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Frontiers in Microbiology 2023High hydrostatic pressure (HHP) regulated gene expression is one of the most commonly adopted strategies for microbial adaptation to the deep-sea environments....
High hydrostatic pressure (HHP) regulated gene expression is one of the most commonly adopted strategies for microbial adaptation to the deep-sea environments. Previously we showed that the HHP-inducible trimethylamine N-oxide (TMAO) reductase improves the pressure tolerance of deep-sea strain QY27. Here, we investigated the molecular mechanism of HHP-responsive regulation of TMAO reductase TorA. By constructing and deletion mutants, we demonstrated that the two-component regulator TorR and sensor TorS are responsible for the HHP-responsive regulation of . Unlike known HHP-responsive regulatory system, the abundance of and was not affected by HHP. Complementation of the Δ mutant with TorS altered at conserved phosphorylation sites revealed that the three sites were indispensable for substrate-induced regulation, but only the histidine located in the alternative transmitter domain was involved in pressure-responsive regulation. Taken together, we demonstrated that the induction of TMAO reductase by HHP is mediated through the TorRS system and proposed a bifurcation of signal transduction in pressure-responsive regulation from the substrate-induction. This work provides novel knowledge of the pressure regulated gene expression and will promote the understanding of the microbial adaptation to the deep-sea HHP environment.
PubMed: 38029070
DOI: 10.3389/fmicb.2023.1291578 -
Journal of Global Antimicrobial... Dec 2023
Topics: Genomic Islands; Salmonella; beta-Lactamases
PubMed: 37748579
DOI: 10.1016/j.jgar.2023.09.014 -
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 -
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 -
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 -
NPJ Biofilms and Microbiomes Apr 2024Increasing evidence infers that some complex diseases are attributed to co-infection with multiple pathogens, such as shrimp white feces syndrome (WFS); however, there...
Increasing evidence infers that some complex diseases are attributed to co-infection with multiple pathogens, such as shrimp white feces syndrome (WFS); however, there is a lack of experimental evidence to validate such causal link. This deficiency further impedes rational design of probiotics to elicit desired benefits to shrimp WFS resistance. Herein, we validated the causal roles of Vibrio fluvialis, V. coralliilyticus and V. tubiashii (in a ratio of 7:2:1) in shrimp WFS etiology, which fully satisfied Koch's postulates. Correspondingly, we precisely designed four antagonistic strains: Ruegeria lacuscaerulensis, Nioella nitratireducens, Bacillus subtilis and Streptomyces euryhalinus in a ratio of 4:3:2:1, which efficiently guarded against WFS. Dietary supplementation of the probiotics stimulated beneficial gut populations, streptomycin, short chain fatty acids, taurine metabolism potentials, network stability, tight junction, and host selection, while reducing turnover rate and average variation degree of gut microbiota, thereby facilitating ecological and mechanical barriers against pathogens. Additionally, shrimp immune pathways, such as Fcγ R-mediated phagocytosis, Toll-like receptor and RIG-I-like receptor signaling pathways conferring immune barrier, were activated by probiotics supplementation. Collectively, we establish an updated framework for precisely validating co-infection with multiple pathogens and rationally designing antagonistic probiotics. Furthermore, our findings uncover the underlying beneficial mechanisms of designed probiotics from the probiotics-gut microbiome-host immunity axis.
Topics: Humans; Gastrointestinal Microbiome; Coinfection; Feces; Probiotics
PubMed: 38605016
DOI: 10.1038/s41522-024-00509-5