A Reverse Ecology Approach Based on a Biological Definition of Microbial Populations.

Delineating ecologically meaningful populations among microbes is important for identifying their roles in environmental and host-associated microbiomes. Here, we introduce a metric of recent gene flow, which when applied to co-existing microbes, identifies congruent genetic and ecological units separated by strong gene flow discontinuities from their next of kin. We then develop a pipeline to identify genome regions within these units that show differential adaptation and allow mapping of populations onto environmental variables or host associations. Using this reverse ecology approach, we show that the human commensal bacterium Ruminococcus gnavus breaks up into sharply delineated populations that show different associations with health and disease. Defining populations by recent gene flow in this way will facilitate the analysis of bacterial and archaeal genomes using ecological and evolutionary theory developed for plants and animals, thus allowing for testing unifying principles across all biology.

A qrr noncoding RNA deploys four different regulatory mechanisms to optimize quorum-sensing dynamics.

Quorum sensing is a cell-cell communication process that bacteria use to transition between individual and social lifestyles. In vibrios, homologous small RNAs called the Qrr sRNAs function at the center of quorum-sensing pathways. The Qrr sRNAs regulate multiple mRNA targets including those encoding the quorum-sensing regulatory components luxR, luxO, luxM, and aphA. We show that a representative Qrr, Qrr3, uses four distinct mechanisms to control its particular targets: the Qrr3 sRNA represses luxR through catalytic degradation, represses luxM through coupled degradation, represses luxO through sequestration, and activates aphA by revealing the ribosome binding site while the sRNA itself is degraded. Qrr3 forms different base-pairing interactions with each mRNA target, and the particular pairing strategy determines which regulatory mechanism occurs. Combined mathematical modeling and experiments show that the specific Qrr regulatory mechanism employed governs the potency, dynamics, and competition of target mRNA regulation, which in turn, defines the overall quorum-sensing response.

The coral pathogen Vibrio coralliilyticus uses a T6SS to secrete a group of novel anti-eukaryotic effectors that contribute to virulence.

Vibrio coralliilyticus is a pathogen of coral and shellfish, leading to devastating economic and ecological consequences worldwide. Although rising ocean temperatures correlate with increased V. coralliilyticus pathogenicity, the specific molecular mechanisms and determinants contributing to virulence remain poorly understood. Here, we systematically analyzed the type VI secretion system (T6SS), a contact-dependent toxin delivery apparatus, in V. coralliilyticus. We identified 2 omnipresent T6SSs that are activated at temperatures in which V. coralliilyticus becomes virulent; T6SS1 is an antibacterial system mediating interbacterial competition, whereas T6SS2 mediates anti-eukaryotic toxicity and contributes to mortality during infection of an aquatic model organism, Artemia salina. Using comparative proteomics, we identified the T6SS1 and T6SS2 toxin arsenals of 3 V. coralliilyticus strains with distinct disease etiologies. Remarkably, T6SS2 secretes at least 9 novel anti-eukaryotic toxins comprising core and accessory repertoires. We propose that T6SSs differently contribute to V. coralliilyticus's virulence: T6SS2 plays a direct role by targeting the host, while T6SS1 plays an indirect role by eliminating competitors.

Frequent nonhomologous replacement of replicative helicase loaders by viruses in Vibrionaceae.

Several microbial genomes lack textbook-defined essential genes. If an essential gene is absent from a genome, then an evolutionarily independent gene of unknown function complements its function. Here, we identified frequent nonhomologous replacement of an essential component of DNA replication initiation, a replicative helicase loader gene, in Vibrionaceae. Our analysis of Vibrionaceae genomes revealed two genes with unknown function, named vdhL1 and vdhL2, that were substantially enriched in genomes without the known helicase-loader genes. These genes showed no sequence similarities to genes with known function but encoded proteins structurally similar with a viral helicase loader. Analyses of genomic syntenies and coevolution with helicase genes suggested that vdhL1/2 encodes a helicase loader. The in vitro assay showed that Vibrio harveyi VdhL1 and Vibrio ezurae VdhL2 promote the helicase activity of DnaB. Furthermore, molecular phylogenetics suggested that vdhL1/2 were derived from phages and replaced an intrinsic helicase loader gene of Vibrionaceae over 20 times. This high replacement frequency implies the host's advantage in acquiring a viral helicase loader gene.

Identification of promoter activity in gene-less cassettes from Vibrionaceae superintegrons.

Integrons are genetic platforms that acquire new genes encoded in integron cassettes (ICs), building arrays of adaptive functions. ICs generally encode promoterless genes, whose expression relies on the platform-associated Pc promoter, with the cassette array functioning as an operon-like structure regulated by the distance to the Pc. This is relevant in large sedentary chromosomal integrons (SCIs) carrying hundreds of ICs, like those in Vibrio species. We selected 29 gene-less cassettes in four Vibrio SCIs, and explored whether their function could be related to the transcription regulation of adjacent ICs. We show that most gene-less cassettes have promoter activity on the sense strand, enhancing the expression of downstream cassettes. Additionally, we identified the transcription start sites of gene-less ICs through 5'-RACE. Accordingly, we found that most of the superintegron in Vibrio cholerae is not silent. These promoter cassettes can trigger the expression of a silent dfrB9 cassette downstream, increasing trimethoprim resistance >512-fold in V. cholerae and Escherichia coli. Furthermore, one cassette with an antisense promoter can reduce trimethoprim resistance when cloned downstream. Our findings highlight the regulatory role of gene-less cassettes in the expression of adjacent cassettes, emphasizing their significance in SCIs and their clinical importance if captured by mobile integrons.

Recovering high-quality bacterial genomes from cross-contaminated cultures: a case study of marine Vibrio campbellii.

BACKGROUND: Environmental monitoring of bacterial pathogens is critical for disease control in coastal marine ecosystems to maintain animal welfare and ecosystem function and to prevent significant economic losses. This requires accurate taxonomic identification of environmental bacterial pathogens, which often cannot be achieved by commonly used genetic markers (e.g., 16S rRNA gene), and an understanding of their pathogenic potential based on the information encoded in their genomes. The decreasing costs of whole genome sequencing (WGS), combined with newly developed bioinformatics tools, now make it possible to unravel the full potential of environmental pathogens, beyond traditional microbiological approaches. However, obtaining a high-quality bacterial genome, requires initial cultivation in an axenic culture, which is a bottleneck in environmental microbiology due to cross-contamination in the laboratory or isolation of non-axenic strains. RESULTS: We applied WGS to determine the pathogenic potential of two Vibrio isolates from coastal seawater. During the analysis, we identified cross-contamination of one of the isolates and decided to use this dataset to evaluate the possibility of bioinformatic contaminant removal and recovery of bacterial genomes from a contaminated culture. Despite the contamination, using an appropriate bioinformatics workflow, we were able to obtain high quality and highly identical genomes (Average Nucleotide Identity value 99.98%) of one of the Vibrio isolates from both the axenic and the contaminated culture. Using the assembled genome, we were able to determine that this isolate belongs to a sub-lineage of Vibrio campbellii associated with several diseases in marine organisms. We also found that the genome of the isolate contains a novel Vibrio plasmid associated with bacterial defense mechanisms and horizontal gene transfer, which may offer a competitive advantage to this putative pathogen. CONCLUSIONS: Our study shows that, using state-of-the-art bioinformatics tools and a sufficient sequencing effort, it is possible to obtain high quality genomes of the bacteria of interest and perform in-depth genomic analyses even in the case of a contaminated culture. With the new isolate and its complete genome, we are providing new insights into the genomic characteristics and functional potential of this sub-lineage of V. campbellii. The approach described here also highlights the possibility of recovering complete bacterial genomes in the case of non-axenic cultures or obligatory co-cultures.

Prophages carrying Zot toxins on different Vibrio genomes: A comprehensive assessment using multilayer networks.

Vibrios, a group of bacteria that are among the most abundant in marine environments, include several species such as Vibrio cholerae and Vibrio parahaemolyticus, which can be pathogenic to humans. Some species of Vibrio contain prophages within their genomes. These prophages can carry genes that code for toxins, such as the zonula occludens toxin (Zot), which contribute to bacterial virulence. Understanding the association between different Vibrio species, prophages and Zot genes can provide insights into their ecological interactions. In this study, we evaluated 4619 Vibrio genomes from 127 species to detect the presence of prophages carrying the Zot toxin. We found 2030 potential prophages with zot-like genes in 43 Vibrio species, showing a non-random association within a primarily modular interaction network. Some prophages, such as CTX or Vf33, were associated with specific species. In contrast, prophages phiVCY and VfO3K6 were found in 28 and 20 Vibrio species, respectively. We also identified six clusters of Zot-like sequences in prophages, with the ZOT2 cluster being the most frequent, present in 34 Vibrio species. This analysis helps to understand the distribution patterns of zot-containing prophages across Vibrio genomes and the potential routes of Zot-like toxin dissemination.

Identification of putative coral pathogens in endangered Caribbean staghorn coral using machine learning.

Coral diseases contribute to the rapid decline in coral reefs worldwide, and yet coral bacterial pathogens have proved difficult to identify because 16S rRNA gene surveys typically identify tens to hundreds of disease-associate bacteria as putative pathogens. An example is white band disease (WBD), which has killed up to 95% of the now-endangered Caribbean Acropora corals since 1979, yet the pathogen is still unknown. The 16S rRNA gene surveys have identified hundreds of WBD-associated bacterial amplicon sequencing variants (ASVs) from at least nine bacterial families with little consensus across studies. We conducted a multi-year, multi-site 16S rRNA gene sequencing comparison of 269 healthy and 143 WBD-infected Acropora cervicornis and used machine learning modelling to accurately predict disease outcomes and identify the top ASVs contributing to disease. Our ensemble ML models accurately predicted disease with greater than 97% accuracy and identified 19 disease-associated ASVs and five healthy-associated ASVs that were consistently differentially abundant across sampling periods. Using a tank-based transmission experiment, we tested whether the 19 disease-associated ASVs met the assumption of a pathogen and identified two pathogenic candidate ASVs-ASV25 Cysteiniphilum litorale and ASV8 Vibrio sp. to target for future isolation, cultivation, and confirmation of Henle-Koch's postulate via transmission assays.

A three-step "ping-pong" mechanism of a GH20 ß-N-acetylglucosaminidase from Vibrio campbellii belonging to a major Clade A-I of the phylogenetic tree of the enzyme superfamily.

ß-N-acetylglucosaminidase (GlcNAcase) is an essential biocatalyst in chitin assimilation by marine Vibrio species, which rely on chitin as their main carbon source. Structure-based phylogenetic analysis of the GlcNAcase superfamily revealed that a GlcNAcase from Vibrio campbellii, formerly named V. harveyi, (VhGlcNAcase) belongs to a major clade, Clade A-I, of the phylogenetic tree. Pre-steady-state and steady-state kinetic analysis of the reaction catalysed by VhGlcNAcase with the fluorogenic substrate 4-methylumbelliferyl N-acetyl-ß-D-glucosaminide suggested the following mechanism: (1) the Michaelis-Menten complex is formed in a rapid enzyme-substrate equilibrium with a Kd of 99.1 ± 1 µM. (2) The glycosidic bond is cleaved by the action of the catalytic residue Glu438, followed by the rapid release of the aglycone product with a rate constant (k2) of 53.3 ± 1 s-1. (3) After the formation of an oxazolinium ion intermediate with the assistance of Asp437, the anomeric carbon of the transition state is attacked by a catalytic water, followed by release of the glycone product with a rate constant (k3) of 14.6 s-1, which is rate-limiting. The result clearly indicated a three-step "ping-pong" mechanism for VhGlcNAcase.

Deciphering nutritional stress responses via knowledge-enriched transcriptomics for microbial engineering.

Understanding diverse bacterial nutritional requirements and responses is foundational in microbial research and biotechnology. In this study, we employed knowledge-enriched transcriptomic analytics to decipher complex stress responses of Vibrio natriegens to supplied nutrients, aiming to enhance microbial engineering efforts. We computed 64 independently modulated gene sets that comprise a quantitative basis for transcriptome dynamics across a comprehensive transcriptomics dataset containing a broad array of nutrient conditions. Our approach led to the i) identification of novel transporter systems for diverse substrates, ii) a detailed understanding of how trace elements affect metabolism and growth, and iii) extensive characterization of nutrient-induced stress responses, including osmotic stress, low glycolytic flux, proteostasis, and altered protein expression. By clarifying the relationship between the acetate-associated regulon and glycolytic flux status of various nutrients, we have showcased its vital role in directing optimal carbon source selection. Our findings offer deep insights into the transcriptional landscape of bacterial nutrition and underscore its significance in tailoring strain engineering strategies, thereby facilitating the development of more efficient and robust microbial systems for biotechnological applications.

Osmotic stress response of the coral and oyster pathogen Vibrio coralliilyticus: acquisition of catabolism gene clusters for the compatible solute and signaling molecule myo-inositol.

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 Vibrio coralliilyticus were unknown. In this study, we showed that to alleviate osmotic stress V. coralliilyticus biosynthesized glycine betaine (GB) and transported into the cell choline, GB, ectoine, dimethylglycine, and dimethylsulfoniopropionate, but not myo-inositol. Myo-inositol is a stress protectant and a signaling molecule that is biosynthesized and used by algae. Bioinformatics identified myo-inositol (iol) catabolism clusters in V. coralliilyticus and other Vibrio, Photobacterium, Grimontia, and Enterovibrio species. Growth pattern analysis demonstrated that V. coralliilyticus utilized myo-inositol as a sole carbon source, with a short lag time of 3 h. An iolG deletion mutant, which encodes an inositol dehydrogenase, was unable to grow on myo-inositol. Within the iol clusters were an MFS-type (iolT1) and an ABC-type (iolXYZ) transporter and analyses showed that both transported myo-inositol. IolG and IolA phylogeny among Vibrionaceae species showed different evolutionary histories indicating multiple acquisition events. Outside of Vibrionaceae, IolG was most closely related to IolG from a small group of Aeromonas fish and human pathogens and Providencia species. However, IolG from hypervirulent A. hydrophila strains clustered with IolG from Enterobacter, and divergently from Pectobacterium, Brenneria, and Dickeya plant pathogens. The iol cluster was also present within Aliiroseovarius, Burkholderia, Endozoicomonas, Halomonas, Labrenzia, Marinomonas, Marinobacterium, Cobetia, Pantoea, and Pseudomonas, of which many species were associated with marine flora and fauna.IMPORTANCEHost associated bacteria such as Vibrio coralliilyticus encounter competition for nutrients and have evolved metabolic strategies to better compete for food. Emerging studies show that myo-inositol is exchanged in the coral-algae symbiosis, is likely involved in signaling, but is also an osmolyte in algae. The bacterial consumption of myo-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 myo-inositol metabolism is complex, acquired multiple times in Vibrio, but acquired once in many bacterial plant pathogens. Further analysis also showed that a conserved iol cluster is prevalent among many marine species (commensals, mutualists, and pathogens) associated with marine flora and fauna, algae, sponges, corals, molluscs, crustaceans, and fish.

Mobile-CRISPRi as a powerful tool for modulating Vibrio gene expression.

CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) is a gene knockdown method that uses a deactivated Cas9 protein (dCas9) that binds a specific gene target locus dictated by an encoded guide RNA (sgRNA) to block transcription. Mobile-CRISPRi is a suite of modular vectors that enable CRISPRi knockdowns in diverse bacteria by integrating IPTG-inducible dcas9 and sgRNA genes into the genome using Tn7 transposition. Here, we show that the Mobile-CRISPRi system functions robustly and specifically in multiple Vibrio species: Vibrio cholerae, Vibrio fischeri, Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio campbellii. We demonstrate efficacy by targeting both essential and non-essential genes that function to produce defined, measurable phenotypes: bioluminescence, quorum sensing, cell division, and growth arrest. We anticipate that Mobile-CRISPRi will be used in Vibrio species to systematically probe gene function and essentiality in various behaviors and native environments.IMPORTANCEThe genetic manipulation of bacterial genomes is an invaluable tool in experimental microbiology. The development of CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) tools has revolutionized genetics in many organisms, including bacteria. Here, we optimized the use of Mobile-CRISPRi in five Vibrio species, each of which has significant impacts on marine environments and organisms that include squid, shrimp, shellfish, finfish, corals, and multiple of which pose direct threats to human health. The Mobile-CRISPRi technology is easily adaptable, moveable from strain to strain, and enables researchers to selectively turn off gene expression. Our experiments demonstrate Mobile-CRISPRi is effective and robust at repressing gene expression of both essential and non-essential genes in Vibrio species.

Vibrio natriegens genome-scale modeling reveals insights into halophilic adaptations and resource allocation.

Vibrio natriegens is a Gram-negative bacterium with an exceptional growth rate that has the potential to become a standard biotechnological host for laboratory and industrial bioproduction. Despite this burgeoning interest, the current lack of organism-specific qualitative and quantitative computational tools has hampered the community's ability to rationally engineer this bacterium. In this study, we present the first genome-scale metabolic model (GSMM) of V. natriegens. The GSMM (iLC858) was developed using an automated draft assembly and extensive manual curation and was validated by comparing predicted yields, central metabolic fluxes, viable carbon substrates, and essential genes with empirical data. Mass spectrometry-based proteomics data confirmed the translation of at least 76% of the enzyme-encoding genes predicted to be expressed by the model during aerobic growth in a minimal medium. iLC858 was subsequently used to carry out a metabolic comparison between the model organism Escherichia coli and V. natriegens, leading to an analysis of the model architecture of V. natriegens' respiratory and ATP-generating system and the discovery of a role for a sodium-dependent oxaloacetate decarboxylase pump. The proteomics data were further used to investigate additional halophilic adaptations of V. natriegens. Finally, iLC858 was utilized to create a Resource Balance Analysis model to study the allocation of carbon resources. Taken together, the models presented provide useful computational tools to guide metabolic engineering efforts in V. natriegens.

Microbial and transcriptional response of Acropora valida and Turbinaria peltata to Vibrio coralliilyticus challenge: insights into corals disease resistance.

BACKGROUND: Coral diseases are significant drivers of global coral reef degradation, with pathogens dominated by Vibrio coralliilyticus playing a prominent role in the development of coral diseases. Coral phenotype, symbiotic microbial communities, and host transcriptional regulation have been well-established as factors involved in determining coral disease resistance, but the underlying mechanisms remain incompletely understood. METHODS: This study employs high-throughput sequencing to analyse the symbiotic microbial and transcriptional response of the hosts in order to evaluate the disease resistance of Acropora valida and Turbinaria peltata exposed to Vibrio coralliilyticus. RESULTS: A. valida exhibited pronounced bleaching and tissue loss within 7 h of pathogen infection, whereas T. peltata showed no signs of disease throughout the experiment. Microbial diversity analyses revealed that T. peltata had a more flexible microbial community and a higher relative abundance of potential beneficial bacteria compared to A. valida. Although Vibrio inoculation resulted in a more significant decrease in the Symbiodiniaceae density of A. valida compared to that of T. peltata, it did not lead to recombination of the coral host and Symbiodiniaceae in either coral species. RNA-seq analysis revealed that the interspecific differences in the transcriptional regulation of hosts after Vibrio inoculation. Differentially expressed genes in A. valida were mainly enriched in the pathways associated with energy supply and immune response, such as G protein-coupled receptor signaling, toll-like receptor signaling, regulation of TOR signaling, while these genes in T. peltata were mainly involved in the pathway related to immune homeostasis and ion transport, such as JAK-STAT signaling pathway and regulation of ion transport. CONCLUSIONS: Pathogenic challenges elicit different microbial and transcriptional shifts across coral species. This study offers novel insights into molecular mechanisms of coral resistance to disease.

Deletion of speA and aroC genes impacts the pathogenicity of Vibrio anguillarum in spotted sea bass.

Vibrio anguillarum is one of the major pathogens responsible for bacterial infections in marine environments, causing significant impacts on the aquaculture industry. The misuse of antibiotics leads to bacteria developing multiple drug resistances, which is detrimental to the development of the fisheries industry. In contrast, live attenuated vaccines are gradually gaining acceptance and widespread recognition. In this study, we constructed a double-knockout attenuated strain, V. anguillarum ΔspeA-aroC, to assess its potential for preparing a live attenuated vaccine. The research results indicate a significant downregulation of virulence-related genes, including Type VI secretion system, Type II secretion system, biofilm synthesis, iron uptake system, and other related genes, in the mutant strain. Furthermore, the strain lacking the genes exhibited a 67.47% reduction in biofilm formation ability and increased sensitivity to antibiotics. The mutant strain exhibited significantly reduced capability in evading host immune system defenses and causing in vivo infections in spotted sea bass (Lateolabrax maculatus), with an LD50 that was 13.93 times higher than that of the wild-type V. anguillarum. Additionally, RT-qPCR analysis of immune-related gene expression in spotted sea bass head kidney and spleen showed a weakened immune response triggered by the knockout strain. Compared to the wild-type V. anguillarum, the mutant strain caused reduced levels of tissue damage. The results demonstrate that the deletion of speA and aroC significantly reduces the biosynthesis of biofilms in V. anguillarum, leading to a decrease in its pathogenicity. This suggests a crucial role of biofilms in the survival and invasive capabilities of V. anguillarum.

Δfur mutant as a potential live attenuated vaccine (LAV) candidate protects American eels (Anguilla rostrata) from Vibrio harveyi infection.

The eel farming industry is highly susceptible to Vibriosis. Although various types of vaccines against Vibriosis have been investigated, there is limited research on decreasing the virulence of Vibrions through gene knockout and utilizing it as live attenuated vaccines (LAV). In this study, we aim to develop a LAV candidate against Vibrio harveyi infection in American eels (Anguilla rostrata) using a ferric uptake regulator (fur) gene mutant strain of V. harveyi (Δfur mutant). After the eels were administrated with the Δfur mutant at the dose of 4 × 102 cfu/g body weight, the phagocytic activity of the leucocytes, plasma IgM antibody titers, activity of lysozyme and Superoxide Dismutase (SOD) enzyme, and gene expression levels of 18 immune related proteins were detected to evaluate the protection effect of the LAV. Preliminary findings suggest that the LAV achieved over 60% relative percent survival (RPS) after the American eels were challenged by a wild-type strain of V. harveyi infection on 28 and 42 days post the immunization (dpi). The protection was mainly attributed to increased plasma IgM antibody titers, higher levels of lysozyme, enhanced activity of SOD and some regulated genes encoded immune related proteins. Together, the Δfur mutant strain of V. harveyi, as a novel LAV vaccine, demonstrates promising protective effects against V. harveyi infection in American eels, thus presenting a potential candidate vaccine for fish farming.

Metagenomic analysis of pathogenic bacteria and virulence factor genes in coastal sediments from highly urbanized cities of India.

A metagenomic approach was employed to investigate the diversity and distribution of Virulence Factors Genes (VFGs) and Pathogenic Bacteria (PB) in sediment samples collected from highly urbanized cities along the Indian coastline. Among the study locations, Mumbai, Veraval and Paradeep showed a higher abundance of PB, with Vibrio and Pseudomonas as dominant at the genus level, and Escherichia coli and Pseudomonas aeruginosa at the species level. In total, 295 VFGs were detected across all sediment samples, of which 40 VFGs showed a similarity of ≥90 % with the Virulence Database (VFDB) and were focused in this study. Among the virulent proteins, twitching motility protein and flagellar P-ring were found to be prevalent and significantly associated with Vibrio spp., and Pseudomonas spp., indicating potential bacterial pathogenicity. This investigation serves as the basis for future studies and provides insights into the comprehensive taxonomic profiles of PB, VFGs and their associated PB in the coastal sediments of India.

Identification, characterization and complete genome analysis of a Vibrio anguillarum isolated from Sebastes schlegelii.

Vibrio anguillarum is an important fish pathogen in mariculture, which can infect fish with great economic losses. In this study, a Vibrio anguillarum isolated from Sebastes schlegelii was named VA1 and was identified and characterized from aspects of morphology, physiological and biochemical characteristics, 16SRNA, virulence genes, drug sensitivity, and extracellular enzyme activity. At the same time, The VA1 was investigated at the genomic level. The results showed that a Gram-negative was isolated from the diseased fish. The VA1 was characterized with uneven surface and visible flagella wrapped in a sheath and microbubble structures. The VA1 was identified as Vibrio anguillarum based on the 16S RNA sequence and physiological and biochemical characteristics. The VA1 carried most of the virulence genes (24/29) and was resistant to penicillin, oxacillin, ampicillin, cefradine, neomycin, pipemidic acid, ofloxacin, and norfloxacin. The pathogenicity of the isolated strain was confirmed by an experimental analysis, and its LD50 was 6.43 × 106 CFU/ml. The VA1 had the ability to secrete gelatinase, protease, and amylase, and it had α-hemolysis. The whole genome size of the VA1 was 4232328bp and the G + C content was 44.95 %, consisting of two circular chromosomes, Chromosome1 and Chromosome2, with no plasmid. There were 1006 predicted protein coding sequences (CDSs). A total of 526 genes were predicted as virulence-related genes which could be classified as type IV pili, flagella, hemolysin, siderophore, and type VI secretion system. Virulence genes and correlation data were supported with the histopathological examination of the affected organs and tissues. 194 genes were predicted as antibiotic resistance genes, including fluoroquinolone antibiotic, aminoglycoside antibiotic, and beta-lactam resistant genes, which agreed with the results of the above drug sensitivity, indicating VA1 to be a multidrug-resistant bacterium. This study provided a theoretical basis for a better understanding of pathogenicity and antibiotic resistance, which might contribute to the prevention of V. anguillarum in the future.

Chromosome recombination and modification by LoxP-mediated evolution in Vibrio natriegens using CRISPR-associated transposases.

Chromosome rearrangement by LoxP-mediated evolution has emerged as a powerful approach to studying how chromosome architecture impacts phenotypes. However, it relies on the in vitro synthesis of artificial chromosomes. The recently reported CRISPR-associated transposases (CASTs) held great promise for the efficient insertion of abundant LoxP sites directly onto the genome of wild-type strains. In this study, with the fastest-growing bacterium Vibrio natrigens (V. natriegens) as an object, a multiplex genome integration tool derived from CASTs was employed to achieve the insertion of cargo genes at eight specific genomic loci within 2 days. Next, we introduced 30 LoxP sites onto chromosome 2 (Chr2) of V. natriegens. Rigorously induced Cre recombinase was used to demonstrate Chromosome Rearrangement and Modification by LoxP-mediated Evolution (CRaMbLE). Growth characterization and genome sequencing showed that the ~358 kb fragment on Chr2 was accountable for the rapid growth of V. natriegens. The enabling tools we developed can help identify genomic regions that influence the rapid growth of V. natriegens without a prior understanding of genome mechanisms. This groundbreaking demonstration may also be extended to other organisms such as Escherichia coli, Pseudomonas putida, Bacillus subtilis, and so on.

Engineering xylose induction in Vibrio natriegens for biomanufacturing applications.

Xylose is an abundant, inexpensive and readily available carbohydrate common in minimally processed feedstocks such as seaweed and algae. While a wide variety of marine microbes have evolved to utilize seaweed and algae, only a few currently have the requisite characteristics and genetic engineering tools necessary to entertain the use of these underutilized feedstocks. The rapidly growing Gram-negative halophilic bacterium Vibrio natriegens is one such chassis. In this study, we engineered and tested xylose induction in V. natriegens as a tool for scalable bioproduction applications. First, we created a sensing construct based on the xylose operon from Escherichia coli MG1665 and measured its activity using a fluorescent reporter and identified that cellular import plays a key role in induction strength and that expression required the XylR transcription factor. Next, we identified that select deletions of the promoter region enhance gene expression, limiting the effect of carbohydrate repression when xylose is used as an inducer in the presence of industrially relevant carbon sources. Lastly, we used the optimized constructs to produce the biopolymer melanin using seawater mimetic media. One of these formulations utilized a nori-based seaweed extract as an inducer and demonstrated melanin yields comparable to previously optimized methods using a more traditional and costly inducer. Together, the results demonstrate that engineering xylose induction in V. natriegens can provide an effective and lower cost option for timed biosynthesis in scalable biomanufacturing applications using renewable feedstocks.