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Nature Communications Jun 2024Although CRISPR-dCas13, the RNA-guided RNA-binding protein, was recently exploited as a translation-level gene expression modulator, it has still been difficult to...
Although CRISPR-dCas13, the RNA-guided RNA-binding protein, was recently exploited as a translation-level gene expression modulator, it has still been difficult to precisely control the level due to the lack of detailed characterization. Here, we develop a synthetic tunable translation-level CRISPR interference (Tl-CRISPRi) system based on the engineered guide RNAs that enable precise and predictable down-regulation of mRNA translation. First, we optimize the Tl-CRISPRi system for specific and multiplexed repression of genes at the translation level. We also show that the Tl-CRISPRi system is more suitable for independently regulating each gene in a polycistronic operon than the transcription-level CRISPRi (Tx-CRISPRi) system. We further engineer the handle structure of guide RNA for tunable and predictable repression of various genes in Escherichia coli and Vibrio natriegens. This tunable Tl-CRISPRi system is applied to increase the production of 3-hydroxypropionic acid (3-HP) by 14.2-fold via redirecting the metabolic flux, indicating the usefulness of this system for the flux optimization in the microbial cell factories based on the RNA-targeting machinery.
Topics: Escherichia coli; CRISPR-Cas Systems; RNA, Guide, CRISPR-Cas Systems; Vibrio; Protein Biosynthesis; Gene Expression Regulation, Bacterial; RNA, Messenger; Clustered Regularly Interspaced Short Palindromic Repeats; Operon; Genetic Engineering; Lactic Acid
PubMed: 38909033
DOI: 10.1038/s41467-024-49642-x -
Archives of Microbiology Jun 2024Vibrio parahaemolyticus possesses two distinct type VI secretion systems (T6SS), namely T6SS1 and T6SS2. T6SS1 is predominantly responsible for adhesion to Caco-2 and...
Vibrio parahaemolyticus possesses two distinct type VI secretion systems (T6SS), namely T6SS1 and T6SS2. T6SS1 is predominantly responsible for adhesion to Caco-2 and HeLa cells and for the antibacterial activity of V. parahaemolyticus, while T6SS2 mainly contributes to HeLa cell adhesion. However, it remains unclear whether the T6SS systems have other physiological roles in V. parahaemolyticus. In this study, we demonstrated that the deletion of icmF2, a structural gene of T6SS2, reduced the biofilm formation capacity of V. parahaemolyticus under low salt conditions, which was also influenced by the incubation time. Nonetheless, the deletion of icmF2 did not affect the biofilm formation capacity in marine-like growth conditions, nor did it impact the flagella-driven swimming and swarming motility of V. parahaemolyticus. IcmF2 was found to promote the production of the main components of the biofilm matrix, including extracellular DNA (eDNA) and extracellular proteins, and cyclic di-GMP (c-di-GMP) in V. parahaemolyticus. Additionally, IcmF2 positively influenced the transcription of cpsA, mfpA, and several genes involved in c-di-GMP metabolism, including scrJ, scrL, vopY, tpdA, gefA, and scrG. Conversely, the transcription of scrA was negatively impacted by IcmF2. Therefore, IcmF2-dependent biofilm formation was mediated through its effects on the production of eDNA, extracellular proteins, and c-di-GMP, as well as its impact on the transcription of cpsA, mfpA, and genes associated with c-di-GMP metabolism. This study confirmed new physiological roles for IcmF2 in promoting biofilm formation and c-di-GMP production in V. parahaemolyticus.
Topics: Vibrio parahaemolyticus; Biofilms; Type VI Secretion Systems; Bacterial Proteins; Cyclic GMP; Humans; Gene Expression Regulation, Bacterial; HeLa Cells
PubMed: 38907796
DOI: 10.1007/s00203-024-04060-x -
BMC Microbiology Jun 2024In Addis Ababa, Ethiopia, open ditches along innner roads in residential areas serve to convey domestic wastewater and rainwater away from residences. Contamination of...
BACKGROUND
In Addis Ababa, Ethiopia, open ditches along innner roads in residential areas serve to convey domestic wastewater and rainwater away from residences. Contamination of drinking water by wastewater through faulty distribution lines could expose households to waterborne illnesses. This prompted the study to assess the microbiological safety of wastewater and drinking water in Addis Ababa, identify the pathogens therein, and determine their antibiotic resistance patterns.
RESULTS VIBRIO CHOLERAE
O1, mainly Hikojima serotype, was isolated from 23 wastewater and 16 drinking water samples. Similarly, 19 wastewater and 10 drinking water samples yielded Escherichia coli O157:H7. V. cholerae O1 were 100% resistant to the penicillins (Amoxacillin and Ampicillin), and 51-82% were resistant to the cephalosporins. About 44% of the V. cholerae O1 isolates in this study were Extended Spectrum Beta-Lactamase (ESBL) producers. Moreover, 26% were resistant to Meropenem. Peperacillin/Tazobactam was the only effective β-lactam antibiotic against V. cholerae O1. V. cholerae O1 isolates showed 37 different patterns of multiple resistance ranging from a minimum of three to a maximum of ten antimicrobials. Of the E. coli O157:H7 isolates, 71% were ESBL producers. About 96% were resistant to Ampicillin. Amikacin and Gentamicin were very effective against E. coli O157:H7 isolates. The isolates from wastewater and drinking water showed multiple antibiotic resistance against three to eight antibiotic drugs.
CONCLUSIONS
Open ditches for wastewater conveyance along innner roads in residence areas and underground faulty municipal water distribution lines could be possible sources for V. cholerae O1 and E. coli O157:H7 infections to surrounding households and for dissemination of multiple drug resistance in humans and, potentially, the environment.
Topics: Ethiopia; Vibrio cholerae O1; Wastewater; Escherichia coli O157; Anti-Bacterial Agents; Drinking Water; Microbial Sensitivity Tests; Drug Resistance, Multiple, Bacterial; beta-Lactamases; Humans; Water Microbiology
PubMed: 38902619
DOI: 10.1186/s12866-024-03302-8 -
Current Microbiology Jun 2024Pyruvate (Pyr) is the end product of the glycolysis pathway. Pyr is also renewable and is further metabolized to produce formate, which is the precursor of H, via...
Pyruvate (Pyr) is the end product of the glycolysis pathway. Pyr is also renewable and is further metabolized to produce formate, which is the precursor of H, via pyruvate formate lyase (PFL) under anaerobic conditions. The formate is excluded and re-imported via the formate channel and is then converted to H via the formate hydrogenlyase (FHL) complex. In H producing marine vibrios, such as Vibrio tritonius and Vibrio porteresiae in the Porteresiae clade of the family Vibrionaceae, apparent but inefficient H production from Pyr has been observed. To elucidate the molecular mechanism of why this inefficient H production is observed in Pry-metabolized marine vibrio cells and how glycolysis affects those H productions of marine vibrios, the "Core Transcriptome" approach to find common gene expressions of those two major H producing Vibrio species in Pyr metabolism was first applied. In the Pyr-metabolized vibrio cells, genes for the "Phosphoenolpyruvate (PEP)-Pyruvate-Oxalate (PPO)" node, due to energy saving, and PhoB-, RhaR-, and DeoR-regulons were regulated. Interestingly, a gene responsible for oxalate/formate family antiporter was up-regulated in Pyr-metabolized cells compared to those of Glc-metabolized cells, which provides new insights into the uses of alternative formate exclusion mechanics due to energy deficiencies in Pyr-metabolized marine vibrios cells. We further discuss the contribution of the Embden-Meyerhof-Parnas (EMP) pathway to efficient H production in marine vibrios.
Topics: Hydrogen; Vibrio; Transcriptome; Glycolysis; Pyruvic Acid; Bacterial Proteins; Seawater; Gene Expression Regulation, Bacterial; Aquatic Organisms
PubMed: 38896159
DOI: 10.1007/s00284-024-03764-z -
International Journal of Molecular... May 2024Alginate lyases cleave the 1,4-glycosidic bond of alginate by eliminating sugar molecules from its bond. While earlier reported alginate lyases were primarily single...
Alginate lyases cleave the 1,4-glycosidic bond of alginate by eliminating sugar molecules from its bond. While earlier reported alginate lyases were primarily single catalytic domains, research on multi-module alginate lyases has been lfiguimited. This study identified VsAly7A, a multi-module alginate lyase present in sp. QY108, comprising a "Pro-Asp-Thr(PDT)" fragment and two PL-7 catalytic domains (CD I and CD II). The "PDT" fragment enhances the soluble expression level and increases the thermostability and binding affinity to the substrate. Moreover, CD I exhibited greater catalytic efficiency than CD II. The incorporation of PDT-CD I resulted in an increase in the optimal temperature of VsAly7A, whereas CD II displayed a preference for polyG degradation. The multi-domain structure of VsAly7A provides a new idea for the rational design of alginate lyase, whilst the "PDT" fragment may serve as a fusion tag in the soluble expression of recombinant proteins.
Topics: Polysaccharide-Lyases; Vibrio; Alginates; Enzyme Stability; Protein Binding; Catalytic Domain; Bacterial Proteins; Solubility; Amino Acid Sequence; Temperature; Recombinant Proteins
PubMed: 38891987
DOI: 10.3390/ijms25115801 -
International Journal of Molecular... May 2024is an emerging foodborne pathogenic bacterium that can cause severe cholera-like diarrhea and various extraintestinal infections, posing challenges to public health and...
is an emerging foodborne pathogenic bacterium that can cause severe cholera-like diarrhea and various extraintestinal infections, posing challenges to public health and food safety worldwide. The arginine deiminase (ADI) pathway plays an important role in bacterial environmental adaptation and pathogenicity. However, the biological functions and regulatory mechanisms of the pathway in remain unclear. In this study, we demonstrate that L-arginine upregulates the expression of the ADI gene cluster and promotes the growth of . The ADI gene cluster, which we proved to be comprised of two operons, and , significantly enhances the survival of in acidic environments both in vitro (in culture medium and in macrophage) and in vivo (in mice). The mRNA level and reporter gene fusion analyses revealed that ArgR, a transcriptional factor, is necessary for the activation of both and transcriptions. Bioinformatic analysis predicted the existence of multiple potential ArgR binding sites at the and promoter regions that were further confirmed by electrophoretic mobility shift assay, DNase I footprinting, or point mutation analyses. Together, our study provides insights into the important role of the ArgR-ADI pathway in the survival of under acidic conditions and the detailed molecular mechanism. These findings will deepen our understanding of how environmental changes and gene expression interact to facilitate bacterial adaptations and virulence.
Topics: Animals; Gene Expression Regulation, Bacterial; Bacterial Proteins; Mice; Hydrolases; Promoter Regions, Genetic; Operon; Repressor Proteins; Vibrio; Arginine; Multigene Family; Virulence; Microbial Viability
PubMed: 38891866
DOI: 10.3390/ijms25115679 -
Journal of Visualized Experiments : JoVE May 2024Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In Vibrio species,...
Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In Vibrio species, the master quorum sensing regulators LuxR/HapR control hundreds of quorum sensing genes, many of which influence virulence, metabolism, motility, and more. Thiophenesulfonamides are potent inhibitors of LuxR/HapR that bind the ligand pocket in these transcription factors and block downstream quorum sensing gene expression. This class of compounds served as the basis for the development of a set of simple, robust, and educational procedures for college students to assimilate their chemistry and biology skills using a CURE model: course-based undergraduate research experience. Optimized protocols are described that comprise three learning stages in an iterative and multi-disciplinary platform to engage students in a year-long CURE: (1) design and synthesize new small molecule inhibitors based on the thiophenesulfonamide core, (2) use structural modeling to predict binding affinity to the target, and (3) assay the compounds for efficacy in microbiological assays against specific Vibrio LuxR/HapR proteins. The described reporter assay performed in E. coli successfully predicts the efficacy of the compounds against target proteins in the native Vibrio species.
Topics: Quorum Sensing; Vibrio; Trans-Activators; Repressor Proteins; Sulfonamides; Thiophenes; Anti-Bacterial Agents; Bacterial Proteins
PubMed: 38884467
DOI: 10.3791/66582 -
Applied and Environmental Microbiology Jun 2024Marine bacteria experience fluctuations in osmolarity that they must adapt to, and most bacteria respond to high osmolarity by accumulating compatible solutes also known...
Marine bacteria experience fluctuations in osmolarity that they must adapt to, and most bacteria respond to high osmolarity by accumulating compatible solutes also known as osmolytes. The osmotic stress response and compatible solutes used by the coral and oyster pathogen were unknown. In this study, we showed that to alleviate osmotic stress biosynthesized glycine betaine (GB) and transported into the cell choline, GB, ectoine, dimethylglycine, and dimethylsulfoniopropionate, but not -inositol. -inositol is a stress protectant and a signaling molecule that is biosynthesized and used by algae. Bioinformatics identified -inositol () catabolism clusters in and other , , , and species. Growth pattern analysis demonstrated that utilized -inositol as a sole carbon source, with a short lag time of 3 h. An deletion mutant, which encodes an inositol dehydrogenase, was unable to grow on -inositol. Within the clusters were an MFS-type () and an ABC-type () transporter and analyses showed that both transported -inositol. IolG and IolA phylogeny among species showed different evolutionary histories indicating multiple acquisition events. Outside of , IolG was most closely related to IolG from a small group of fish and human pathogens and species. However, IolG from hypervirulent strains clustered with IolG from and divergently from , , and plant pathogens. The cluster was also present within , , , , , , , , , and , of which many species were associated with marine flora and fauna.IMPORTANCEHost associated bacteria such as encounter competition for nutrients and have evolved metabolic strategies to better compete for food. Emerging studies show that -inositol is exchanged in the coral-algae symbiosis, is likely involved in signaling, but is also an osmolyte in algae. The bacterial consumption of -inositol could contribute to a breakdown of the coral-algae symbiosis during thermal stress or disrupt the coral microbiome. Phylogenetic analyses showed that the evolutionary history of -inositol metabolism is complex, acquired multiple times in , but acquired once in many bacterial plant pathogens. Further analysis also showed that a conserved cluster is prevalent among many marine species (commensals, mutualists, and pathogens) associated with marine flora and fauna, algae, sponges, corals, molluscs, crustaceans, and fish.
PubMed: 38874337
DOI: 10.1128/aem.00920-24 -
Proceedings of the National Academy of... Jun 2024causes life-threatening wound and gastrointestinal infections, mediated primarily by the production of a Multifunctional-Autoprocessing Repeats-In-Toxin (MARTX) toxin....
causes life-threatening wound and gastrointestinal infections, mediated primarily by the production of a Multifunctional-Autoprocessing Repeats-In-Toxin (MARTX) toxin. The most commonly present MARTX effector domain, the Makes Caterpillars Floppy-like (MCF) toxin, is a cysteine protease stimulated by host adenosine diphosphate (ADP) ribosylation factors (ARFs) to autoprocess. Here, we show processed MCF then binds and cleaves host s-related proteins in rain (Rab) guanosine triphosphatases within their C-terminal tails resulting in Rab degradation. We demonstrate MCF binds Rabs at the same interface occupied by ARFs. Moreover, we show MCF preferentially binds to ARF1 prior to autoprocessing and is active to cleave Rabs only subsequent to autoprocessing. We then use structure prediction algorithms to demonstrate that structural composition, rather than sequence, determines Rab target specificity. We further determine a crystal structure of aMCF as a swapped dimer, revealing an alternative conformation we suggest represents the open, activated state of MCF with reorganized active site residues. The cleavage of Rabs results in Rab1B dispersal within cells and loss of Rab1B density in the intestinal tissue of infected mice. Collectively, our work describes an extracellular bacterial mechanism whereby MCF is activated by ARFs and subsequently induces the degradation of another small host guanosine triphosphatase (GTPase), Rabs, to drive organelle damage, cell death, and promote pathogenesis of these rapidly fatal infections.
Topics: Animals; Female; Humans; Mice; ADP-Ribosylation Factors; Bacterial Toxins; HEK293 Cells; Mice, Inbred ICR; Proteolysis; rab GTP-Binding Proteins; Vibrio Infections; Vibrio vulnificus
PubMed: 38861595
DOI: 10.1073/pnas.2316143121 -
The Science of the Total Environment Sep 2024The final point-of-drinking water (FPODW) exposure to Vibrio and waterborne pathogens remains a misaim surveillance target. Therefore, the current study purposed to... (Meta-Analysis)
Meta-Analysis
Global and regional final point-of-drinking water prevalence of Vibrio pathogens: a systematic analysis with socioeconomic, global health security, and WASH indices-guided meta-regressions.
The final point-of-drinking water (FPODW) exposure to Vibrio and waterborne pathogens remains a misaim surveillance target. Therefore, the current study purposed to estimate the global and regional prevalence of Vibrio pathogens in FPODW. Vibrio-FPODW data derived from integrated databases per PRISMA protocol were fitted to a random-intercept-logistic mixed-effects and meta-regression models. The global FPODW Vibrio prevalence was 5.13% (95%CI: 2.24-11.30) with 7.76% (6.84-8.78) cross-validated value. Vibrio prevalence in different FPODW varied with the highest in unclassified (13.98%, 3.98-38.95), household stored (6.42%, 1.16-28.69), municipal (4.39%, 1.54-11.90), and bottled (1.06%, 0.00-98.57) FPODW. Regionally, FPODW Vibrio prevalence varied significantly with highest in Africa (6.31%, 0.49-47.88), then Asia (4.83%, 2.01-11.18). Similarly, it varied significantly among income classification with the highest from low-income (8.77%, 0.91-50.05), then lower-middle-income (6.16%, 2.75-13.20), upper-middle-income (0.23%, 0.00-82.04), and 0.94% (0.19-2.72) in high-income economies. Among the WHO region, it varied significantly from 1.41% (0.17-10.45) in Eastern Mediterranean, 6.31% (0.49-47.88) in Africa to 8.86% (3.85-19.06) in South-East Asia and declining among SDI-quintiles from 11.64% (3.29-33.83) in Low-SDI, 10.59% (4.58-22.61) in High-middle-SDI to 0.26% (0.01-9.09) in Middle-SDI. FPODW Vibrio prevalence was 7.31% (2.94-17.03) in the low-GHSIG, followed by 4.55% (0.00-100.00) in the upper-GHSIG, and 2.21% (0.31-14.24) in middle-GHSIG; rural (4.18%, 0.06-76.17) and urban (5.28%, 2.35-11.44) settings. Also, sample size, SDI, SDI-quintiles, and nation significantly explained 14.12%, 10.91%, 30.35%, and 87.65% variance in FPODW Vibrio prevalence, respectively as a univariate influence. Additionally, 11.90% variance in FPODW Vibrio prevalence explained mortality rate attributed to unsafe WASH services. In conclusion, the study revealed a substantial high FPODW prevalence of Vibrio calling for initiative-taking and intentional surveillances of waterborne pathogens at the neglected stage across nations in order to achieve sustainably the SDG 3.
Topics: Vibrio; Drinking Water; Prevalence; Water Microbiology; Global Health; Humans; Socioeconomic Factors
PubMed: 38852862
DOI: 10.1016/j.scitotenv.2024.173818