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One Health (Amsterdam, Netherlands) Dec 2021In this study, a phylogenic analysis was performed on pathogens previously identified in Hong Kong wet markets' cutting boards. Phylogenetic comparisons were made...
In this study, a phylogenic analysis was performed on pathogens previously identified in Hong Kong wet markets' cutting boards. Phylogenetic comparisons were made between phylotypes obtained in this study and environmental and clinical phylotypes for establishing the possible origin of selected bacterial species isolated from wet market cutting board ecosystems. The results reveal a strong relationship between wet market bacterial assemblages and environmental and clinically relevant phylotypes. However, our poor knowledge of potential cross-contamination sources within these wet markets is further exacerbated by failing to determine the exact or presumed origin of its identified pathogens. In this study, several clinically relevant bacterial pathogens such as , and were linked to cutting boards associated with pork; , , , and in those associated with poultry; and , , , and in those associated with seafood. Identifying non-foodborne clinically relevant pathogens in wet market cutting boards in this study confirms the need for safety approaches for wet market meat, including cold storage. The presented study justifies the need for future systematic epidemiological studies to determine identified microbial pathogens. Such studies should bring about significant improvements in the management of hygienic practices in Hong Kong's wet markets and work towards a One Health goal by recognizing the importance of wet markets as areas interconnecting food processing with animal and clinical environments.
PubMed: 34409148
DOI: 10.1016/j.onehlt.2021.100300 -
Biochemical and Biophysical Research... Jun 2021V. cholerae, the causative agent of cholera epidemic, and V. fluvialis, the emerging foodborne pathogen, share highly homologous T6SS consisting of one large cluster...
V. cholerae, the causative agent of cholera epidemic, and V. fluvialis, the emerging foodborne pathogen, share highly homologous T6SS consisting of one large cluster and two small orphan or auxiliary clusters, and each of which was generally recognized as one operon. Here, we showed that the genes in each of the small clusters are organized into two transcriptional units. Specifically, the inner tube coding gene hcp/tssD is highly transcribed as one monocistron, while the tip component vgrG/tssI and its downstream effector and immunity genes are in one polycistron with very low transcriptional level. This conclusion is supported by qPCR analysis of mRNA abundance, reporter fusion analysis and transcriptional unit definition with RT-PCR analysis. Taking tssI2_a of V. fluvialis as an example, we further demonstrated that quorum sensing (QS) regulator HapR and global regulator IHF activate vgrG/tssI transcription by directly binding to its promoter region. Taken together, current studies deepen our understanding of T6SS system, highlighting its regulatory complexity during functional execution process.
Topics: Bacterial Proteins; Gene Expression Regulation, Bacterial; Humans; Quorum Sensing; Transcriptional Activation; Type VI Secretion Systems; Vibrio; Vibrio Infections; Vibrio cholerae
PubMed: 33932896
DOI: 10.1016/j.bbrc.2021.04.092 -
Archives of Microbiology Aug 2021Vibrio fluvialis is a halophilic bacterium frequently found in estuarine and coastal waters environments. The strain 362.3 was isolated from Mussismilia braziliensis...
Vibrio fluvialis is a halophilic bacterium frequently found in estuarine and coastal waters environments. The strain 362.3 was isolated from Mussismilia braziliensis coral of Abrolhos Bank. In this study, to gain insights into the marine adaptation in V. fluvialis, we sequenced the genome of 362.3 strain, which comprised 4,607,294 bp with a G + C content of 50.2%. In silico analysis showed that V. fluvialis 362.2 encodes genes related to chitin catabolic pathway, iron metabolism, osmotic stress and membrane transport.
Topics: Adaptation, Physiological; Animals; Anthozoa; Base Sequence; Genome, Bacterial; Phylogeny; Vibrio; Water Microbiology
PubMed: 33829291
DOI: 10.1007/s00203-021-02279-6 -
Biochemistry Feb 2024Globin-coupled sensors constitute an important family of heme-based gas sensors, an emerging class of heme proteins. In this study, we have identified and characterized...
Globin-coupled sensors constitute an important family of heme-based gas sensors, an emerging class of heme proteins. In this study, we have identified and characterized a globin-coupled sensor phosphodiesterase containing an HD-GYP domain (GCS-HD-GYP) from the human pathogen , which is an emerging foodborne pathogen of increasing public health concern. The amino acid sequence encoded by the gene from indicated the presence of an N-terminal globin domain and a C-terminal HD-GYP domain, with HD-GYP domains shown previously to display phosphodiesterase activity toward bis(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP), a bacterial second messenger that regulates numerous important physiological functions in bacteria, including in bacterial pathogens. Optical absorption spectral properties of GCS-HD-GYP were found to be similar to those of myoglobin and hemoglobin and of other bacterial globin-coupled sensors. The binding of O to the Fe(II) heme iron complex of GCS-HD-GYP promoted the catalysis of the hydrolysis of c-di-GMP to its linearized product, 5'-phosphoguanylyl-(3',5')-guanosine (pGpG), whereas CO and NO binding did not enhance the catalysis, indicating a strict discrimination of these gaseous ligands. These results shed new light on the molecular mechanism of gas-selective catalytic regulation by globin-coupled sensors, with these advances apt to lead to a better understanding of the family of globin-coupled sensors, a still growing family of heme-based gas sensors. In addition, given the importance of c-di-GMP in infection and virulence, our results suggested that GCS-HD-GYP could play an important role in the ability of to sense O and NO in the context of host-pathogen interactions.
Topics: Humans; Phosphoric Diester Hydrolases; Globins; Bacterial Proteins; Catalysis; Cyclic GMP; Heme; Vibrio
PubMed: 38264987
DOI: 10.1021/acs.biochem.3c00484 -
Journal of Chemical Information and... Nov 2021ω-Transaminases (ω-TAs) catalyze the conversion of ketones to chiral amines, often with high enantioselectivity and specificity, which makes them attractive for...
ω-Transaminases (ω-TAs) catalyze the conversion of ketones to chiral amines, often with high enantioselectivity and specificity, which makes them attractive for industrial production of chiral amines. Tailoring ω-TAs to accept non-natural substrates is necessary because of their limited substrate range. We present a computational protocol for predicting the enantioselectivity and catalytic selectivity of an ω-TA from with different substrates and benchmark it against 62 compounds gathered from the literature. Rosetta-generated complexes containing an external aldimine intermediate of the transamination reaction are used as starting conformations for multiple short independent molecular dynamics (MD) simulations. The combination of molecular docking and MD simulations ensures sufficient and accurate sampling of the relevant conformational space. Based on the frequency of near-attack conformations observed during the MD trajectories, enantioselectivities can be quantitatively predicted. The predicted enantioselectivities are in agreement with a benchmark dataset of experimentally determined % values. The substrate-range predictions can be based on the docking score of the external aldimine intermediate. The low computational cost required to run the presented framework makes it feasible for use in enzyme design to screen thousands of enzyme variants.
Topics: Molecular Docking Simulation; Molecular Dynamics Simulation; Substrate Specificity; Transaminases; Vibrio
PubMed: 34653331
DOI: 10.1021/acs.jcim.1c00617 -
Angewandte Chemie (International Ed. in... Apr 2020Long-chain aliphatic amines such as (S,Z)-heptadec-9-en-7-amine and 9-aminoheptadecane were synthesized from ricinoleic acid and oleic acid, respectively, by whole-cell...
Long-chain aliphatic amines such as (S,Z)-heptadec-9-en-7-amine and 9-aminoheptadecane were synthesized from ricinoleic acid and oleic acid, respectively, by whole-cell cascade reactions using the combination of an alcohol dehydrogenase (ADH) from Micrococcus luteus, an engineered amine transaminase from Vibrio fluvialis (Vf-ATA), and a photoactivated decarboxylase from Chlorella variabilis NC64A (Cv-FAP) in a one-pot process. In addition, long chain aliphatic esters such as 10-(heptanoyloxy)dec-8-ene and octylnonanoate were prepared from ricinoleic acid and oleic acid, respectively, by using the combination of the ADH, a Baeyer-Villiger monooxygenase variant from Pseudomonas putida KT2440, and the Cv-FAP. The target compounds were produced at rates of up to 37 U g dry cells with conversions up to 90 %. Therefore, this study contributes to the preparation of industrially relevant long-chain aliphatic chiral amines and esters from renewable fatty acid resources.
Topics: Alcohol Dehydrogenase; Amines; Carboxy-Lyases; Chlorella; Esters; Micrococcus luteus; Molecular Structure; Oleic Acid; Photochemical Processes; Ricinoleic Acids
PubMed: 31957098
DOI: 10.1002/anie.201915108 -
Chembiochem : a European Journal of... Oct 2023An enzyme cascade was established previously consisting of a recycling system with an l-amino acid oxidase (hcLAAO4) and a catalase (hCAT) for different α-keto acid...
An enzyme cascade was established previously consisting of a recycling system with an l-amino acid oxidase (hcLAAO4) and a catalase (hCAT) for different α-keto acid co-substrates of (S)-selective amine transaminases (ATAs) in kinetic resolutions of racemic amines. Only 1 mol % of the co-substrate was required and l-amino acids instead of α-keto acids could be applied. However, soluble enzymes cannot be reused easily. Immobilization of hcLAAO4, hCAT and the (S)-selective ATA from Vibrio fluvialis (ATA-Vfl) was addressed here. Immobilization of the enzymes together rather than on separate beads showed higher reaction rates most likely due to fast co-substrate channeling between ATA-Vfl and hcLAAO4 due to their close proximity. Co-immobilization allowed further reduction of the co-substrate amount to 0.1 mol % most likely due to a more efficient H O -removal caused by the stabilized hCAT and its proximity to hcLAAO4. Finally, the co-immobilized enzyme cascade was reused in 3 cycles of preparative kinetic resolutions to produce (R)-1-PEA with high enantiomeric purity (97.3 %ee). Further recycling was inefficient due to the instability of ATA-Vfl, while hcLAAO4 and hCAT revealed high stability. An engineered ATA-Vfl-8M was used in the co-immobilized enzyme cascade to produce (R)-1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethanamine, an apremilast-intermediate, with a 1,000 fold lower input of the co-substrate.
Topics: Amines; Transaminases; L-Amino Acid Oxidase; Enzymes, Immobilized; Catalase; Keto Acids
PubMed: 37368451
DOI: 10.1002/cbic.202300425 -
Engineering in Life Sciences Oct 2019The increasing demand for freshwater and the continued depletion of available resources has led to a deepening global water crisis. Significant water consumption...
The increasing demand for freshwater and the continued depletion of available resources has led to a deepening global water crisis. Significant water consumption required by many biotechnological processes contributes to both the environmental and economic cost of this problem. Relatively few biocatalytic processes have been developed to utilize the more abundant supply of seawater, with seawater composition and salinity limiting its use with many mesophilic enzymes. We recently reported a salt tolerant ω-transaminase enzyme, Ad2-TAm, isolated from the genome of a halophilic bacterium, sp. CSM-2, from a Triassic period salt mine. In this study we aimed to demonstrate its applicability to biocatalytic reactions carried out in a seawater-based medium. Ad2-TAm was examined for its ability to aminate the industrially relevant substrate, furfural, in both seawater and freshwater-based reaction systems. Furfural was aminated with 53.6% conversion in a buffered seawater system, displaying improved function versus freshwater. Ad2-TAm outperformed the commonly employed commercial ω-TAms from and , both of which showed decreased conversion in seawater. Given the increasingly precarious availability of global freshwater, such applications of enzymes from halophiles have the ability to reduce demand for freshwater in large-scale industrial processes, delivering considerable environmental and economic benefits.
PubMed: 32624965
DOI: 10.1002/elsc.201900053 -
Zhonghua Liu Xing Bing Xue Za Zhi =... Apr 2024To explore the regulation mechanism of the quorum sensing regulator AphA on the functional activity of type Ⅵ secretion system VflT6SS2 in . Western Blot analysis...
To explore the regulation mechanism of the quorum sensing regulator AphA on the functional activity of type Ⅵ secretion system VflT6SS2 in . Western Blot analysis was used to detect the relative expression and secretion of VflT6SS2 signature component hemolysin-coregulated protein (Hcp) in wild type (WT), Δ and corresponding complementary strains. Quantitative reverse transcription PCR and luminescence activity assay of the promoter- fusion system was used to measure the mRNA expression levels and promoter activity of the VflT6SS2 core and accessory gene-cluster representative genes 2, (2) and (2), and the quorum sensing regulator HapR in WT and Δ strains. A point mutation experiment combined with a luminescence activity assay was used to verify the regulatory binding site of AphA in the 2b promoter region. Electrophoretic mobility shift assay (EMSA) was used to determine AphA binding to the promoter. The mRNA expression levels of 2, (2), (2), and as well as the protein expression and secretion levels of Hcp in Δ strain, were significantly higher than those in the WT strain. The promoter activities of the VflT6SS2 core cluster, 2a, 2a, and were higher in Δ strain than in the WT strain, while the promoter activity of 2b showed the opposite trend. The promoter sequence analysis of 2a and 2b found significant differences in the region from -335 bp to -229 bp, and two potential AphA binding sites on 2b. The promoter activity of 2b decreased significantly after the point mutation of the two potential AphA binding sites. EMSA results showed that AphA binds directly to the promoter region of . AphA indirectly inhibits the regulation of the VflT6SS2 core and accessory gene clusters at the promoter level by directly repressing the expression of . AphA showed opposite regulation patterns for 2a and 2b, and AphA could positively regulate the expression of 2b by directly binding to the 2b promoter region (-335 bp to -229 bp).
Topics: Quorum Sensing; Vibrio; Bacterial Proteins; Promoter Regions, Genetic; Gene Expression Regulation, Bacterial; Type VI Secretion Systems; Multigene Family
PubMed: 38678354
DOI: 10.3760/cma.j.cn112338-20231215-00354 -
Frontiers in Microbiology 2021Bacterial pathogens are a major cause of infectious diseases in aquatic animals. The abuse of antibiotics in the aquatic industry has led to the proliferation of...
Bacterial pathogens are a major cause of infectious diseases in aquatic animals. The abuse of antibiotics in the aquatic industry has led to the proliferation of antibiotic resistance. It is therefore essential to develop more effective and safer strategies to increase the efficacy and extend the life span of the antibiotics used in aquaculture. In this study, we show that six aquaculture bacterial pathogens (i.e., , , , , , and ) in the stationary phase can be rapidly killed after immersion in gentamicin- or neomycin-containing, ion-free solutions for a few minutes. Such hypoionic shock treatment enhances the bacterial uptake of gentamicin in an ATP-dependent manner. Importantly, we demonstrate, as a proof of concept, that gentamicin under hypoionic shock conditions can effectively kill in a skin infection model of zebrafish (), completely curing the infected fish. Given that pathogenic bacteria generally adhere to the skin surface and gills of aquatic animals, our strategy is of potential significance for bacterial infection control, especially for small-scale economic fish farming and ornamental fish farming. Further, the combined treatment can be completed within 5 min with a relatively small volume of solution, thus minimizing the amount of residual antibiotics in both animals and the environment.
PubMed: 33889141
DOI: 10.3389/fmicb.2021.641846