Regulatory mechanisms of dopamine metabolism in a marine Meyerozyma guilliermondii GXDK6 under NaCl stress as revealed by integrative multi-omics analysis.

Dopamine can be used to treat depression, myocardial infarction, and other diseases. However, few reports are available on the de novo microbial synthesis of dopamine from low-cost substrate. In this study, integrated omics technology was used to explore the dopamine metabolism of a novel marine multi-stress-tolerant aromatic yeast Meyerozyma guilliermondii GXDK6. GXDK6 was found to have the ability to biosynthesize dopamine when using glucose as the substrate. 14 key genes for the biosynthesis of dopamine were identified by whole genome-wide analysis. Transcriptomic and proteomic data showed that the expression levels of gene AAT2 encoding aspartate aminotransferase (regulating dopamine anabolism) were upregulated, while gene AO-I encoding copper amine oxidase (involved in dopamine catabolism) were downregulated under 10 % NaCl stress compared with non-NaCl stress, thereby contributing to biosynthesis of dopamine. Further, the amount of dopamine under 10 % NaCl stress was 2.51-fold higher than that of zero NaCl, which was consistent with the multi-omics results. Real-time fluorescence quantitative PCR (RT-qPCR) and high-performance liquid chromatography (HPLC) results confirmed the metabolic model of dopamine. Furthermore, by overexpressing AAT2, AST enzyme activity was increased by 24.89 %, the expression of genes related to dopamine metabolism was enhanced, and dopamine production was increased by 56.36 % in recombinant GXDK6AAT2. In conclusion, Meyerozyma guilliermondii GXDK6 could utilize low-cost carbon source to synthesize dopamine, and NaCl stress promoted the biosynthesis of dopamine.

Effect of ammonium stress on phosphorus solubilization of a novel marine mangrove microorganism Bacillus aryabhattai NM1-A2 as revealed by integrated omics analysis.

BACKGROUND: Phosphorus is one of the essential nutrients for plant growth. Phosphate-solubilizing microorganisms (PSMs) can alleviate available P deficiency and enhance plant growth in an eco-friendly way. Although ammonium toxicity is widespread, there is little understanding about the effect of ammonium stress on phosphorus solubilization (PS) of PSMs. RESULTS: In this study, seven PSMs were isolated from mangrove sediments. The soluble phosphate concentration in culture supernatant of Bacillus aryabhattai NM1-A2 reached a maximum of 196.96 mg/L at 250 mM (NH4)2SO4. Whole-genome analysis showed that B. aryabhattai NM1-A2 contained various genes related to ammonium transporter (amt), ammonium assimilation (i.e., gdhA, gltB, and gltD), organic acid synthesis (i.e., ackA, fdhD, and idh), and phosphate transport (i.e., pstB and pstS). Transcriptome data showed that the expression levels of amt, gltB, gltD, ackA and idh were downregulated, while gdhA and fdhD were upregulated. The inhibition of ammonium transporter and glutamine synthetase/glutamate synthase (GS/GOGAT) pathway contributed to reducing energy loss. For ammonium assimilation under ammonium stress, accompanied by protons efflux, the glutamate dehydrogenase pathway was the main approach. More 2-oxoglutarate (2-OG) was induced to provide abundant carbon skeletons. The downregulation of formate dehydrogenase and high glycolytic rate resulted in the accumulation of formic acid and acetic acid, which played key roles in PS under ammonium stress. CONCLUSIONS: The accumulation of 2-OG and the inhibition of GS/GOGAT pathway played a key role in ammonium detoxification. The secretion of protons, formic acid and acetic acid was related to PS. Our work provides new insights into the PS mechanism, which will provide theoretical guidance for the application of PSMs.

Model of shrimp pond-mediated spatiotemporal dynamic distribution of antibiotic resistance genes in the mangrove habitat of a subtropical gulf.

Aquacultures are the main reason for the environmental selection of antibiotic resistance genes (ARGs), resulting in the enrichment of ARGs. As a filter, a marine mangrove ecosystem can reduce antimicrobial resistance (AMR) or eliminate ARGs; however, its elimination mechanism remains unclear. This study investigated the spatiotemporal dynamic distribution of ARGs in two different types of mangrove habitats (shrimp ponds and virgin forests), within a subtropical gulf located in the Beibu Gulf, China, during dry and wet seasons by using metagenomics and real time quantitative polymerase chain reaction (RT-qPCR) analysis. As the key environmental factors, sulfide, salinity, and mobile genetic elements significantly were found to contribute to ARGs distribution, respectively. Wet and dry seasons influenced the dispersal of ARGs but did not affect the microbial community structure. Three potential biomarkers, TEM-116, smeD, and smeE, played key roles in seasonal differences. The key different genes in the biological relevance of absolute abundance were demonstrated by RT-qPCR. Co-occurrence network analysis indicated that high-abundance ARGs were distributed in a modular manner. For the first time, a risk index weighted by risk rank (RIR) was proposed and used to quantify the human risk of ARGs in the mangrove metagenome. The shrimp ponds during the wet season showed the highest RIR detected. In addition to offering a perspective on reducing AMR in mangrove wetlands, this study constructed the first spatiotemporal dynamic model of ARGs in the Beibu Gulf, China and contributed to revealing the global spread of ARGs. Meanwhile, this study proposes a new pipeline for assessing the risk of ARGs, while also exploring the concept of "One Health."

Analogous assembly mechanisms and functional guilds govern prokaryotic communities in mangrove ecosystems of China and South America.

As an important coastal "blue carbon sink," mangrove ecosystems contain microbial communities with an as-yet-unknown high species diversity. Exploring the assemblage and structure of sediment microbial communities therein can aid in a better understanding of their ecosystem functioning, such as carbon sequestration and other biogeochemical cycles in mangrove wetlands. However, compared to other biomes, the study of mangrove sediment microbiomes is limited, especially in diverse mangrove ecosystems at a large spatial scale, which may harbor microbial communities with distinct compositions and functioning. Here, we analyzed 380 sediment samples from 13 and 8 representative mangrove ecosystems, respectively, in China and South America and compared their microbial features. Although the microbial community compositions exhibited strong distinctions, the community assemblage in the two locations followed analogous patterns: the assemblages of the entire community, abundant taxa, rare taxa, and generalists were predominantly driven by stochastic processes with significant distance-decay patterns, while the assembly of specialists was more likely related to the behaviors of other organisms in or surrounding the mangrove ecosystems. In addition, co-occurrence and topological network analysis of mangrove sediment microbiomes underlined the dominance of sulfate-reducing prokaryotes in both the regions. Moreover, we found that more than 70% of the keystone and hub taxa were sulfate-reducing prokaryotes, implying their important roles in maintaining the linkage and stability of the mangrove sediment microbial communities. This study fills a gap in the large-scale analysis of microbiome features covering distantly located and diverse mangrove ecosystems. Here, we propose a suggestion to the Mangrove Microbiome Initiative that 16S rRNA sequencing protocols should be standardized with a unified primer to facilitate the global-scale analysis of mangrove microbiomes and further comparisons with the reference data sets from other biomes.IMPORTANCEMangrove wetlands are important ecosystems possessing valuable ecological functions for carbon storage, species diversity maintenance, and coastline stabilization. These functions are greatly driven or supported by microorganisms that make essential contributions to biogeochemical cycles in mangrove ecosystems. The mechanisms governing the microbial community assembly, structure, and functions are vital to microbial ecology but remain unclear. Moreover, studying these mechanisms of mangrove microbiomes at a large spatial scale can provide a more comprehensive insight into their universal features and can help untangle microbial interaction patterns and microbiome functions. In this study, we compared the mangrove microbiomes in a large spatial range and found that the assembly patterns and key functional guilds of the Chinese and South American mangrove microbiomes were analogous. The entire communities exhibited significant distance-decay patterns and were strongly governed by stochastic processes, while the assemblage of specialists may be merely associated with the behaviors of the organisms in mangrove ecosystems. Furthermore, our results highlight the dominance of sulfate-reducing prokaryotes in mangrove microbiomes and their key roles in maintaining the stability of community structure and functions.

Salt stress perception and metabolic regulation network analysis of a marine probiotic Meyerozyma guilliermondii GXDK6.

Introduction: Extremely salt-tolerant microorganisms play an important role in the development of functional metabolites or drug molecules. Methods: In this work, the salt stress perception and metabolic regulation network of a marine probiotic Meyerozyma guilliermondii GXDK6 were investigated using integrative omics technology. Results: Results indicated that GXDK6 could accept the salt stress signals from signal transduction proteins (e.g., phosphorelay intermediate protein YPD1), thereby contributing to regulating the differential expression of its relevant genes (e.g., CTT1, SOD) and proteins (e.g., catalase, superoxide dismutase) in response to salt stress, and increasing the salt-tolerant viability of GXDK6. Omics data also suggested that the transcription (e.g., SMD2), translation (e.g., MRPL1), and protein synthesis and processing (e.g., inner membrane protein OXA1) of upregulated RNAs may contribute to increasing the salt-tolerant survivability of GXDK6 by improving protein transport activity (e.g., Small nuclear ribonucleoprotein Sm D2), anti-apoptotic ability (e.g., 54S ribosomal protein L1), and antioxidant activity (e.g., superoxide dismutase). Moreover, up to 65.9% of the differentially expressed genes/proteins could stimulate GXDK6 to biosynthesize many salt tolerant-related metabolites (e.g., ß-alanine, D-mannose) and drug molecules (e.g., deoxyspergualin, calcitriol), and were involved in the metabolic regulation of GXDK6 under high NaCl stress. Discussion: This study provided new insights into the exploration of novel functional products and/or drugs from extremely salt-tolerant microorganisms.Graphical Abstract.

Sulfur metabolism in subtropical marine mangrove sediments fundamentally differs from other habitats as revealed by SMDB.

Shotgun metagenome sequencing provides the opportunity to recover underexplored rare populations and identify difficult-to-elucidate biochemical pathways. However, information on sulfur genes, including their sequences, is scattered in public databases. Here, we introduce SMDB ( https://smdb.gxu.edu.cn/ )-a manually curated database of sulfur genes based on an in-depth review of the scientific literature and orthology database. The SMDB contained a total of 175 genes and covered 11 sulfur metabolism processes with 395,737 representative sequences affiliated with 110 phyla and 2340 genera of bacteria/archaea. The SMDB was applied to characterize the sulfur cycle from five habitats and compared the microbial diversity of mangrove sediments with that of other habitats. The structure and composition of microorganism communities and sulfur genes were significantly different among the five habitats. Our results show that microorganism alpha diversity in mangrove sediments was significantly higher than in other habitats. Genes involved in dissimilatory sulfate reduction were abundant in subtropical marine mangroves and deep-sea sediments. The neutral community model results showed that microbial dispersal was higher in the marine mangrove ecosystem than in others habitats. The Flavilitoribacter of sulfur-metabolizing microorganism becomes a reliable biomarker in the five habitats. SMDB will assist researchers to analyze genes of sulfur cycle from the metagenomic efficiently.

Deciphering the biodesulfurization pathway employing marine mangrove Bacillus aryabhattai strain NM1-A2 according to whole genome sequencing and transcriptome analyses.

In the biogeochemical cycle, sulfur oxidation plays a vital role and is typically referred to as the elemental sulfur or reductive sulfide oxidation process. This study aimed to characterize a subtropical mangrove-isolated bacterial strain using biochemical, whole-genome, and transcriptome sequencing analyses to enhance our understanding of sulfur metabolism and biodegradation from a molecular genetic perspective. Strain NM1-A2 was characterized as Gram-positive and found to have a close molecular phylogenetic relationship with Bacillus aryabhattai. NM1-A2 efficiently converted dibenzothiophene (DBT) into 2-hydroxybiphenyl (2-HBP) via a 4S pathway with 95% efficiency, using enzymes encoded by the dsz operon (dszA, dszB, and dszC), which determine monooxygenases (DszA & DszC) and desulfinase (DszB). The whole-genome sequence of NM1-A2 had a length of approximately 5,257,678 bp and included 16 sulfur metabolism-related genes, featuring the ABC transport system, small subunit (ssu) and cysteine (cys) gene families, and adenosine 5'-phosphosulfate (APS) and 3'-phosphoadenosine-5'-phosphosulfate (PAPS) biosynthesis-related genes. Transcriptomic analysis of NM1-A2 using three sulfur groups-magnesium sulfate (MS), sulfur powder (SP), and sodium thiosulfate (ST) resulted in a significant number of differentially expressed genes (1200, 2304, and 2001, respectively). This analysis revealed that intracellular cysteine concentration directly regulated the expression of cys and ssu genes. Sulfate did not directly affect cys gene expression but repressed ssu gene expression. The cys gene expression levels decreased during the conversion of sulfate to sulfide and cysteine. The transcriptomic data was validated by analyzing the expression patterns of NM1-A2 using real-time quantitative PCR validation analysis. The expression levels of cysl, mccB, and nrnA were significantly upregulated, while cysH, metB, and sat were downregulated in the SP, ST, and MS groups, respectively. This research contributes to our understanding of marine mangrove microorganisms' bacterial efficiency through characterization, whole-genome, and transcriptome sequencing-based molecular degradation of organic compounds in the mangrove ecosystem, which may enhance nutrient availability.

Integration of Microbial Transformation Mechanism of Polyphosphate Accumulation and Sulfur Cycle in Subtropical Marine Mangrove Ecosystems with Spartina alterniflora Invasion.

Excessive phosphorus can lead to eutrophication in marine and coastal ecosystems. Sulfur metabolism-associated microorganisms stimulate biological phosphorous removal. However, the integrating co-biotransformation mechanism of phosphorus and sulfur in subtropical marine mangrove ecosystems with Spartina alterniflora invasion is poorly understood. In this study, an ecological model of the coupling biotransformation of sulfur and phosphorus is constructed using metagenomic analysis and quantitative polymerase chain reaction strategies. Phylogenetic analysis profiling, a distinctive microbiome with high frequencies of Gammaproteobacteria and Deltaproteobacteria, appears to be an adaptive characteristic of microbial structures in subtropical mangrove ecosystems. Functional analysis reveals that the levels of sulfate reduction, sulfur oxidation, and poly-phosphate (Poly-P) aggregation decrease with increasing depth. However, at depths of 25-50 cm in the mangrove ecosystems with S. alterniflora invasion, the abundance of sulfate reduction genes, sulfur oxidation genes, and polyphosphate kinase (ppk) significantly increased. A strong positive correlation was found among ppk, sulfate reduction, sulfur oxidation, and sulfur metabolizing microorganisms, and the content of sulfide was significantly and positively correlated with the abundance of ppk. Further microbial identification suggested that Desulfobacterales, Anaerolineales, and Chromatiales potentially drove the coupling biotransformation of phosphorus and sulfur cycling. In particular, Desulfobacterales exhibited dominance in the microbial community structure. Our findings provided insights into the simultaneous co-biotransformation of phosphorus and sulfur bioconversions in subtropical marine mangrove ecosystems with S. alterniflora invasion.

Coupling effects of nitrate reduction and sulfur oxidation in a subtropical marine mangrove ecosystem with Spartina alterniflora invasion.

The mangrove ecosystem has a high nitrate reduction capacity, which significantly alleviates severe nitrogen pollution. However, current research on nitrate reduction mechanisms in the mangrove ecosystem is limited. Furthermore, Spartina alterniflora invasion has disrupted the balance of the mangrove ecosystem and the effect of S. alterniflora on nitrate reduction has not yet been fully elucidated. Nitrate reduction was comprehensively investigated in a subtropical mangrove ecosystem in this study, which has been invaded by S. alterniflora for 40 years. Results showed that S. alterniflora significantly increased the relative and absolute abundance of nitrate reduction genes, especially nirS (nitrite reductase), in the mangrove ecosystem. Dissimilatory nitrate reduction to ammonium was the main pathway of nitrate reduction in the mangrove ecosystem. Nitrate reduction was mainly performed by Desulfobacterales and occurred in the shallow layers (0-10 cm) of mangrove sediments. A strong positive correlation was found between nitrate reduction and sulfur oxidation (especially sulfide oxidation), and the sulfide content was significantly positively correlated with the relative abundance of nitrate reduction genes. Moreover, 207 metagenomic assembled genomes (MAGs) were constructed, including 50 MAGs with high numbers (≥ 10) of nitrate reduction genes. This finding indicates that the dominant microbes had strong nitrate reduction potential in mangrove sediments. Our findings highlight the impact of S. alterniflora invasion on nitrate reduction in a subtropical marine mangrove ecosystem. This study provides new insights into our understanding of nitrogen pollution control and contributes to the exploration of new nitrogen-degrading microbes in mangrove ecosystems.

Effects of Spartina alterniflora Invasion on Nitrogen Fixation and Phosphorus Solubilization in a Subtropical Marine Mangrove Ecosystem.

Nitrogen fixation (NF) and phosphorus solubilization (PS) play a key role in maintaining the stability of mangrove ecosystems. In China, the invasion of Spartina alterniflora has brought a serious threat to the mangrove ecosystem. However, systematic research on NF and PS in mangrove sediments has not been conducted, and limited studies have focused on the response of NF and PS to S. alterniflora invasion, particularly at different sediment depths. In the present study, shotgun metagenomics and quantitative PCR were used to study the 0- to 100-cm sediment profile of the mangrove ecosystem in the Beibu Gulf of China. Results showed that the PS potential of mangrove sediments was primarily caused by enzymes encoded by phoA, phoD, ppx, ppa, and gcd genes. S. alterniflora changed environmental factors, such as total nitrogen, total phosphorus, and total organic carbon, and enhanced the potential of NF and PS in sediments. Moreover, most microorganisms involved in NF or PS (NFOPSMs) responded positively to the invasion of S. alterniflora. Cd, available iron, and salinity were the key environmental factors that affected the distribution of NF and PS genes (NFPSGs) and NFOPSMs. A strong coupling effect was observed between NF and PS in the mangrove ecosystem. S. alterniflora invasion enhanced the coupling of NF and PS and the interaction of microorganisms involved in NF and PS (NFAPSM), thereby promoting the turnover of NP and improving sediment quality. Finally, 108 metagenome-assembled genomes involved in NF or PS were reconstructed to further evaluate NFOPSMs. IMPORTANCE This study revealed the efficient nutrient cycling mechanism of mangroves. Positive coupling effects were observed in sediment quality, NF and PS processes, and NFOPSMs with the invasion of S. alterniflora. This research contributed to the understanding of the effects of S. alterniflora invasion on the subtropical mangrove ecosystem and provided theoretical guidance for mangrove protection, restoration, and soil management. Additionally, novel NFOPSMs provided a reference for the development of marine biological fertilizers.

MicrobioSee: A Web-Based Visualization Toolkit for Multi-Omics of Microbiology.

With the upgrade and development of the high-throughput sequencing technology, multi-omics data can be obtained at a low cost. However, mapping tools that existed for microbial multi-omics data analysis cannot satisfy the needs of data description and result in high learning costs, complex dependencies, and high fees for researchers in experimental biology fields. Therefore, developing a toolkit for multi-omics data is essential for microbiologists to save effort. In this work, we developed MicrobioSee, a real-time interactive visualization tool based on web technologies, which could visualize microbial multi-omics data. It includes 17 modules surrounding the major omics data of microorganisms such as the transcriptome, metagenome, and proteome. With MicrobioSee, methods for plotting are simplified in multi-omics studies, such as visualization of diversity, ROC, and enrichment pathways for DEGs. Subsequently, three case studies were chosen to represent the functional application of MicrobioSee. Overall, we provided a concise toolkit along with user-friendly, time-saving, cross-platform, and source-opening for researchers, especially microbiologists without coding experience. MicrobioSee is freely available at https://microbiosee.gxu.edu.cn.

Whole-Genome and Transcriptome Sequencing-Based Characterization of Bacillus Cereus NR1 From Subtropical Marine Mangrove and Its Potential Role in Sulfur Metabolism.

Sulfur, organosulfur compounds, and sulfides are essential parts of life. Microbial sulfate assimilation is among the most active and ancient metabolic activities in the sulfur cycle that operates in various ecosystems. We analyzed the molecular basis of bacterial characterization. NR1 was isolated and purified from mangrove sediments. Whole-genome sequencing indicated that the NR1 isolate was closely related to Bacillus cereus. The genome contained 5,305 functional genes with a total length of 5,420,664 bp, a GC content of 35.62%, 42 rRNA, and 107 tRNA. DBT-grown cultures exhibited DBT utilization, fleeting emergence of DBT sulfone (DBTO2), and formation of 2-hydroxybiphenyl (2-HBP). Molecular analysis of the PCR products' dsz operon revealed the presence of dszA, dszB, and dszC genes, which encoded for NR1's 90% DBT desulfurization activity. Furthermore, 17 sulfur metabolism-related genes, including genes involved in assimilation sulfate reduction, APS and PAPS, and the cys, ssu, and TST gene families, were identified. In sulfate media, alkenesulfonate was converted to sulfite and inhibited ssu enzymes. Downregulated cysK variants were associated with nrnA expression and the regulation of L-cysteine synthesis. These findings established a scientific foundation for further research and application of bacteria to mangrove rehabilitation and ecological treatment by evaluating the bacterial characterization and sulfur degradation metabolic pathway. We used whole-genome and transcriptome sequencing to examine their genetic characteristics.

Copper Tolerance Mechanism of the Novel Marine Multi-Stress Tolerant Yeast Meyerozyma guilliermondii GXDK6 as Revealed by Integrated Omics Analysis.

Various agricultural products used in food fermentation are polluted by heavy metals, especially copper, which seriously endangers human health. Methods to remove copper with microbial strategies have gained interests. A novel Meyerozyma guilliermondii GXDK6 could survive independently under high stress of copper (1400 ppm). The copper tolerance mechanism of GXDK6 was revealed by integrated omics in this work. Whole-genome analysis showed that nine genes (i.e., CCC2, CTR3, FRE2, GGT, GST, CAT, SOD2, PXMP4, and HSP82) were related to GXDK6 copper tolerance. Copper stress elevated glutathione metabolism-related gene expression, glutathione content, and glutathione sulfur transferase activity, suggesting enhanced copper conjugation and detoxification in cells. The inhibited copper uptake by Ctr3 and enhanced copper efflux by Ccc2 contributed to the decrease in intracellular copper concentration. The improved expression of antioxidant enzyme genes (PXMP4, SOD2, and CAT), accompanied by the enhanced activities of antioxidant enzymes (peroxidase, superoxide dismutase, and catalase), decreased copper-induced reactive oxygen species production, protein carbonylation, lipid peroxidation, and cell death. The metabolite D-mannose against harsh stress conditions was beneficial to improving copper tolerance. This study contributed to understanding the copper tolerance mechanism of M. guilliermondii and its application in removing copper during fermentation.

Desulfobacterales stimulates nitrate reduction in the mangrove ecosystem of a subtropical gulf.

The amount of nitrogen compounds discharged into the natural environment has increased drastically due to frequent human activities and led to worsening pollution. The mangrove ecosystem can remove nitrogen pollution, in this regard, few studies had focused on the relationship among nitrogen cycling genes, environmental factors, and taxonomic composition. In this study, shotgun metagenomic sequencing and quantitative polymerase chain reaction were used to understand the nitrogen cycle in the subtropical mangrove ecosystem in the Beibu Gulf of China. Eight nitrogen cycling pathways were annotated. Nitrogen metabolism activities were significantly higher in the wet season than those in the dry season. The most abundant genes were those related to the synthesis and degradation of organic nitrogen, followed by the genes involved in nitrate reduction (denitrification, dissimilation/assimilation nitrate reduction). Furthermore, dissimilation nitrate reduction was the main nitrate reduction pathway. Desulfobacterales plays an important role in nitrogen cycling and contributes 12% of the genes of nitrogen pathways on average; as such, a strong coupling relationship exists among nitrogen cycling, sulfur cycling, and carbon cycling in the mangrove ecosystem. Nitrogen pollution in the mangrove wetland can be efficiently alleviated by nitrate reduction of Desulfobacterales. Nevertheless, only 50% of genes can be matched among the known species, suggesting that many unknown microorganisms in the mangrove ecosystem can perform nitrogen cycling. Total phosphorus, available iron, and total organic carbon are the key environmental factors that influence the distribution of nitrogen cycling genes, related pathways, and the taxonomic composition. Our study clearly illustrates how the mangrove ecosystem mitigates nitrogen pollution through Desulfobacterales. This finding could provide a research reference for the whole nitrogen cycling in the mangrove ecosystem.

Whole genome sequencing and metabolomics analyses reveal the biosynthesis of nerol in a multi-stress-tolerant Meyerozyma guilliermondii GXDK6.

BACKGROUND: Nerol (C10H18O), an acyclic monoterpene, naturally presents in plant essential oils, and is used widely in food, cosmetics and pharmaceuticals as the valuable fragrance. Meanwhile, chemical synthesis is the only strategy for large-scale production of nerol, and the disadvantages of chemical synthesis greatly limit the production and its application. These defects drive the interests of researchers shift to the production of nerol by eco-friendly methods known as biosynthesis methods. However, the main technical bottleneck restricting the biosynthesis of nerol is the lacking of corresponding natural aroma-producing microorganisms. RESULTS: In this study, a novel multi-stress-tolerant probiotics Meyerozyma guilliermondii GXDK6 with aroma-producing properties was identified by whole genome sequencing and metabolomics technology. GXDK6 showed a broad pH tolerance in the range of 2.5-10.0. The species also showed salt tolerance with up to 12% NaCl and up to 18% of KCl or MgCl2. GXDK6 exhibited heavy-metal Mn2+ tolerance of up to 5494 ppm. GXDK6 could also ferment with a total of 21 kinds of single organic matter as the carbon source, and produce abundant aromatic metabolites. Results from the gas chromatography-mass spectrometry indicated the production of 8-14 types of aromatic metabolites (isopentanol, nerol, geraniol, phenylethanol, isobutanol, etc.) when GXDK6 was fermented up to 72 h with glucose, sucrose, fructose, or xylose as the single carbon source. Among them, nerol was found to be a novel aromatic metabolite from GXDK6 fermentation, and its biosynthesis mechanism had also been further revealed. CONCLUSION: A novel aroma-producing M. guilliermondii GXDK6 was identified successfully by whole genome sequencing and metabolomics technology. GXDK6 showed high multi-stress-tolerant properties with acid-base, salty, and heavy-metal environments. The aroma-producing mechanism of nerol in GXDK6 had also been revealed. These findings indicated the aroma-producing M. guilliermondii GXDK6 with multi-stress-tolerant properties has great potential value in the fermentation industry.

Multi-Omics Analysis of Lipid Metabolism for a Marine Probiotic Meyerozyma guilliermondii GXDK6 Under High NaCl Stress.

Investigating microbial lipid regulation contributes to understanding the lipid-dependent signal transduction process of cells and helps to improve the sensitivity of microorganisms to environmental factors by interfering with lipid metabolism, thus beneficial for constructing advanced cell factories of novel molecular drugs. Integrated omics technology was used to systematically reveal the lipid metabolism mechanism of a marine Meyerozyma guilliermondii GXDK6 under high NaCl stress and test the sensitivity of GXDK6 to antibiotics when its lipid metabolism transformed. The omics data showed that when GXDK6 perceived 10% NaCl stress, the expression of AYR1 and NADPH-dependent 1-acyldihydroxyacetone phosphate reductase was inhibited, which weaken the budding and proliferation of cell membranes. This finding was further validated by decreased 64.39% of OD600 under 10% NaCl stress when compared with salt-free stress. In addition, salt stress promoted a large intracellular accumulation of glycerol, which was also verified by exogenous addition of glycerol. Moreover, NaCl stress remarkably inhibited the expression of drug target proteins (such as lanosterol 14-alpha demethylase), thereby increasing sensitivity to fluconazole. This study provided new insights into the molecular mechanism involved in the regulation of lipid metabolism in Meyerozyma guilliermondii strain and contributed to developing new methods to improve the effectiveness of killing fungi with lower antibiotics.

Characterization of NMCR-2, a new non-mobile colistin resistance enzyme: implications for an MCR-8 ancestor.

MCR-4 and MCR-8 are two recently identified members of an ongoing MCR family of colistin resistance. Although that aquatic reservoir for MCR-4 is proposed, the origin and mechanism of MCR-8 is poorly understood. Here we report a previously unrecognized non-mobile colistin resistance enzyme, termed NMCR-2, originating from the plant pathogen Kosakonia pseudosacchari. NMCR-2 (551aa) gives 67.3% identity to MCR-8 (565aa). NMCR-2 is placed as a progenitor/ancestor for MCR-8 in phylogeny of MCR members. Genetic study reveals that nmcr-2 is comparable to mcr-8 in the ability of producing phenotypic colistin resistance. Biochemical analyses determine that these two enzymes catalyse the transfer of PEA from the donor PE lipid substrate to the recipient lipid A molecule by a putative 'ping-pong' trade-off. Further experiment of protein engineering demonstrates that the two motifs (TM region and catalytic domain) of NMCR-2 are functionally exchangeable with that of MCR-8, rather than MCR-1. Physiological impacts of nmcr-2 and/or mcr-8 are detected in Escherichia coli, featuring with fitness cost. Evidently, the action and mechanism of NMCR-2 is analogous to that of MCR-8. Therefore, our finding underlines that NMCR-2 might be a possible progenitor of MCR-8.

VV Reduction by Polaromonas spp. in Vanadium Mine Tailings.

Vanadium (V) is an important metal with critical industrial and medical applications. Elevated V contamination, however, can be a threat to the environment and human health. Microorganisms can reduce the more toxic and mobile VV to the less toxic and immobile VIV, which could be a detoxification and energy metabolism strategy adopted by V-reducing bacteria (VRB). The limited understanding of microbial responses to V contamination and the mechanisms for VV reduction, however, hamper our capability to attenuate V contamination. This study focused on determining the microbial responses to elevated V concentration and the mechanisms of VV reduction in V tailings. The bacterial communities were characterized and compared between the V tailings and the less contaminated adjacent mineral soils. Further, VV-reducing enrichments indicated that bacteria associated with Polaromonas, a genus belonging to the family Burkholderiaceae, were potentially responsible for VV reduction. Retrieved metagenome-assembled genomes (MAGs) suggested that the Polaromonas spp. encoded genes (cymA, omcA, and narG) were responsible for VV reduction. Additionally, Polaromonas spp. was metabolically versatile and could use both organic and inorganic electron donors. The metabolic versatility of Polaromonas spp. may be important for its ability to flourish in the V tailings.

Patterns and drivers of Vibrio isolates phylogenetic diversity in the Beibu Gulf, China.

Members of the genus Vibrio are ubiquitous in aquatic environments and can be found either in a culturable or a viable but nonculturable (VBNC) state. Despite widespread concerns as to how to define the occurrence and dynamics of Vibrio populations by culture-independent approaches, further physiological research and relevant biotechnological developments will require the isolation and cultivation of the microbes from various environments. The present work provides data and perspectives on our understanding of culturable Vibrio community structure and diversity in the Beibu Gulf. Finally, we isolated 1,037 strains of Vibrio from 45 samples and identified 18 different species. Vibrio alginolyticus, V. cyclitrophicus, V. tasmaniensis, V. brasiliensis, and V. splendidus were the dominant species that had regional distribution characteristics. The correlation between the quantitative distribution and community structure of culturable Vibrio and environmental factors varied with the Vibrio species and geographical locations. Among them, salinity, nitrogen, and phosphorus were the main factors affecting the diversity of culturable Vibrio. These results help to fill a knowledge gap on Vibrio diversity and provide data for predicting and controlling pathogenic Vibrio outbreaks in the Beibu Gulf.

Harnessing efficient multiplex PCR methods to detect the expanding Tet(X) family of tigecycline resistance genes.

A growing number of tet(X)-type tigecycline resistance determinants [tet(X1) to tet(X5)] constitutes an expanding family of tetracycline-inactivating enzymes, posing a potential risk to global public health. Here, we report the development of an efficient multiplex PCR method to detect the family of tet(X) variants. This method is successfully applied in the screen and validation of tet(X) genes in the field and clinic bacterial samples. In addition, we found that the formerly proposed tet(X1) is a premature truncated version by the inappropriate annotation, and fixed this error. Overall, it might be the first genetic tool for the detection of different Tet(X) members.