16S rRNA high-throughput sequencing to analyze the impact of plateau uranium mining on the microbial diversity and community structure of tailings soil, tunnel soil, and soil at different depths in an open pit. The results showed that uranium mining significantly reduced soil microbial community richness and diversity indicators, including Chao1, Pielou evenness, and Shannon index (P < 0.05). Uranium mining activities significantly reduced the abundance of RB41, Vicinamidactaceae, and Nitrospira (P < 0.05). Interestingly, the abundance of Thiobacillus, Sphingomonas, and Sulfuriferula significantly increased in the soil samples from various environments and depths during uranium mining (P < 0.05). Beta diversity analysis found that uranium mining resulted in the differentiation of soil microbial communities. Functional enrichment analysis found that uranium mining resulted in the functional enrichment of DNA binding response regulator, DNA helicase, methyl-accepting chemotaxis protein, and Helicase conserved C-terminal domain, whereas cell wall synthesis, nonspecific serine/threonine protein kinase, RNA polymerase sigma-70 factor, and ATP binding cassette transporter were significantly affected by uranium mining (P < 0.05). In addition, we also found that different uranium mining environments and soil depths enriched diverse microbial populations and functions to cope with the environmental pressures that were elicited by uranium mining, including Gaiella, Gemmatimonas, Lysobacter, Pseudomonas, signal transformation histidine kinase, DNA-directed DNA polymerase, and iron complex outer membrane receptor protein functions (P < 0.05). The results have enhanced our understanding of the impact of uranium mining on plateau soil microecological stability and the mechanism of microbial response to uranium mining activities for the first time and aided us in screening microbial strains that can promote the environmental remediation of uranium mining in plateaus.

Metabolism-linked methylotaxis sensors responsible for plant colonization in Methylobacterium aquaticum strain 22A.

Motile bacteria take a competitive advantage in colonization of plant surfaces to establish beneficial associations that eventually support plant health. Plant exudates serve not only as primary growth substrates for bacteria but also as bacterial chemotaxis attractants. A number of plant-derived compounds and corresponding chemotaxis sensors have been documented, however, the sensors for methanol, one of the major volatile compounds released by plants, have not been identified. Methylobacterium species are ubiquitous plant surface-symbiotic, methylotrophic bacteria. A plant-growth promoting bacterium, M. aquaticum strain 22A exhibits chemotaxis toward methanol (methylotaxis). Its genome encodes 52 methyl-accepting chemotaxis proteins (MCPs), among which we identified three MCPs (methylotaxis proteins, MtpA, MtpB, and MtpC) responsible for methylotaxis. The triple gene mutant of the MCPs exhibited no methylotaxis, slower gathering to plant tissues, and less efficient colonization on plants than the wild type, suggesting that the methylotaxis mediates initiation of plant-Methylobacterium symbiosis and engages in proliferation on plants. To examine how these MCPs are operating methylotaxis, we generated multiple gene knockouts of the MCPs, and Ca2+-dependent MxaFI and lanthanide (Ln3+)-dependent XoxF methanol dehydrogenases (MDHs), whose expression is regulated by the presence of Ln3+. MtpA was found to be a cytosolic sensor that conducts formaldehyde taxis (formtaxis), as well as methylotaxis when MDHs generate formaldehyde. MtpB contained a dCache domain and exhibited differential cellular localization in response to La3+. MtpB expression was induced by La3+, and its activity required XoxF1. MtpC exhibited typical cell pole localization, required MxaFI activity, and was regulated under MxbDM that is also required for MxaF expression. Strain 22A methylotaxis is realized by three independent MCPs, two of which monitor methanol oxidation by Ln3+-regulated MDHs, and one of which monitors the common methanol oxidation product, formaldehyde. We propose that methanol metabolism-linked chemotaxis is the key factor for the efficient colonization of Methylobacterium on plants.

Gut Dysbiosis: A New Avenue for Stroke Prevention and Therapeutics.

A stroke is a serious life-threatening condition and a leading cause of death and disability that happens when the blood vessels to part of the brain are blocked or burst. While major advances in the understanding of the ischemic cascade in stroke was made over several decades, limited therapeutic options and high mortality and disability have caused researchers to extend the focus toward peripheral changes beyond brain. The largest proportion of microbes in human body reside in the gut and the interaction between host and microbiota in health and disease is well known. Our study aimed to explore the gut microbiota in patients with stroke with comparison to control group. Fecal samples were obtained from 51 subjects: 25 stroke patients (18 hemorrhagic, 7 ischemic) and 26 healthy control subjects. The variable region V3-V4 of the 16S rRNA gene was sequenced using the Illumina MiSeq platform. PICRUSt2 was used for prediction of metagenomics functions. Our results show taxonomic dysbiosis in stroke patients in parallel with functional dysbiosis. Here, we show that stroke patients have (1) increased Parabacteroides and Escherichia_Shigella, but decreased Prevotella and Fecalibacterium; (2) higher transposase and peptide/nickel transport system substrate-binding protein, but lower RNA polymerase sigma-70 factor and methyl-accepting chemotaxis protein, which are suggestive of malnutrition. Nutrients are essential regulators of both host and microbial physiology and function as key coordinators of host-microbe interactions. Manipulation of nutrition is expected to alleviate gut dysbiosis and prognosis and improve disability and mortality in the management of stroke.

The structural analysis of the periplasmic domain of Sinorhizobium meliloti chemoreceptor McpZ reveals a novel fold and suggests a complex mechanism of transmembrane signaling.

Chemotaxis is a fundamental process whereby bacteria seek out nutrient sources and avoid harmful chemicals. For the symbiotic soil bacterium Sinorhizobium meliloti, the chemotaxis system also plays an essential role in the interaction with its legume host. The chemotactic signaling cascade is initiated through interactions of an attractant or repellent compound with chemoreceptors or methyl-accepting chemotaxis proteins (MCPs). S. meliloti possesses eight chemoreceptors to mediate chemotaxis. Six of these receptors are transmembrane proteins with periplasmic ligand-binding domains (LBDs). The specific functions of McpW and McpZ are still unknown. Here, we report the crystal structure of the periplasmic domain of McpZ (McpZPD) at 2.7 Å resolution. McpZPD assumes a novel fold consisting of three concatenated four-helix bundle modules. Through phylogenetic analyses, we discovered that this helical tri-modular domain fold arose within the Rhizobiaceae family and is still evolving rapidly. The structure, offering a rare view of a ligand-free dimeric MCP-LBD, reveals a novel dimerization interface. Molecular dynamics calculations suggest ligand binding will induce conformational changes that result in large horizontal helix movements within the membrane-proximal domains of the McpZPD dimer that are accompanied by a 5 Å vertical shift of the terminal helix toward the inner cell membrane. These results suggest a mechanism of transmembrane signaling for this family of MCPs that entails both piston-type and scissoring movements. The predicted movements terminate in a conformation that closely mirrors those observed in related ligand-bound MCP-LBDs.

Expanded presentation of Lyme autoantigens and identification of a novel immunogenic CD4+ T cell epitope from Borrelia burgdorferiMCP4 in murine Lyme arthritis

Abstract Lyme arthritis (LA), caused by Borrelia burgdorferi (Bb), is often accompanied by autoimmune T and B cell responses, but the mechanisms of infection-induced autoimmunity are unclear. We used an immunopeptidomics approach to identify potential Lyme autoantigens and immunogenic Bb antigens, which were validated by histology and testing of T cell reactivity. C57BL/6 (B6) mice, which develop mild inflammatory LA, and B6 Il10−/− (IL10 KO) mice, which develop severe, persistent LA, were infected with Bb for 4 or 16 weeks, and MHCII-bound peptides from inguinal and popliteal lymph nodes were identified by LC-MS/MS. Joint inflammation, fibrosis, and vascular remodeling were analyzed by H&amp;E, Masson’s trichrome, and anti-CD31 immunohistochemistry, respectively. Six Bb peptides were identified by LC-MS/MS, of which one epitope from methyl-accepting chemotaxis protein 4 (MCP4) was an immunogenic CD4+ T cell antigen. Over 10,000 self peptides, particularly from proteins involved in cholesterol metabolism, tissue damage, and vascular inflammation were identified in infected mice. Presentation of peptides from previously identified human Lyme autoantigens apolipoprotein B-100, fibronectin, and type V collagen were expanded in infected mice, suggestive of epitope spreading. Consistent with immunopeptidomics data, joints showed increased inflammatory infiltrate, fibrosis and neovascularization in infected mice, compared with joints from uninfected mice. In conclusion, this immunopeptidomics approach revealed key insights into potential mechanisms of infection-induced autoimmunity in LA and indentified a novel immunogenic CD4+ T cell antigen from Bb MCP4. Supported by grants from NIAID (R21AI148982)

Localization and pathogenic role of the cysteine protease dentipain in Treponema denticola.

The Msp protein complex and the serine protease dentilisin are the best-characterized virulence factors in Treponema denticola, the major etiological agent of chronic periodontitis. In addition to these outer sheath factors, the cysteine protease dentipain contributes to pathogenicity, but its secretion, processing, cellular localization, and role in T. denticola virulence are not fully understood. In this study, we found that full-sized dentipain (74-kDa) and the 52-kDa truncated form of the enzyme are located, respectively, in the outer sheath derived from T. denticola dentilisin- and the Msp-deficient mutants. Furthermore, dentipain was barely detected in the wild-type strain. These results suggest that dentilisin and Msp, the major outer sheath proteins, are involved in the secretion and maturation of dentipain. Inactivation of the dentipain gene slowed the growth of T. denticola, and the effect was more profound in serum-free medium than in serum-containing medium. Several genes, including those encoding transporters and methyl-accepting chemotaxis proteins, were differentially expressed in the dentipain-deficient mutant. Furthermore, the mutant strain was more hydrophobic than the wild-type strain. Finally, the mutant showed less autoaggregation activity and adhesion to IgG in a serum-free medium than the wild-type strain. These findings suggest that dentipain contributes to the virulence of T. denticola by facilitating adhesion and acquisition of nutrients essential for colonization and proliferation in the gingival crevice under serum-rich conditions.

RECONSTRUCTION AND ANALYSIS OF THE TRANSCRIPTOME REGULATORY NETWORK OF CLOSTRIDIUM BOTULINUM TYPE A3 STR. LOCH MAREE

Clostridium botulinum type A3 str. Loch Maree is a clinically important strain that produces botulinum neurotoxin type A3 and causes foodborne, infant, and wound botulism worldwide. Studying the mechanism underlying the virulence of this organism is imperative to understand its antibacterial resistance and discovering new drugs or inhibitors. The biochemical and molecular characteristics of this organism have been intensively studied, but their gene regulatory mechanisms are unclear. Hence, we reconstructed the transcriptional regulatory network from the complete genome of this organism and analyzed interactive genes from the identified hub module using a knowledge-based bottom-up approach. The biological reliability, topological properties, and robustness of the regulatory network model were validated with network parameters, followed by gene ontology terms and literature support. The reconstructed regulatory network consisted of 12 transcriptional regulators associated with 2369 coding genes. ResD, SpoOA, ComK, CcpC, DinR, DegU, CitT, CodY, GerE, GltC, GltR, IolR, and LevR were identified as transcriptional regulators from this organism homologous to Bacillus subtilis 168. These regulators have been shown to control beta-lactamase, methyl-accepting chemotaxis protein, DNA replication protein DnaD, sensor histidine kinase, and putative membrane proteins of this organism. This study also predicted all possible promoter sites in regulated genes and their associated molecular functions. We conclude that a global regulatory network model of this organism provides insights into its growth physiology and virulence elicitation in the human intestinal environment.

Abstract 2376: The potential role of novel Small Protein 21 interacting with methyl-accepting chemotaxis proteins in P. aeruginosa

Abstract 2376: The potential role of novel Small Protein 21 interacting with methyl-accepting chemotaxis proteins in P. aeruginosa

High-throughput genomic and proteomic interpretation of gene duplication in Vibrio cholera genomes: An in silico study

BackgroundVibrio cholerae leads to severe acute watery diarrhea with a high mortality rate; owing to several virulence factors, especially the cholera toxin. Generally, gene duplication is a major evolutionary process that affects the environmental adaptation of bacteria. In this study, we aimed to investigate the repetitive virulence factors in V. cholera genomes. MethodTotally, 1530 genomes were collected and the possible duplication of 25 known virulence factors was assessed. Next, the genomes carrying duplicated cholera toxins (e.g. ctxA and ctxB) were further analyzed for the identification of novel repetitive virulence factors. All duplicated potential virulence proteins were classified based on clusters of orthologous groups (COGs) and their functions in different pathways were assessed. ResultsGenome-wide analysis showed eight repetitive known virulence factors including rstA, ctxA, ctxB, zot, ace, espA, VC_RS09110, and mshA in V. cholerae genomes. It seems that the majority of the duplicated virulence factors are encoded on the CTXφ such as cholera toxin. Interestingly, our analysis revealed that fourteen genomes had simultaneously two copy numbers of ctxA and ctxB. The proteome of these strains harbored some repetitive proteins that resembled virulence factors of other bacteria. These duplicated proteins were mainly involved in replication, transcription, metabolism, and pathogenesis including Elongation factor Tu, Transketolase, Methyl-accepting chemotaxis protein, GNAT family N-acetyltransferases, cGAMP-activated phospholipase of Vibrio (CapV), HigA, and Helix-turn-helix transcriptional regulator. ConclusionSurprisingly, a considerable number of duplicated genes have essential roles in the virulence and pathogenesis of V. cholerae. Moreover, it seems that novel duplicated genes had different roles in cellular processes or metabolic pathways.

Characterization of Changes in Penile Microbiome Following Pediatric Circumcision

BackgroundWhile microbiome and host regulation contribute independently to many disease states, it is unclear how circumcision in pediatric population influences subsequent changes in penile microbiome. ObjectiveOur study aims to analyze jointly paired taxonomic profiles and assess pathways implicated in inflammation, barrier protection, and energy metabolism. Design, setting, and participantsWe analyzed 11 paired samples, periurethral collection, before and after circumcision, to generate microbiome and mycobiome profiling. Sample preparation of 16S ribosomal RNA and internal transcribed spacer sequencing was adapted from the methods developed by the National Institutes of Health Human Microbiome Project. Outcome measurements and statistical analysisWe obtained the predictive functional attributes of the microbial communities between samples using Silva-Tax4Fun and the Greengenes–Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) approach. The predictive functioning of the microbial communities was determined by linearly combining the normalized taxonomic abundances into the precomputed association matrix of Kyoto Encyclopedia of Genes and Genomes orthology reference profiles. Results and limitationsSeveral notable microbiome and mycobiome compositional differences were observed between pre- and postcircumcision patients. Pairwise comparisons across taxa revealed a significant decrease (p < 0.05, false discovery rate corrected) of microbiome organisms (Clostridiales, Bacteroidales, and Campylobacterales) and mycobiome (Saccharomycetales and Pleosporales) following circumcision. A total of 14 pathways were found to differ in abundance between the pre- and postcircumcision groups (p < 0.005, false discovery rate <0.1 and linear discriminant analysis score >3; five enriched and nine depleted). The pathways reduced after circumcision were mostly involved with amino acid and glucose metabolism, while pathways prior to circumcision were enriched in genetic information processing and transcription processes. As expected, enrichment in methyl-accepting chemotaxis protein, an integral membrane protein involved in directed motility of microbes to chemical cues and environment, occurred prior to circumcision, while the filamentous hemagglutinin pathway (a strong immunogenic protein) was depleted after circumcision ConclusionsOur results offer greater insight into the host-microbiota relationship of penile circumcision and may serve to lay the groundwork for future studies focused on drivers of inflammation, infection, and oncogenesis. Patient summaryOur study showed a significant reduction in bacteria and fungi after circumcision, particularly anaerobic bacteria, which are known to be potential inducers of inflammation and cancer. This is the first study of its kind showing the changes in microbiome after circumcision, and some of the changes that occur in healthy infants after circumcision that may explain the differences in cancer and inflammatory disorders in adulthood.

PAS Domain-Containing Chemoreceptors Influence the Signal Sensing and Intestinal Colonization of Vibrio cholerae.

Bacterial chemotaxis is the phenomenon in which bacteria migrate toward a more favorable niche in response to chemical cues in the environment. The methyl-accepting chemotaxis proteins (MCPs) are the principal sensory receptors of the bacterial chemotaxis system. Aerotaxis is a special form of chemotaxis in which oxygen serves as the signaling molecule; the process is dependent on the aerotaxis receptors (Aer) containing the Per-Arnt-Sim (PAS) domain. Over 40 MCPs are annotated on the genome of Vibrio cholerae; however, little is known about their functions. We investigated six MCPs containing the PAS domain in V. cholerae El Tor C6706, namely aer2, aer3, aer4, aer5, aer6, and aer7. Deletion analyses of each aer homolog gene indicated that these Aer receptors are involved in aerotaxis, chemotaxis, biofilm formation, and intestinal colonization. Swarming motility assay indicated that the aer2 gene was responsible for sensing the oxygen gradient independent of the other five homologs. When bile salts and mucin were used as chemoattractants, each Aer receptor influenced the chemotaxis differently. Biofilm formation was enhanced by overexpression of the aer6 and aer7 genes. Moreover, deletion of the aer2 gene resulted in better bacterial colonization of the mutant in adult mice; however, virulence gene expression was unaffected. These data suggest distinct roles for different Aer homologs in V. cholerae physiology.

Redox properties and PAS domain structure of the Escherichia coli energy sensor Aer indicate a multistate sensing mechanism

The Per-Arnt-Sim (PAS; named for the representative proteins: Period, Aryl hydrocarbon receptor nuclear translocator protein and Single-minded) domain of the dimeric Escherichia coli aerotaxis receptor Aer monitors cellular respiration through a redox-sensitive flavin adenine dinucleotide (FAD) cofactor. Conformational shifts in the PAS domain instigated by the oxidized FAD (FADOX)/FAD anionic semiquinone (FADASQ) redox couple traverse the HAMP (histidine kinases, adenylate cyclases, methyl-accepting chemotaxis proteins, and phosphatases) and kinase control domains of the Aer dimer to regulate CheA kinase activity. The PAS domain of Aer is unstable and has not been previously purified. Here, residue substitutions that rescue FAD binding in an FAD binding-deficient full-length Aer variant were used in combination to stabilize the Aer PAS domain. We solved the 2.4Å resolution crystal structure of this variant, Aer-PAS-GVV, and revealed a PAS fold that contains distinct features associated with FAD-based redox sensing, such as a close contact between the Arg115 side chain and N5 of the isoalloxazine ring and interactions of the flavin with the side chains of His53 and Asn85 that are poised to convey conformational signals from the cofactor to the protein surface. In addition, we determined the FADox/FADASQ formal potentials of Aer-PAS-GVV and full-length Aer reconstituted into nanodiscs. The Aer redox couple is remarkably low at -289.6± 0.4mV. In conclusion, we propose a model for Aer energy sensing based on the low potential of Aer-PAS-FADox/FADASQ couple and the inability of Aer-PAS to bind to the fully reduced FAD hydroquinone.

Exploring AI-2-mediated interspecies communications within rumen microbial communities

BackgroundThe rumen is an ecosystem with a complex microbial microflora in which microbes initiate biofilm formation by attaching to plant surfaces for plant degradation and are capable of converting feed to nutrients and energy via microbial processes. Quorum sensing (QS) is a cell-to-cell communication mechanism that allows microbes to synchronize the expression of multiple genes in the group to perform social behaviors such as chemotaxis and biofilm formation using self-synthesized QS signaling molecules. Whereas QS has been extensively studied in model microorganisms under pure culture conditions, QS mechanisms are poorly understood in complex bacterial communities, such as the rumen microflora, in which cell-to-cell communication may be common.ResultsHere, we analyzed 981 rumens bacterial and archaeal genomes from the Joint Genome Institute (JGI) and GenBank databases and identified 15 types of known QS signaling molecule-related genes. The analysis of the prevalence and abundance of genes involved in QS showed that 767 microbial genomes appeared to possess QS-related genes, including 680 bacterial genomes containing autoinducer-2 (AI-2) synthase- or receptor-encoding genes. Prevotella, Butyivibrio, Ruminococcus, Oribacterium, Selenomonas, and Treponema, known abundant bacterial genera in the rumen, possessed the greatest numbers of AI-2-related genes; these genes were highly expressed within the metatranscriptome dataset, suggesting that intra- and interspecies communication mediated by AI-2 among rumen microbes was universal in the rumen. The QS processes mediated by the dCache_1-containing AI-2 receptors (CahRs) with various functional modules may be essential for degrading plants, digesting food, and providing energy and nutrients to the host. Additionally, a universal natural network based on QS revealed how rumen microbes coordinate social behaviors via the AI-2-mediated QS system, most of which may potentially function via AI-2 binding to the extracellular sensor dCache_1 domain to activate corresponding receptors involved in different signal transduction pathways, such as methyl-accepting chemotaxis proteins, histidine kinases, serine phosphatases, c-di-GMP synthases and phosphodiesterases, and serine/threonine kinases in the rumen.ConclusionsThe exploration of AI-2-related genes, especially CahR-type AI-2 receptors, greatly increased our insight into AI-2 as a potentially “universal” signal mediating social behaviors and will help us better understand microbial communication networks and the function of QS in plant-microbe interactions in complex microecosystems.DGdc6xosz2q-6EKgKy6aZ-Video

Structural signatures of Escherichia coli chemoreceptor signaling states revealed by cellular crosslinking

The chemotaxis machinery of Escherichia coli has served as a model for exploring the molecular signaling mechanisms of transmembrane chemoreceptors known as methyl-accepting chemotaxis proteins (MCPs). Yet, fundamental questions about signal transmission through MCP molecules remain unanswered. Our work with the E. coli serine chemoreceptor Tsr has developed invivo reporters that distinguish kinase-OFF and kinase-ON structures in the cytoplasmic methylation helix (MH) cap, which receives stimulus signals from an adjoining, membrane-proximal histidine kinase, adenylyl cyclases, MCPs, and phosphatases (HAMP) domain. The cytoplasmic helices of the Tsr homodimer interact mainly through packing interactions of hydrophobic residues at a and d heptad positions. We investigated the invivo crosslinking properties of Tsr molecules bearing cysteine replacements at functionally tolerant g heptad positions in the N-terminal and C-terminal cap helices. Upon treatment of cells with bismaleimidoethane (BMOE), a bifunctional thiol-reagent, Tsr-G273C/Q504C readily formed a doubly crosslinked product in the presence of serine but not in its absence. Moreover, a serine stimulus combined with BMOE treatment during invivo Förster resonance energy transfer-based kinase assays locked Tsr-G273C/Q504C in kinase-OFF output. An OFF-shifting lesion in MH1 (D269P) promoted the formation of the doubly crosslinked species in the absence of serine, whereas an ON-shifting lesion (G268P) suppressed the formation of the doubly crosslinked species. Tsr-G273C/Q504C also showed output-dependent crosslinking patterns in combination with ON-shifting and OFF-shifting adaptational modifications. Our results are consistent with a helix breathing-axial rotation-bundle repacking signaling mechanism and imply that invivo crosslinking tools could serve to probe helix-packing transitions and their output consequences in other regions of the receptor molecule.

Multiple detection of both attractants and repellents by the dCache-chemoreceptor SO_1056 of Shewanella oneidensis.

Chemoreceptors are usually transmembrane proteins dedicated to the detection of compound gradients or signals in the surroundings of a bacterium. After detection, they modulate the activation of CheA-CheY, the core of the chemotactic pathway, to allow cells to move upwards or downwards depending on whether the signal is an attractant or a repellent, respectively. Environmental bacteria such as Shewanella oneidensis harbour dozens of chemoreceptors or MCPs (methyl-accepting chemotaxis proteins). A recent study revealed that MCP SO_1056 of S. oneidensis binds chromate. Here, we show that this MCP also detects an additional attractant (l-malate) and two repellents (nickel and cobalt). The experiments were performed in vivo by the agarose-in-plug technique after overproducing MCP SO_1056 and in vitro, when possible, by submitting the purified ligand-binding domain (LBD) of SO_1056 to a thermal shift assay (TSA) coupled to isothermal titration calorimetry (ITC). ITC assays revealed a KD of 3.4 μm for l-malate and of 47.7 μm for nickel. We conclude that MCP SO_1056 binds attractants and repellents of unrelated composition. The LBD of SO_1056 belongs to the double Cache_1 family and is highly homologous to PctA, a chemoreceptor from Pseudomonas aeruginosa that detects several amino acids. Therefore, LBDs of the same family can bind diverse compounds, confirming that experimental approaches are required to define accurate LBD-binding molecules or signals.

Posttranscriptional Regulation in Response to Different Environmental Stresses in Campylobacter jejuni.

ABSTRACTThe survival strategies that Campylobacter jejuni (C. jejuni) employ throughout its transmission and infection life cycles remain largely elusive. Specifically, there is a lack of understanding about the posttranscriptional regulation of stress adaptations resulting from small noncoding RNAs (sRNAs). Published C. jejuni sRNAs have been discovered in specific conditions but with limited insights into their biological activities. Many more sRNAs are yet to be discovered as they may be condition-dependent. Here, we have generated transcriptomic data from 21 host- and transmission-relevant conditions. The data uncovered transcription start sites, expression patterns and posttranscriptional regulation during various stress conditions. This data set helped predict a list of putative sRNAs. We further explored the sRNAs’ biological functions by integrating differential gene expression analysis, coexpression analysis, and genome-wide sRNA target prediction. The results showed that the C. jejuni gene expression was influenced primarily by nutrient deprivation and food storage conditions. Further exploration revealed a putative sRNA (CjSA21) that targeted tlp1 to 4 under food processing conditions. tlp1 to 4 are transcripts that encode methyl-accepting chemotaxis proteins (MCPs), which are responsible for chemosensing. These results suggested CjSA21 inhibits chemotaxis and promotes survival under food processing conditions. This study presents the broader research community with a comprehensive data set and highlights a novel sRNA as a potential chemotaxis inhibitor.IMPORTANCE The foodborne pathogen C. jejuni is a significant challenge for the global health care system. It is crucial to investigate C. jejuni posttranscriptional regulation by small RNAs (sRNAs) in order to understand how it adapts to different stress conditions. However, limited data are available for investigating sRNA activity under stress. In this study, we generate gene expression data of C. jejuni under 21 stress conditions. Our data analysis indicates that one of the novel sRNAs mediates the adaptation to food processing conditions. Results from our work shed light on the posttranscriptional regulation of C. jejuni and identify an sRNA associated with food safety.