| 1. |
Le Roux F,
Binesse J,
Saulnier D,
Mazel D,
( 2007 ) Construction of a Vibrio splendidus mutant lacking the metalloprotease gene vsm by use of a novel counterselectable suicide vector. PMID : 17122399 : DOI : 10.1128/AEM.02147-06 PMC : PMC1800747 Abstract >>
Vibrio splendidus is a dominant culturable Vibrio in seawater, and strains related to this species are also associated with mortality in a variety of marine animals. The determinants encoding the pathogenic properties of these strains are still poorly understood; however, the recent sequencing of the genome of V. splendidus LGP32, an oyster pathogen, provides an opportunity to decipher the basis of the virulence properties by disruption of candidate genes. We developed a novel suicide vector based on the pir-dependent R6K replicative origin, which potentially can be transferred by RP4-based conjugation to any Vibrio strain and which also carries the plasmid F toxin ccdB gene under control of the PBAD promoter. We demonstrated that this genetic system allows efficient counterselection of integrated plasmids in the presence of arabinose in both V. splendidus and Vibrio cholerae and thus permits efficient markerless allelic replacement in these species. We used this technique to construct several mutants of V. splendidus LGP32, including a derivative with a secreted metalloprotease gene, vsm, deleted. We found that this gene is essential for LGP32 extracellular product toxicity when the extracellular products are injected into oysters but is not necessary for virulence of bacteria in the oyster infection model when bacteria are injected.
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2. |
Le Roux F,
Goubet A,
Thompson FL,
Faury N,
Gay M,
Swings J,
Saulnier D,
( 2005 ) Vibrio gigantis sp. nov., isolated from the haemolymph of cultured oysters (Crassostrea gigas). PMID : 16280478 : DOI : 10.1099/ijs.0.63666-0 Abstract >>
Polyphasic analysis of four new Vibrio isolates originating from the haemolymph of diseased cultured oysters is described. The new isolates were closely related to Vibrio splendidus, having 98 % 16S rRNA gene sequence similarity. Phylogenetic analysis based on DNA gyrase subunit B (gyrB), RNA polymerase sigma70 factor (rpoD), replication origin-binding protein (rctB) and transmembrane regulatory protein (toxR) genes, fluorescent amplified fragment length polymorphism and DNA-DNA hybridization experiments clearly showed that the new isolates form a tight genomic group that is different from the currently known Vibrio species. It is proposed that these new isolates should be accommodated in a novel species, Vibrio gigantis sp. nov. Phenotypic features that differentiate V. gigantis from other known Vibrio species include arginine dihydrolase, gelatinase and beta-galactosidase activities, NO(2) production, growth at 35 degrees C, and utilization of sucrose, melibiose, amygdalin, glycerol, galactose, starch and glycogen. The type strain is LGP 13T (=LMG 22741T=CIP 108656T).
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3. |
Thompson FL,
Gevers D,
Thompson CC,
Dawyndt P,
Naser S,
Hoste B,
Munn CB,
Swings J,
( 2005 ) Phylogeny and molecular identification of vibrios on the basis of multilocus sequence analysis. PMID : 16151093 : DOI : 10.1128/AEM.71.9.5107-5115.2005 PMC : PMC1214639 Abstract >>
We analyzed the usefulness of rpoA, recA, and pyrH gene sequences for the identification of vibrios. We sequenced fragments of these loci from a collection of 208 representative strains, including 192 well-documented Vibrionaceae strains and 16 presumptive Vibrio isolates associated with coral bleaching. In order to determine the intraspecies variation among the three loci, we included several representative strains per species. The phylogenetic trees constructed with the different genetic loci were roughly in agreement with former polyphasic taxonomic studies, including the 16S rRNA-based phylogeny of vibrios. The families Vibrionaceae, Photobacteriaceae, Enterovibrionaceae, and Salinivibrionaceae were all differentiated on the basis of each genetic locus. Each species clearly formed separated clusters with at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively. The genus Vibrio was heterogeneous and polyphyletic, with Vibrio fischeri, V. logei, and V. wodanis grouping closer to the Photobacterium genus. V. halioticoli-, V. harveyi-, V. splendidus-, and V. tubiashii-related species formed groups within the genus Vibrio. Overall, the three genetic loci were more discriminatory among species than were 16S rRNA sequences. In some cases, e.g., within the V. splendidus and V. tubiashii group, rpoA gene sequences were slightly less discriminatory than recA and pyrH sequences. In these cases, the combination of several loci will yield the most robust identification. We can conclude that strains of the same species will have at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively.
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4. |
Thompson CC,
Thompson FL,
Vandemeulebroecke K,
Hoste B,
Dawyndt P,
Swings J,
( 2004 ) Use of recA as an alternative phylogenetic marker in the family Vibrionaceae. PMID : 15143042 : DOI : 10.1099/ijs.0.02963-0 Abstract >>
This study analysed the usefulness of recA gene sequences as an alternative phylogenetic and/or identification marker for vibrios. The recA sequences suggest that the genus Vibrio is polyphyletic. The high heterogeneity observed within vibrios was congruent with former polyphasic taxonomic studies on this group. Photobacterium species clustered together and apparently nested within vibrios, while Grimontia hollisae was apart from other vibrios. Within the vibrios, Vibrio cholerae and Vibrio mimicus clustered apart from the other genus members. Vibrio harveyi- and Vibrio splendidus-related species formed compact separated groups. On the other hand, species related to Vibrio tubiashii appeared scattered in the phylogenetic tree. The pairs Vibrio coralliilyticus and Vibrio neptunius, Vibrio nereis and Vibrio xuii and V. tubiashii and Vibrio brasiliensis clustered completely apart from each other. There was a correlation of 0.58 between recA and 16S rDNA pairwise similarities. Strains of the same species have at least 94 % recA sequence similarity. recA gene sequences are much more discriminatory than 16S rDNA. For 16S rDNA similarity values above 98 % there was a wide range of recA similarities, from 83 to 99 %.
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5. |
Preheim SP,
Timberlake S,
Polz MF,
( 2011 ) Merging taxonomy with ecological population prediction in a case study of Vibrionaceae. PMID : 21873482 : DOI : 10.1128/AEM.00665-11 PMC : PMC3194886 Abstract >>
We synthesized population structure data from three studies that assessed the fine-scale distribution of Vibrionaceae among temporally and spatially distinct environmental categories in coastal seawater and animals. All studies used a dynamic model (AdaptML) to identify phylogenetically cohesive and ecologically distinct bacterial populations and their predicted habitats without relying on a predefined genetic cutoff or relationships to previously named species. Across the three studies, populations were highly overlapping, displaying similar phylogenetic characteristics (identity and diversity), and were predominantly congruent with taxonomic Vibrio species previously characterized as genotypic clusters by multilocus sequence analysis (MLSA). The environmental fidelity of these populations appears high, with 9 out of 12 reproducibly associating with the same predicted (micro)habitats when similar environmental categories were sampled. Overall, this meta-analysis provides information on the habitat predictability and structure of previously described species, demonstrating that MLSA-based taxonomy can, at least in some cases, serve to approximate ecologically cohesive populations.
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6. |
Yang Q,
Han Y,
Zhang XH,
( 2011 ) Detection of quorum sensing signal molecules in the family Vibrionaceae. PMID : 21395950 : DOI : 10.1111/j.1365-2672.2011.04998.x Abstract >>
The aim of this study was to detect the production of three kinds of quorum sensing (QS) signal molecules, i.e. the N-acyl-homoserine lactone (AHL), the autoinducer-2 (AI-2) and the cholerae autoinducer-1-like (CAI-1-like) molecules in 25 Vibrionaceae strains. The QS signal molecules in 25 Vibrionaceae strains were detected with different biosensors. Except Salinivibrio costicola VIB288 and Vibrio natriegens VIB299, all the other 23 Vibrionaceae strains could produce one or more kinds of detectable QS signal molecules. Twenty-one of the 25 strains were found to produce AHL signal molecules by using Vibrio harveyi JMH612 and Agrobacterium tumefaciens KYC55 (pJZ372; pJZ384; pJZ410) as biosensors. The AHL fingerprints of eight strains were detected by thin-layer chromatography with Ag. tumefaciens KYC55, and two of them, i.e. V. mediterranei VIB296 and Aliivibrio logei VIB414 had a high diversity of AHLs. Twenty of the 25 strains were found to have the AI-2 activity, and the luxS gene sequences in 18 strains were proved to be conserved by PCR amplification and sequencing. Only six (five Vibrio strains and A. logei VIB414) of the 25 strains possessed the CAI-1-like activity. A. logei VIB414, V. campbellii VIB285, V. furnissii VIB293, V. pomeroyi LMG20537 and two V. harveyi strains VIB571 and VIB645 were found to produce all the three kinds of QS signal molecules. The results indicated that the QS signal molecules, especially AHL and AI-2 molecules, were widespread in the family Vibrionaceae. In response to a variety of environmental conditions and selection forces, the family Vibrionaceae produced QS signal molecules with great diversity and complexity. The knowledge we obtained from this study will be useful for further research on the roles of different QS signal molecules in this family.
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7. |
Beaz-Hidalgo R,
Doce A,
Pascual J,
Toranzo AE,
Romalde JL,
( 2009 ) Vibrio gallaecicus sp. nov. isolated from cultured clams in north-western Spain. PMID : 19186015 : DOI : 10.1016/j.syapm.2008.12.002 Abstract >>
A group of three motile facultative anaerobic marine bacteria were isolated from cultured Manila clams (Ruditapes philippinarum) in Galicia, north-western Spain. The strains were characterized phenotypically and genotypically. Phylogenetic analysis of the 16S rRNA gene and four housekeeping genes, RNA polymerase alpha-chain (rpoA), RecA protein (recA), the alpha-subunit of bacterial ATP synthase (atpA) and the uridine monophosphate (UMP) kinase (pyrH), indicated that these strains were closely related to the Vibrio splendidus clade. The amplified fragment length polymorphism (AFLP) fingerprints, DNA-DNA hybridizations and phylogenies of the housekeeping and 16S rRNA gene sequences showed that the three strains represented a different species from all currently described vibrios. The new species could be differentiated from its nearest neighbours on the basis of several phenotypic features. The three strains are therefore a novel species within the genus Vibrio, for which the name Vibrio gallaecicus is proposed, with the type strain being VB 8.9T(=CECT 7244T=LMG 24045T).
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8. |
Binesse J,
Delsert C,
Saulnier D,
Champomier-Vergès MC,
Zagorec M,
Munier-Lehmann H,
Mazel D,
Le Roux F,
( 2008 ) Metalloprotease vsm is the major determinant of toxicity for extracellular products of Vibrio splendidus. PMID : 18836018 : DOI : 10.1128/AEM.01261-08 PMC : PMC2592927 Abstract >>
Genomic data combined with reverse genetic approaches have contributed to the characterization of major virulence factors of Vibrio species; however, these studies have targeted primarily human pathogens. Here, we investigate virulence factors in the oyster pathogen Vibrio splendidus LGP32 and show that toxicity is correlated to the presence of a metalloprotease and its corresponding vsm gene. Comparative genomics showed that an avirulent strain closely related to LGP32 lacked the metalloprotease. The toxicity of LGP32 metalloprotease was confirmed by exposing mollusk and mouse fibroblastic cell lines to extracellular products (ECPs) of the wild type (wt) and a vsm deletion mutant (Deltavsm mutant). The ECPs of the wt induced a strong cytopathic effect whose severity was cell type dependent, while those of the Deltavsm mutant were much less toxic, and exposure to purified protein demonstrated the direct toxicity of the Vsm metalloprotease. Finally, to investigate Vsm molecular targets, a proteomic analysis of the ECPs of both LGP32 and the Deltavsm mutant was performed, revealing a number of differentially expressed and/or processed proteins. One of these, the VSA1062 metalloprotease, was found to have significant identity to the immune inhibitor A precursor, a virulence factor of Bacillus thuringiensis. Deletion mutants corresponding to several of the major proteins were constructed by allelic exchange, and the ECPs of these mutants proved to be toxic to both cell cultures and animals. Taken together, these data demonstrate that Vsm is the major toxicity factor in the ECPs of V. splendidus.
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9. |
Urbanczyk H,
Ast JC,
Kaeding AJ,
Oliver JD,
Dunlap PV,
( 2008 ) Phylogenetic analysis of the incidence of lux gene horizontal transfer in Vibrionaceae. PMID : 18359809 : DOI : 10.1128/JB.00101-08 PMC : PMC2394989 Abstract >>
Horizontal gene transfer (HGT) is thought to occur frequently in bacteria in nature and to play an important role in bacterial evolution, contributing to the formation of new species. To gain insight into the frequency of HGT in Vibrionaceae and its possible impact on speciation, we assessed the incidence of interspecies transfer of the lux genes (luxCDABEG), which encode proteins involved in luminescence, a distinctive phenotype. Three hundred three luminous strains, most of which were recently isolated from nature and which represent 11 Aliivibrio, Photobacterium, and Vibrio species, were screened for incongruence of phylogenies based on a representative housekeeping gene (gyrB or pyrH) and a representative lux gene (luxA). Strains exhibiting incongruence were then subjected to detailed phylogenetic analysis of horizontal transfer by using multiple housekeeping genes (gyrB, recA, and pyrH) and multiple lux genes (luxCDABEG). In nearly all cases, housekeeping gene and lux gene phylogenies were congruent, and there was no instance in which the lux genes of one luminous species had replaced the lux genes of another luminous species. Therefore, the lux genes are predominantly vertically inherited in Vibrionaceae. The few exceptions to this pattern of congruence were as follows: (i) the lux genes of the only known luminous strain of Vibrio vulnificus, VVL1 (ATCC 43382), were evolutionarily closely related to the lux genes of Vibrio harveyi; (ii) the lux genes of two luminous strains of Vibrio chagasii, 21N-12 and SB-52, were closely related to those of V. harveyi and Vibrio splendidus, respectively; (iii) the lux genes of a luminous strain of Photobacterium damselae, BT-6, were closely related to the lux genes of the lux-rib(2) operon of Photobacterium leiognathi; and (iv) a strain of the luminous bacterium Photobacterium mandapamensis was found to be merodiploid for the lux genes, and the second set of lux genes was closely related to the lux genes of the lux-rib(2) operon of P. leiognathi. In none of these cases of apparent HGT, however, did acquisition of the lux genes correlate with phylogenetic divergence of the recipient strain from other members of its species. The results indicate that horizontal transfer of the lux genes in nature is rare and that horizontal acquisition of the lux genes apparently has not contributed to speciation in recipient taxa.
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10. |
Kamruzzaman M,
Nishibuchi M,
( 2008 ) Detection and characterization of a functional insertion sequence, ISVpa2, in Vibrio parahaemolyticus. PMID : 18164873 : DOI : 10.1016/j.gene.2007.11.012 Abstract >>
PCR analysis of the pandemic strain of Vibrio parahaemolyticus, KX-V237 (total genome sequenced) showed a subculture where the size of the amplicons had increased. The purpose of this study was to analyze the mechanism of this change. We found a 1,243-bp DNA sequence inserted in one of the pandemic marker genes in this strain. The inserted DNA sequence possessed the genetic structures shared by insertion sequences (ISs) of the IS3 family. This IS had 26-bp imperfect terminal inverted repeats (IRs) and two partially overlapping reading frames, orfA and orfB. OrfA codes for a helix-turn-helix, OrfA and OrfAB produced by translational frameshifting code for leucine zipper motifs, and OrfB codes for a DDE motif. orfA and orfB were homologous to those in the IS3 family. This IS was named ISVpa2. Southern blot analysis showed the copy number of ISVpa2 in our stock culture and its subculture of KX-V237 was three and four, respectively; whereas it was only one in the reported genome sequence. Analysis of the flanking sequences for seven ISVpa2 copies showed ISVpa2 is capable of inserting at multiple sites and ISVpa2 causes genetic rearrangements including insertional inactivation of the target gene and adjacent deletion. ISVpa2 created 3-base duplications upon insertion. PCR, hybridization, and nucleotide sequence analyses showed ISVpa2 homologs were detected in all of the 62 other strains of V. parahaemolyticus examined; and in some strains of Vibrio vulnificus (98% identity), Vibrio penaeicida (86% identity), and Vibrio splendidus (87% identity); but was not in 25 other species in the genus Vibrio. The data demonstrate that ISVpa2 is a transpositionally active IS discovered for the first time in V. parahaemolyticus and suggest that ISVpa2 may be transferred among the species of the genus Vibrio.
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11. |
Thompson CC,
Thompson FL,
Vicente AC,
Swings J,
( 2007 ) Phylogenetic analysis of vibrios and related species by means of atpA gene sequences. PMID : 17978204 : DOI : 10.1099/ijs.0.65223-0 Abstract >>
We investigated the use of atpA gene sequences as alternative phylogenetic and identification markers for vibrios. A fragment of 1322 bp (corresponding to approximately 88% of the coding region) was analysed in 151 strains of vibrios. The relationships observed were in agreement with the phylogeny inferred from 16S rRNA gene sequence analysis. For instance, the Vibrio cholerae, Vibrio halioticoli, Vibrio harveyi and Vibrio splendidus species groups appeared in the atpA gene phylogenetic analyses, suggesting that these groups may be considered as separate genera within the current Vibrio genus. Overall, atpA gene sequences appeared to be more discriminatory for species differentiation than 16S rRNA gene sequences. 16S rRNA gene sequence similarities above 97% corresponded to atpA gene sequences similarities above 80%. The intraspecies variation in the atpA gene sequence was about 99% sequence similarity. The results showed clearly that atpA gene sequences are a suitable alternative for the identification and phylogenetic study of vibrios.
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12. |
Ast JC,
Urbanczyk H,
Dunlap PV,
( 2007 ) Natural merodiploidy of the lux-rib operon of Photobacterium leiognathi from coastal waters of Honshu, Japan. PMID : 17586644 : DOI : 10.1128/JB.00672-07 PMC : PMC1951928 Abstract >>
Sequence analysis of the bacterial luminescence (lux) genes has proven effective in helping resolve evolutionary relationships among luminous bacteria. Phylogenetic analysis using lux genes, however, is based on the assumptions that the lux genes are present as single copies on the bacterial chromosome and are vertically inherited. We report here that certain strains of Photobacterium leiognathi carry multiple phylogenetically distinct copies of the entire operon that codes for luminescence and riboflavin synthesis genes, luxCDABEG-ribEBHA. Merodiploid lux-rib strains of P. leiognathi were detected during sequence analysis of luxA. To define the gene content, organization, and sequence of each lux-rib operon, we constructed a fosmid library of genomic DNA from a representative merodiploid strain, lnuch.13.1. Sequence analysis of fosmid clones and genomic analysis of lnuch.13.1 defined two complete, physically separate, and apparently functional operons, designated lux-rib1 and lux-rib2. P. leiognathi strains lelon.2.1 and lnuch.21.1 were also found to carry lux-rib1 and lux-rib2, whereas ATCC 25521T apparently carries only lux-rib1. In lnuch.13.1, lelon.2.1, lnuch.21.1, and ATCC 25521T, lux-rib1 is flanked upstream by lumQ and putA and downstream by a gene for a hypothetical multidrug efflux pump. In contrast, transposase genes flank lux-rib2 of lnuch.13.1, and the chromosomal location of lux-rib2 apparently differs in lnuch.13.1, lelon.2.1, and lnuch.21.1. Phylogenetic analysis demonstrated that lux-rib1 and lux-rib2 are more closely related to each other than either one is to the lux and rib genes of other bacterial species, which rules out interspecies lateral gene transfer as the origin of lux-rib2 in P. leiognathi; lux-rib2 apparently arose within a previously unsampled or extinct P. leiognathi lineage. Analysis of 170 additional strains of P. leiognathi, for a total of 174 strains examined from coastal waters of Japan, Taiwan, the Philippine Islands, and Thailand, identified 106 strains that carry only a single lux-rib operon and 68 that carry multiple lux-rib operons. Strains bearing a single lux-rib operon were obtained throughout the geographic sampling range, whereas lux-rib merodiploid strains were found only in coastal waters of central Honshu. This is the first report of merodiploidy of lux or rib genes in a luminous bacterium and the first indication that a natural merodiploid state in bacteria can correlate with geography.
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13. |
Wendling CC,
Piecyk A,
Refardt D,
Chibani C,
Hertel R,
Liesegang H,
Bunk B,
Overmann J,
Roth O,
( 2017 ) Tripartite species interaction: eukaryotic hosts suffer more from phage susceptible than from phage resistant bacteria. PMID : 28399796 : DOI : 10.1186/s12862-017-0930-2 PMC : PMC5387238 Abstract >>
Evolutionary shifts in bacterial virulence are often associated with a third biological player, for instance temperate phages, that can act as hyperparasites. By integrating as prophages into the bacterial genome they can contribute accessory genes, which can enhance the fitness of their prokaryotic carrier (lysogenic conversion). Hyperparasitic influence in tripartite biotic interactions has so far been largely neglected in empirical host-parasite studies due to their inherent complexity. Here we experimentally address whether bacterial resistance to phages and bacterial harm to eukaryotic hosts is linked using a natural tri-partite system with bacteria of the genus Vibrio, temperate vibriophages and the pipefish Syngnathus typhle. We induced prophages from all bacterial isolates and constructed a three-fold replicated, fully reciprocal 75 �� 75 phage-bacteria infection matrix. According to their resistance to phages, bacteria could be grouped into three distinct categories: highly susceptible (HS-bacteria), intermediate susceptible (IS-bacteria), and resistant (R-bacteria). We experimentally challenged pipefish with three selected bacterial isolates from each of the three categories and determined the amount of viable Vibrio counts from infected pipefish and the expression of pipefish immune genes. While the amount of viable Vibrio counts did not differ between bacterial groups, we observed a significant difference in relative gene expression between pipefish infected with phage susceptible and phage resistant bacteria. These findings suggest that bacteria with a phage-susceptible phenotype are more harmful against a eukaryotic host, and support the importance of hyperparasitism and the need for an integrative view across more than two levels when studying host-parasite evolution.
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14. |
Datta MS,
Sliwerska E,
Gore J,
Polz MF,
Cordero OX,
( 2016 ) Microbial interactions lead to rapid micro-scale successions on model marine particles. PMID : 27311813 : DOI : 10.1038/ncomms11965 PMC : PMC4915023 Abstract >>
In the ocean, organic particles harbour diverse bacterial communities, which collectively digest and recycle essential nutrients. Traits like motility and exo-enzyme production allow individual taxa to colonize and exploit particle resources, but it remains unclear how community dynamics emerge from these individual traits. Here we track the taxon and trait dynamics of bacteria attached to model marine particles and demonstrate that particle-attached communities undergo rapid, reproducible successions driven by ecological interactions. Motile, particle-degrading taxa are selected for during early successional stages. However, this selective pressure is later relaxed when secondary consumers invade, which are unable to use the particle resource but, instead, rely on carbon from primary degraders. This creates a trophic chain that shifts community metabolism away from the particle substrate. These results suggest that primary successions may shape particle-attached bacterial communities in the ocean and that rapid community-wide metabolic shifts could limit rates of marine particle degradation.
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15. |
Wendling CC,
Wegner KM,
( 2015 ) Adaptation to enemy shifts: rapid resistance evolution to local Vibrio spp. in invasive Pacific oysters. PMID : 25716784 : DOI : 10.1098/rspb.2014.2244 PMC : PMC4375854 Abstract >>
One hypothesis for the success of invasive species is reduced pathogen burden, resulting from a release from infections or high immunological fitness of invaders. Despite strong selection exerted on the host, the evolutionary response of invaders to newly acquired pathogens has rarely been considered. The two independent and genetically distinct invasions of the Pacific oyster Crassostrea gigas into the North Sea represent an ideal model system to study fast evolutionary responses of invasive populations. By exposing both invasion sources to ubiquitous and phylogenetically diverse pathogens (Vibrio spp.), we demonstrate that within a few generations hosts adapted to newly encountered pathogen communities. However, local adaptation only became apparent in selective environments, i.e. at elevated temperatures reflecting patterns of disease outbreaks in natural populations. Resistance against sympatric and allopatric Vibrio spp. strains was dominantly inherited in crosses between both invasion sources, resulting in an overall higher resistance of admixed individuals than pure lines. Therefore, we suggest that a simple genetic resistance mechanism of the host is matched to a common virulence mechanism shared by local Vibrio strains. This combination might have facilitated a fast evolutionary response that can explain another dimension of why invasive species can be so successful in newly invaded ranges.
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16. |
Gulla S,
Sørum H,
Vågnes ?,
Colquhoun DJ,
( 2015 ) Phylogenetic analysis and serotyping of Vibrio splendidus-related bacteria isolated from salmon farm cleaner fish. PMID : 26648104 : DOI : 10.3354/dao02938 Abstract >>
Cleaner fish, i.e. various wrasse (Labridae) species and lumpsucker Cyclopterus lumpus, are to an increasing extent used for biocontrol of the salmon louse Lepeophtheirus salmonis in European salmon farming. Although efficient de-licers, cleaner fish mortality levels in salmon farms are often high. Bacterial infections are common, and Vibrio splendidus-related strains are frequently identified during diagnostic investigations. The population structure of 112 V. splendidus-related isolates, derived primarily from wrasse species, was investigated by means of multilocus sequence analysis using 5 housekeeping genes (rpoD, ftsZ, pyrH, rpoA and atpA). Most isolates were found to be closely related to the V. splendidus type strain, yet displayed extensive genetic microdiversity. Slide agglutination testing using polyclonal rabbit antisera further indicated O-antigen variability. Intra-outbreak genetic and antigenic diversity suggests direct infection from seawater, rather than fish-to-fish transmission, as the main route of infection. The variable nature of isolates involved complicates qualified selection of representative candidate strains, e.g. for infection and vaccine trials.
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17. |
Machado H,
Gram L,
( 2015 ) The fur gene as a new phylogenetic marker for Vibrionaceae species identification. PMID : 25662978 : DOI : 10.1128/AEM.00058-15 PMC : PMC4375339 Abstract >>
Microbial taxonomy is essential in all areas of microbial science. The 16S rRNA gene sequence is one of the main phylogenetic species markers; however, it does not provide discrimination in the family Vibrionaceae, where other molecular techniques allow better interspecies resolution. Although multilocus sequence analysis (MLSA) has been used successfully in the identification of Vibrio species, the technique has several limitations. They include the fact that several locus amplifications and sequencing have to be performed, which still sometimes lead to doubtful identifications. Using an in silico approach based on genomes from 103 Vibrionaceae strains, we demonstrate here the high resolution of the fur gene in the identification of Vibrionaceae species and its usefulness as a phylogenetic marker. The fur gene showed within-species similarity higher than 95%, and the relationships inferred from its use were in agreement with those observed for 16S rRNA analysis and MLSA. Furthermore, we developed a fur PCR sequencing-based method that allowed identification of Vibrio species. The discovery of the phylogenetic power of the fur gene and the development of a PCR method that can be used in amplification and sequencing of the gene are of general interest whether for use alone or together with the previously suggested loci in an MLSA.
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18. |
Wendling CC,
Batista FM,
Wegner KM,
( 2014 ) Persistence, seasonal dynamics and pathogenic potential of Vibrio communities from Pacific oyster hemolymph. PMID : 24728233 : DOI : 10.1371/journal.pone.0094256 PMC : PMC3984124 Abstract >>
Bacteria of the genus Vibrio occur at a continuum from free-living to symbiotic life forms, including opportunists and pathogens, that can contribute to severe diseases, for instance summer mortality events of Pacific oysters Crassostrea gigas. While most studies focused on Vibrio isolated from moribund oysters during mortality outbreaks, investigations of the Vibrio community in healthy oysters are rare. Therefore, we characterized the persistence, diversity, seasonal dynamics, and pathogenicity of the Vibrio community isolated from healthy Pacific oysters. In a reciprocal transplant experiment we repeatedly sampled hemolymph from adult Pacific oysters to differentiate population from site-specific effects during six months of in situ incubation in the field. We characterized virulence phenotypes and genomic diversity based on multilocus sequence typing in a total of 70 Vibrio strains. Based on controlled infection experiments we could show that strains with the ability to colonize healthy adult oysters can also have the potential to induce high mortality rates on larvae. Diversity and abundance of Vibrio varied significantly over time with highest values during and after spawning season. Vibrio communities from transplanted and stationary oysters converged over time, indicating that communities were not population specific, but rather assemble from the surrounding environment forming communities, some of which can persist over longer periods.
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19. |
Rahman MS,
Martino ME,
Cardazzo B,
Facco P,
Bordin P,
Mioni R,
Novelli E,
Fasolato L,
( 2014 ) Vibrio trends in the ecology of the Venice lagoon. PMID : 24487545 : DOI : 10.1128/AEM.04133-13 PMC : PMC3993166 Abstract >>
Vibrio is a very diverse genus that is responsible for different human and animal diseases. The accurate identification of Vibrio at the species level is important to assess the risks related to public health and diseases caused by aquatic organisms. The ecology of Vibrio spp., together with their genetic background, represents an important key for species discrimination and evolution. Thus, analyses of population structure and ecology association are necessary for reliable characterization of bacteria and to investigate whether bacterial species are going through adaptation processes. In this study, a population of Vibrionaceae was isolated from shellfish of the Venice lagoon and analyzed in depth to study its structure and distribution in the environment. A multilocus sequence analysis (MLSA) was developed on the basis of four housekeeping genes. Both molecular and biochemical approaches were used for species characterization, and the results were compared to assess the consistency of the two methods. In addition, strain ecology and the association between genetic information and environment were investigated through statistical models. The phylogenetic and population analyses achieved good species clustering, while biochemical identification was demonstrated to be imprecise. In addition, this study provided a fine-scale overview of the distribution of Vibrio spp. in the Venice lagoon, and the results highlighted a preferential association of the species toward specific ecological variables. These findings support the use of MLSA for taxonomic studies and demonstrate the need to consider environmental information to obtain broader and more accurate bacterial characterization.
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20. |
Xue H,
Cordero OX,
Camas FM,
Trimble W,
Meyer F,
Guglielmini J,
Rocha EP,
Polz MF,
( 2015 ) Eco-Evolutionary Dynamics of Episomes among Ecologically Cohesive Bacterial Populations. PMID : 25944863 : DOI : 10.1128/mBio.00552-15 PMC : PMC4436056 Abstract >>
Although plasmids and other episomes are recognized as key players in horizontal gene transfer among microbes, their diversity and dynamics among ecologically structured host populations in the wild remain poorly understood. Here, we show that natural populations of marine Vibrionaceae bacteria host large numbers of families of episomes, consisting of plasmids and a surprisingly high fraction of plasmid-like temperate phages. Episomes are unevenly distributed among host populations, and contrary to the notion that high-density communities in biofilms act as hot spots of gene transfer, we identified a strong bias for episomes to occur in free-living as opposed to particle-attached cells. Mapping of episomal families onto host phylogeny shows that, with the exception of all phage and a few plasmid families, most are of recent evolutionary origin and appear to have spread rapidly by horizontal transfer. Such high eco-evolutionary turnover is particularly surprising for plasmids that are, based on previously suggested categorization, putatively nontransmissible, indicating that this type of plasmid is indeed frequently transferred by currently unknown mechanisms. Finally, analysis of recent gene transfer among plasmids reveals a network of extensive exchange connecting nearly all episomes. Genes functioning in plasmid transfer and maintenance are frequently exchanged, suggesting that plasmids can be rapidly transformed from one category to another. The broad distribution of episomes among distantly related hosts and the observed promiscuous recombination patterns show how episomes can offer their hosts rapid assembly and dissemination of novel functions. Plasmids and other episomes are an integral part of bacterial biology in all environments, yet their study is heavily biased toward their role as vectors for antibiotic resistance genes. This study presents a comprehensive analysis of all episomes within several coexisting bacterial populations of Vibrionaceae from the coastal ocean and represents the largest-yet genomic survey of episomes from a single bacterial family. The host population framework allows analysis of the eco-evolutionary dynamics at unprecedented resolution, yielding several unexpected results. These include (i) discovery of novel, nonintegrative temperate phages, (ii) revision of a class of episomes, previously termed nontransmissible," as highly transmissible, and (iii) surprisingly high evolutionary turnover of episomes, manifest as frequent birth, spread, and loss."
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21. |
Rodríguez-Blanco A,
Lemos ML,
Osorio CR,
( 2012 ) Integrating conjugative elements as vectors of antibiotic, mercury, and quaternary ammonium compound resistance in marine aquaculture environments. PMID : 22314526 : DOI : 10.1128/AAC.05997-11 PMC : PMC3346659 Abstract >>
The presence of SXT/R391-related integrating conjugative elements (ICEs) in bacterial strains isolated from fish obtained from marine aquaculture environments in 2001 to 2010 in the northwestern Iberian Peninsula was studied. ICEs were detected in 12 strains taxonomically related to Vibrio scophthalmi (3 strains), Vibrio splendidus (5 strains), Vibrio alginolyticus (1 strain), Shewanella haliotis (1 strain), and Enterovibrio nigricans (2 strains), broadening the known host range able to harbor SXT/R391-like ICEs. Variable DNA regions, which confer element-specific properties to ICEs of this family, were characterized. One of the ICEs encoded antibiotic resistance functions in variable region III, consisting of a tetracycline resistance locus. Interestingly, hot spot 4 included genes providing resistance to rifampin (ICEVspPor2 and ICEValPor1) and quaternary ammonium compounds (QACs) (ICEEniSpa1), and variable region IV included a mercury resistance operon (ICEVspSpa1 and ICEEniSpa1). The S exclusion group was more represented than the R exclusion group, accounting for two-thirds of the total ICEs. Mating experiments allowed ICE mobilization to Escherichia coli strains, showing the corresponding transconjugants' rifampin, mercury, and QAC resistance. These results show the first evidence of ICEs providing rifampin and QAC resistances, suggesting that these mobile genetic elements contribute to the dissemination of antimicrobial, heavy metal, and QAC resistance determinants in aquaculture environments.
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22. |
Figge MJ,
Robertson LA,
Ast JC,
Dunlap PV,
( 2011 ) Historical microbiology: revival and phylogenetic analysis of the luminous bacterial cultures of M. W. Beijerinck. PMID : 22066815 : DOI : 10.1111/j.1574-6941.2011.01177.x Abstract >>
Luminous bacteria isolated by Martinus W. Beijerinck were sealed in glass ampoules in 1924 and 1925 and stored under the names Photobacterium phosphoreum and 'Photobacterium splendidum'. To determine if the stored cultures were viable and to assess their evolutionary relationship with currently recognized bacteria, portions of the ampoule contents were inoculated into culture medium. Growth and luminescence were evident after 13 days of incubation, indicating the presence of viable cells after more than 80 years of storage. The Beijerinck strains are apparently the oldest bacterial cultures to be revived from storage. Multi-locus sequence analysis, based on the 16S rRNA, gapA, gyrB, pyrH, recA, luxA, and luxB genes, revealed that the Beijerinck strains are distant from the type strains of P. phosphoreum, ATCC 11040(T), and Vibrio splendidus, ATCC 33125(T), and instead form an evolutionarily distinct clade of Vibrio. Newly isolated strains from coastal seawater in Norway, France, Uruguay, Mexico, and Japan grouped with the Beijerinck strains, indicating a global distribution for this new clade, designated as the beijerinckii clade. Strains of the beijerinckii clade exhibited little sequence variation for the seven genes and approximately 6300 nucleotides examined despite the geographic distances and the more than 80 years separating their isolation. Gram-negative bacteria therefore can survive for many decades in liquid storage, and in nature, they do not necessarily diverge rapidly over time.
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23. |
Lyu Q,
Zhang K,
Shi Y,
Li W,
Diao X,
Liu W,
( 2019 ) Structural insights into a novel Ca2+-independent PL-6 alginate lyase from Vibrio OU02 identify the possible subsites responsible for product distribution. PMID : 31004719 : DOI : 10.1016/j.bbagen.2019.04.013 Abstract >>
Alginate lyases have a wide range of industrial applications, such as oligosaccharide preparation, medical treatment, and bioconversion. Therefore, the discovery and characterization of novel alginate lyases are extremely important. PL-6 alginate lyases are classified into two groups: those with a single domain or two domains. However, only one structure of a two-domain alginate lyase has been determined to date. In this study, we characterized a novel single-domain PL-6 alginate lyase (named AlyF). According to the biochemical analysis, AlyF possesses unique features compared with other PL-6 enzymes, including (1) a Ca2+-independent catalytic mechanism and (2) a PolyG-specific cleavage specificity that predominantly produces trisaccharides. The structures of AlyF and its complexes described here reveal the structural basis for these unique features and substrate binding mechanisms, which were further confirmed using mutagenesis. More importantly, we determined the possible subsites specifying the predominantly trisaccharide products of AlyF, which may facilitate the rational design of AlyF for potential applications in preparing a single alginate oligomer.
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24. |
Kauffman KM,
Hussain FA,
Yang J,
Arevalo P,
Brown JM,
Chang WK,
VanInsberghe D,
Elsherbini J,
Sharma RS,
Cutler MB,
Kelly L,
Polz MF,
( 2018 ) A major lineage of non-tailed dsDNA viruses as unrecognized killers of marine bacteria. PMID : 29364876 : DOI : 10.1038/nature25474 Abstract >>
The most abundant viruses on Earth are thought to be double-stranded DNA (dsDNA) viruses that infect bacteria. However, tailed bacterial dsDNA viruses (Caudovirales), which dominate sequence and culture collections, are not representative of the environmental diversity of viruses. In fact, non-tailed viruses often dominate ocean samples numerically, raising the fundamental question of the nature of these viruses. Here we characterize a group of marine dsDNA non-tailed viruses with short 10-kb genomes isolated during a study that quantified the diversity of viruses infecting Vibrionaceae bacteria. These viruses, which we propose to name the Autolykiviridae, represent a novel family within the ancient lineage of double jelly roll (DJR) capsid viruses. Ecologically, members of the Autolykiviridae have a broad host range, killing on average 34 hosts in four Vibrio species, in contrast to tailed viruses which kill on average only two hosts in one species. Biochemical and physical characterization of autolykiviruses reveals multiple virion features that cause systematic loss of DJR viruses in sequencing and culture-based studies, and we describe simple procedural adjustments to recover them. We identify DJR viruses in the genomes of diverse major bacterial and archaeal phyla, and in marine water column and sediment metagenomes, and find that their diversity greatly exceeds the diversity that is currently captured by the three recognized families of such viruses. Overall, these data suggest that viruses of the non-tailed dsDNA DJR lineage are important but often overlooked predators of bacteria and archaea that impose fundamentally different predation and gene transfer regimes on microbial systems than on tailed viruses, which form the basis of all environmental models of bacteria-virus interactions.
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25. |
Lyu Q,
Zhang K,
Zhu Q,
Li Z,
Liu Y,
Fitzek E,
Yohe T,
Zhao L,
Li W,
Liu T,
Yin Y,
Liu W,
( 2018 ) Structural and biochemical characterization of a multidomain alginate lyase reveals a novel role of CBM32 in CAZymes. PMID : 29864445 : DOI : 10.1016/j.bbagen.2018.05.024 Abstract >>
Noncatalytic carbohydrate binding modules (CBMs) have been demonstrated to play various roles with cognate catalytic domains. However, for polysaccharide lyases (PLs), the roles of CBMs remain mostly unknown. AlyB is a multidomain alginate lyase that contains CBM32 and a PL7 catalytic domain. The AlyB structure determined herein reveals a noncanonical alpha helix linker between CBM32 and the catalytic domain. More interestingly, CBM32 and the linker does not significantly enhance the catalytic activity but rather specifies that trisaccharides are predominant in the degradation products. Detailed mutagenesis, biochemical and cocrystallization analyses show "weak but important" CBM32 interactions with alginate oligosaccharides. In combination with molecular modeling, we propose that the CBM32 domain serves as a "pivot point" during the trisaccharide release process. Collectively, this work demonstrates a novel role of CBMs in the activity of the appended PL domain and provides a new avenue for the well-defined generation of alginate oligosaccharides by taking advantage of associated CBMs.
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26. |
( 2013 ) Diversity of Vibrio spp. isolated at ambient environmental temperature in the Eastern English Channel as determined by pyrH sequencing. PMID : 23473469 : DOI : 10.1111/jam.12181 Abstract >>
To describe the diversity of the culturable mesophilic and potentially pathogenic vibrios isolated at 22 and 37�XC on TCBS medium, in September 2009 from seawater and surface sediments. q-PCR assays previously selected for the identification of bacterial strains isolated at 37�XC were used in combination with the partial sequencing of two housekeeping genes, pyrH and toxR, to identify 315 strains isolated at 22�XC. The great majority of the 37�XC strains was identified by q-PCR assays, (five of the six species) with the predominance of Vibrio alginolyticus (85�P9%) and V. harveyi (10�P7%). The human pathogens V. parahaemolyticus and V. cholerae were rarely detected (two strains each). The 22�XC strains were successfully identified by the phylogeny analysis of pyrH and toxR genes, revealing 20 Vibrio species, with the predominance of the clam pathogen V. celticus (36�P8%). The Splendidus and the Harveyi groups represented the main Vibrio group at 22�XC (80%) and 37�XC (99�P5%), respectively. The combination of q-PCR assays and the sequencing of pyrH and toxR genes highlighted two different Vibrio communities at 22 and 37�XC both dominated by pathogenic species for marine organisms. The sequencing of the pyrH gene revealed to be a valuable tool to identify environmental Vibrio spp. strains isolated at 22�XC, as 92�P3% of them were identified in this study.
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27. |
( 2013 ) Vibrio hemicentroti sp. nov., an alginate lyase-producing bacterium, isolated from the gut microflora of sea urchin (Hemicentrotus pulcherrimus). PMID : 23625262 : DOI : 10.1099/ijs.0.047951-0 DOI : 10.1099/ijs.0.047951-0 Abstract >>
An alginate lyase-producing bacterium, designated AlyHP32(T), was isolated from the gut of sea urchin (Hemicentrotus pulcherrimus) obtained from the South Sea, Republic of Korea. Cells of strain AlyHP32(T) were Gram-reaction-negative and motile with a single polar flagellum. The strain grew with 1-6 % (w/v) NaCl (optimum 2-4 %) and at 4-30 �XC (optimum 15-25 �XC). Phylogenetic analysis based on sequences of the 16S rRNA gene and five housekeeping genes (atpA, pyrH, recA, rpoA and rpoD) revealed that strain AlyHP32(T) belonged to the genus Vibrio and formed a compact clade with the Vibrio splendidus group. However, DNA-DNA hybridization and fingerprints using the repetitive primers BOX and REP indicated that strain AlyHP32(T) was distinct from closely related species of the genus Vibrio. The major fatty acids were summed feature 3 (C16:1�s7c and/or C16:1�s6c) and C16:0. The DNA G+C content was 44.1 mol%. The predominant quinone was ubiquinone Q-8. Based on genotypic, phenotypic and DNA-DNA hybridization analysis, strain AlyHP32(T) represents a novel species of the genus Vibrio; the name Vibrio hemicentroti sp. nov. (type strain AlyHP32(T) = KCTC 32085(T) = DSM 26178(T)) is proposed for this novel taxon.
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28. |
( 2013 ) Integrating conjugative elements of the SXT/R391 family from fish-isolated Vibrios encode restriction-modification systems that confer resistance to bacteriophages. PMID : 22974320 : DOI : 10.1111/1574-6941.12007 Abstract >>
Integrating conjugative elements (ICEs) of the SXT/R391 family have been described in Vibrios, mainly Vibrio cholerae, and other bacteria as carriers of variable gene content conferring adaptive advantages upon their hosts, including antimicrobial resistance and motility regulation. However, our knowledge on their host range and ecological significance is still limited. Here, we report the identification and characterization of ICEVspPor3 and ICEValSpa1, two novel ICEs of the SXT/R391 family from fish-isolated Vibrio splendidus and Vibrio alginolyticus, respectively. We found that ICEVspPor3 carries tetracycline and HgCl(2) resistance determinants and can be transferred by conjugation to Escherichia coli and to several species of marine bacteria including some of the major bacterial fish pathogens in marine aquaculture, whereas ICEValSpa1 lacks resistance genes. Interestingly, both ICEs harbor genes encoding distinct restriction-modification (RM) systems. We demonstrate here that these RM systems, when expressed in E. coli, confer protection to infection by T1 bacteriophage and by environmental water bacteriophages. Our results provide evidences that the variable gene content of ICEs of the SXT/R391 family encodes fitness functions beyond those related to antimicrobial resistance and motility regulation and suggest that the host range of these elements in the marine environment might be broader than expected.
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