Relationship of the luminous bacterial symbiont of the Caribbean flashlight fish,Kryptophanaron alfredi (family Anomalopidae) to other luminous bacteria based on bacterial luciferase (luxA) genes

Flashlight fishes (family Anomalopidae) have light organs that contain luminous bacterial symbionts. Although the symbionts have not yet been successfully cultured, the luciferase genes have been cloned directly from the light organ of the Caribbean species, Kryptophanaron alfredi. The goal of this project was to evaluate the relationship of the symbiont to free-living luminous bacteria by comparison of genes coding for bacterial luciferase (lux genes). Hybridization of a luxAB probe from the Kryptophanaron alfredi symbiont to DNAs from 9 strains (8 species) of luminous bacteria showed that none of the strains tested had lux genes highly similar to the symbiont. The most similar were a group consisting of Vibrio harveyi, Vibrio splendidus and Vibrio orientalis. The nucleotide sequence of the luciferase subunit gene luxA of the Kryptophanaron alfredi symbiont was determined in order to do a more detailed comparison with published luxA sequences from Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi. The hybridization results, sequence comparisons and the mol% G+C of the Kryptophanaron alfredi symbiont luxA gene suggest that the symbiont may be considered as a new species of luminous Vibrio related to Vibrio harveyi.The nucleotide sequence reported in this article has been deposited in Genbank under accession number M36597     

Characterization of marine luminous bacteria isolated off the Coast of China and description ofVibrio orientalis sp. nov.

Luminous strains of marine bacteria, isolated off the Coast of China, were subjected to a phenotypic characterization, which included a test of their ability to utilize 82 organic compounds as sole or principal sources of carbon and energy. A numerical analysis of the data revealed five clusters which were readily identified asPhotobacterium phosphoreum, P. leiognathi, Vibrio harveyi, andV. splendidus biotype I. The remaining cluster of luminous isolates was phenotypically distinct from all the previously described species ofVibrio andPhotobacterium and was given the species designation,Vibrio orientalis. This species differed from all the other luminous species ofVibrio by its ability to accumulate poly-β-hydroxybutyrate as an intracellular reserve product. Additional distinctive properties were the presence of an arginine dihydrolase system, growth at 4° but not 40°C, and the ability to utilize putrescine and spermine.     

Distribution and Identification of Luminous Bacteria from the Sargasso Sea

Vibrio fischeri and Lucibacterium harveyi constituted 75 of the 83 luminous bacteria isolated from Sargasso Sea surface waters. Photobacterium leiognathi and Photobacterium phosphoreum constituted the remainder of the isolates. Luminescent bacteria were recovered at concentrations of 1 to 63 cells per 100 ml from water samples collected at depths of 160 to 320 m. Two water samples collected at the thermocline yielded larger numbers of viable, aerobic heterotrophic and luminous bacteria. Luminescent bacteria were not recovered from surface microlayer samples. The species distribution of the luminous bacteria reflected previously recognized growth patterns; i.e., L. harveyi and V. fischeri were predominant in the upper, warm waters (only one isolate of P. phosphoreum was obtained from surface tropical waters).     

Detection of the Light Organ Symbiont, Vibrio fischeri, in Hawaiian Seawater by Using lux Gene Probes

Symbiotic bacteria that inhabit the light-emitting organ of the Hawaiian squid Euprymna scolopes are distinctive from typical Vibrio fischeri organisms in that they are not visibly luminous when grown in laboratory culture. Therefore, the abundance of these bacteria in seawater samples cannot be estimated simply by identifying them among luminous colonies that arise on nutrient agar plates. Instead, we have used luxR and polymerase chain reaction generated luxA gene probes to identify both luminous and non-visibly luminous V. fischeri colonies by DNA-DNA hybridization. The probes were specific, hybridizing at least 50 to 100 times more strongly to immobilized DNAs from V. fischeri strains than to those of pure cultures of other related species. Thus, even non-visibly luminous V. fischeri colonies could be identified among colonies obtained from natural seawater samples by their probe-positive reaction. Bacteria in seawater samples, obtained either within or distant from squid habitats, were collected on membrane filters and incubated until colonies appeared. The filters were then observed for visibly luminous V. fischeri colonies and hybridized with the lux gene probes to determine the number of total V. fischeri colonies (both luminous and non-visibly luminous). We detected no significant differences in the abundance of luminous V. fischeri CFU in any of the water samples observed (≤1 to 3 CFU/100 ml). However, probe-positive colonies of V. fischeri (up to 900 CFU/100 ml) were found only in seawater collected from within the natural habitats of the squids. A number of criteria were used to confirm that these probe-positive strains were indistinguishable from symbiotic V. fischeri. Therefore, the luxA and luxR gene probes were species specific and gave a reliable estimate of the number of culturable V. fischeri colonies in natural water samples.     

Development of species-specific hybridization probes for marine luminous bacteria by using in vitro DNA amplification.

By using two highly conserved region of the luxA gene as primers, polymerase chain reaction amplification methods were used to prepare species-specific probes against the luciferase gene from four major groups of marine luminous bacteria. Laboratory studies with test strains indicated that three of the four probes cross-reacted with themselves and with one or more of the other species at low stringencies but were specific for members of their own species at high stringencies. The fourth probe, generated from Vibrio harveyi DNA, cross-reacted with DNAs from two closely related species, V. orientalis and V. vulnificus. When nonluminous cultures were tested with the species-specific probes, no false-positive results were observed, even at low stringencies. Two field isolates were correctly identified as Photobacterium phosphoreum by using the species-specific hybridization probes at high stringency. A mixed probe (four different hybridization probes) used at low stringency gave positive results with all of the luminous bacteria tested, including the terrestrial species, Xenorhabdus luminescens, and the taxonomically distinct marine bacterial species Shewanella hanedai; minimal cross-hybridization with these species was seen at higher stringencies.     

Development of species-specific hybridization probes for marine luminous bacteria by using in vitro DNA amplification.

By using two highly conserved region of the luxA gene as primers, polymerase chain reaction amplification methods were used to prepare species-specific probes against the luciferase gene from four major groups of marine luminous bacteria. Laboratory studies with test strains indicated that three of the four probes cross-reacted with themselves and with one or more of the other species at low stringencies but were specific for members of their own species at high stringencies. The fourth probe, generated from Vibrio harveyi DNA, cross-reacted with DNAs from two closely related species, V. orientalis and V. vulnificus. When nonluminous cultures were tested with the species-specific probes, no false-positive results were observed, even at low stringencies. Two field isolates were correctly identified as Photobacterium phosphoreum by using the species-specific hybridization probes at high stringency. A mixed probe (four different hybridization probes) used at low stringency gave positive results with all of the luminous bacteria tested, including the terrestrial species, Xenorhabdus luminescens, and the taxonomically distinct marine bacterial species Shewanella hanedai; minimal cross-hybridization with these species was seen at higher stringencies.     

Evolutionary relationships in Vibrio and Photobacterium as determined by immunological studies of superoxide dismutase

The amino acid sequence divergence of superoxide dismutases (SODs) from 22 species and five groups of Vibrio, Photobacterium, and a number of related organisms was determined by means of the microcomplement fixation technique and the Ouchterlony double diffusion procedure. Five reference antisera were used which had been prepared against the purified SODs from V. alginolyticus, V. splendidus II, V. fischeri, V. cholerae, and P. leiognathi. With a few exceptions the results were in agreement with past studies of other informational molecules and provided a comprehensive overview of evolutionary relationships in Vibrio and Photobacterium. The genus Vibrio was found to consist of a major group of primarily marine species which included V. fischeri, V. logei, V. splendidus, V. pelagius, V. nereis, V. campbellii, V. harveyi, V. natriegens, V. alginolyticus, V. parahaemolyticus, V. proteolyticus, V. fluvialis, V. vulnificus, V. nigripulchritudo, and V. anguillarum. On the outskirts of this large and relatively heterogeneous group were the fresh water and estuarine species V. cholerae and V. metschnikovii as well as the marine species V. gazogenes. A considerable distance from Vibrio were the related species of Photobacterium: P. phosphoreum, P. leiognathi, and P. angustum. Both genera were distant from species of Aeromonas as well as from Plesiomonas shigelloides, Escherichia coli, and Alteromonas hanedai, a luminous strict aerobe. The agreement between these and previous studies of evolution of informational molecules in Vibrio and Photobacterium is best explained by vertical evolution (involving no genetic exchange between species) rather than by its opposite — horizontal evolution.Non-Standard Abbreviations Anti-Plei, anti-Valg, anti-Vcho, anti-Vfis, anti-Vspl antisera to the Fe-containing superoxide dismutases from Photobacterium leiognathi strain 480, Vibrio alginolyticus strain 90, V. cholerae strain M 13, V. fischeri strain 61, and v. splendidus biotype II strain 2, respectively - AP alkaline phosphatase - ATCC American Type Culture Collection - GS glutamine synthetase - ImD immunological distance - NCMB National Colleciion of Marine Bacteria - NCTC National Collection of Type Cultures - NRC National Research Council of Canada Culture Collection - SDS sodium dodecyl sulfate - SOD superoxide dismutase     

Construction of a Vibrio splendidus Mutant Lacking the Metalloprotease Gene vsm by Use of a Novel Counterselectable Suicide Vector

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 P_BAD 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.     

Investigating the action of the microalgal pigment marennine on Vibrio splendidus by in vivo 2H and 31P solid-state NMR

This work investigates the potential probiotic effect of marennine - a natural pigment produced by the diatom Haslea ostrearia - on Vibrio splendidus. These marine bacteria are often considered a threat for aquaculture; therefore, chemical antibiotics can be required to reduce bacterial outbreaks. In vivo ²H solid-state NMR was used to probe the effects of marennine on the bacterial membrane in the exponential and stationary phases. Comparisons were made with polymyxin B (PxB) - an antibiotic used in aquaculture and known to interact with Gram(?) bacteria membranes. We also investigated the effect of marennine using ³¹P solid-state NMR on model membranes. Our results show that marennine has little effect on phospholipid headgroups dynamics, but reduces the acyl chain fluidity. Our data suggest that the two antimicrobial agents perturb V. splendidus membranes through different mechanisms. While PxB would alter the bacterial outer and inner membranes, marennine would act through a membrane stiffening mechanism, without affecting the bilayer integrity. Our study proposes this microalgal pigment, which is harmless for humans, as a potential treatment against vibriosis.     

Stress and Stress-Induced Neuroendocrine Changes Increase the Susceptibility of Juvenile Oysters (Crassostrea gigas) to Vibrio splendidus

Oysters are permanently exposed to various microbes, and their defense system is continuously solicited to prevent accumulation of invading and pathogenic organisms. Therefore, impairment of the animal's defense system usually results in mass mortalities in cultured oyster stocks or increased bacterial loads in food products intended for human consumption. In the present study, experiments were conducted to examine the effects of stress on the juvenile oyster's resistance to the oyster pathogen Vibrio splendidus. Oysters (Crassostrea gigas) were challenged with a low dose of a pathogenic V. splendidus strain and subjected to a mechanical stress 3 days later. Both mortality and V. splendidus loads increased in stressed oysters, whereas they remained low in unstressed animals. Injection of noradrenaline or adrenocorticotropic hormone, two key components of the oyster neuroendocrine stress response system, also caused higher mortality and increased accumulation of V. splendidus in challenged oysters. These results suggest that the physiological changes imposed by stress, or stress hormones, influenced host-pathogen interactions in oysters and increased juvenile C. gigas vulnerability to Vibrio splendidus.     

Occurrence and Expression of Luminescence in Vibrio cholerae

Several species of the genus Vibrio, including Vibrio cholerae, are bioluminescent or contain bioluminescent strains. Previous studies have reported that only 10% of V. cholerae strains are luminescent. Analysis of 224 isolates of non-O1/non-O139 V. cholerae collected from Chesapeake Bay, MD, revealed that 52% (116/224) were luminescent when an improved assay method was employed and 58% (130/224) of isolates harbored the luxA gene. In contrast, 334 non-O1/non-O139 V. cholerae strains isolated from two rural provinces in Bangladesh yielded only 21 (6.3%) luminescent and 35 (10.5%) luxA⁺ isolates. An additional 270 clinical and environmental isolates of V. cholerae serogroups O1 and O139 were tested, and none were luminescent or harbored luxA. These results indicate that bioluminescence may be a trait specific for non-O1/non-O139 V. cholerae strains that frequently occur in certain environments. Luminescence expression patterns of V. cholerae were also investigated, and isolates could be grouped based on expression level. Several strains with defective expression of the lux operon, including natural K variants, were identified.     

Planktonic Luminous Bacteria of Vellar Estuary,South India

Distribution of planktonic luminous bacteria in relation to environmental parameters was investigated at two stations located in the Vellar Estuary. Luminous microflora showed a pronounced seasonal cycle with very low counts during monsoon months followed by an increase in post monsoon and peak counts during summer. The population density of these procaryotes was remarkably high ranging from 3.5 to 33.1 colony forming units per ml. They constituted 2.1 to 52.1 % of the total bacterial counts. Salinity appeared to govern the distribution of luminous procaryotes as their counts corresponded well with fluctuations in salinity. Taxonomic affiliation of the isolates revealed predominance of Vibrio harveyi. Vibrio fischeri and Photobacterium leiognathi exhibited sparse distribution.     

Mutations in the lux Operon of Natural Dark Mutants in the Genus Vibrio

Bacterial bioluminescence can display a wide range of intensities among strains, from very bright to undetectable, and it has been shown previously that there are nonluminous vibrios that possess lux genes. In this paper, we report the isolation and characterization of completely dark natural mutants in the genus Vibrio. Screening of over 600 Vibrio isolates with a luxA gene probe revealed that approximately 5% carried the luxA gene. Bioluminescence assays of the luxA-positive isolates, followed by repetitive extragenic palindromic-PCR fingerprinting, showed three unique genotypes that are completely dark. The dark mutants show a variety of lesions, including an insertion sequence, point mutations, and deletions. Strain BCB451 has an IS10 insertion sequence in luxA, a mutated luxE stop codon, and a truncated luxH. Strain BCB494 has a 396-bp deletion in luxC, and strain BCB440 has a frameshift in luxC. This paper represents the first molecular characterization of natural dark mutants and the first demonstration of incomplete lux operons in natural isolates.     

Phylogenetic analysis of the incidence of lux gene horizontal transfer in Vibrionaceae

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.     

Growth and luminescence of luminous bacteria promoted by agents of microbial origin

The examination of four species of luminous bacteria Photobacterium leiognathi, Photobacterium phosphoreum, Vibrio fischeri and Vibrio harveyi has enabled us to reveal some nutrient medium components effecting growth, luminescence intensity and luciferase synthesis. These agents are nucleic components (nucleotides, nucleosides and amine bases), amino acids and vitamins, which are part of hydrolysates from the biomass of various lithotrophic microorganisms, hydrogen-oxidizing, ironoxidizing and carboxydobacteria. The effect of promoting agents essentially alters the physiological state and ultrastructure of the cells of luminous bacteria and increases luciferase biosynthesis two- to three-fold compared to a control.     

Specificity of anti-Vibrio immune response through p38 MAPK and PKC activation in the hemocytes of the mussel Mytilusgalloprovincialis

In mussel (Mytilus sp.) hemocytes, differential functional responses to injection with different types of live and heat-killed Vibrio species have been recently demonstrated.In this work, responses of Mytilus hemocytes to heat-killed Vibrio splendidus LGP32 and the mechanisms involved were investigated in vitro and the results were compared with those obtained with Vibrio anguillarum (ATCC 19264). Adhesion of hemocytes after incubation with bacteria was evaluated by flow cytometry: both total hemocyte counts (THC) and percentage of hemocyte sub-populations were determined in non-adherent cells. Functional parameters such as lysosomal membrane stability, lysozyme release, extracellular ROS production and NO production were evaluated, as well as the phosphorylation state of the stress-activated p38 MAPK and PKC. Neither Vibrio affected total hemocyte adhesion, while both induced similar lysosomal destabilization and NO production. However, V. splendidus decreased adhesion of large granulocytes, induced rapid and persistent lysozyme release and stimulated extracellular ROS production: these effects were associated with persistent activation of p38 MAPK and PKC. In contrast, V. anguillarum decreased adhesion of large semigranular hemocytes and increased that of hyalinocytes, had no effect on the extracellular ROS production, and induced significantly lower lysozyme release and phosphorylation of p-38 MAPK and PKC than V. splendidus. These data reinforced the existence of specific interactions between mussel hemocytes and V. splendidus LGP32 and suggest that this Vibrio strain affects bivalve hemocytes through disregulation of immune signaling. The results support the hypothesis that responses of bivalve hemocytes to different bacterial stimuli may depend not only on the nature of the stimulus, but also on the cell subtype, thus leading to differential activation of signaling components.     

Numerical Taxonomy of <Emphasis Type="Italic">Vibrionaceae</Emphasis> Isolated from Cultured Amberjack (<Emphasis Type="Italic">Seriola dumerili</Emphasis>) and Surrounding Water

A numerical taxonomic study was performed on 148 isolates of Gram-negative, heterotrophic, facultative anaerobic bacteria isolated from amberjack (Seriola dumerili) and its surrounding culture water. The study included 30 type and reference strains belonging to genera Vibrio, Listonella, and Photobacterium. The strains were characterized by 109 morphological, biochemical, physiological, and nutritional tests. Cluster analysis of similarity matrices obtained with S_SM and S_J coefficients was carried out. UPGMA (unweighted pair group mathematical average) analysis defined 11 phena at S_SM values ≥ 86%. Nine phena were identified as Vibrio alginolyticus, V. fischeri, V. harveyi, V. carchariae, V. mediterranei, V. splendidus, V. furnissii, V. parahaemolyticus, and Photobacterium damselae subsp. damselae. The two latter comprised strains isolated from diseased fish. Received: 27 March 2002 / Accepted: 24 May 2002     

Isolation and Identification of Photobacterium phosphoreum from an Unexpected Niche: Migrating Salmon

Six luminous bacteria were isolated from migrating salmon in the Yukon River, Alaska. All isolates were identified as Photobacterium phosphoreum. Previous studies suggest that P. phosphoreum is an exclusively marine bacterium, while our Alaskan isolates are from salmon which migrated up to 1,228 km from the marine environment.     

Evidence for the Involvement of Pathogenic Bacteria in Summer Mortalities of the Pacific Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

A study was conducted to investigate the involvement of bacteria in oyster mortalities during summer. Moribund and apparently healthy oysters were sampled during mortality events along the French coast and in rearing facilities, usually when temperature reached 19°C or higher, and oysters were in the gonadal maturation phase. Hemolymph samples were aseptically withdrawn and submitted to bacteriological analysis. In healthy oysters, bacteria colonized hemolymph at low concentrations depending on the location. In most moribund oysters, bacteria were present in hemolymph and other tissues. These bacterial populations were more often diverse in oysters originating from the open sea than from facilities where animals were generally infected by a single type of bacterium. Only the dominant colonies were identified by phenotypic and genotypic characters (RFLP of GyrB gene and partial sequence of 16S rRNA gene). They belonged to a limited number of species including Vibrio aestuarianus, members of the V. splendidus group, V. natriegens, V. parahaemolyticus, and Pseudoalteromonas sp. The most frequently encountered species was V. aestuarianus (56% of isolates), which was composed of several strains closely related by their 16S rRNA gene but diverse by their phenotypic characters. They appeared intimately linked to oysters. The species within the V. splendidus group were less prevalent (25% of isolates) and more taxonomically dispersed. A majority of the dominant strains of V. aestuarianus and V. splendidus group injected to oysters induced mortality, whereas others belonging to the same species, particularly those found in mixture, appeared innocuous.     

Antimicrobial Resistance of the Coral Pathogen Vibrio coralliilyticus and Caribbean Sister Phylotypes Isolated from a Diseased Octocoral

Vibrio coralliilyticus is a global marine pathogen that has been found to cause disease in several marine organisms, including corals. This study is the first report of the isolation of V. coralliilyticus from a diseased Caribbean octocoral, Pseudopterogorgia americana. Five sister phylotypes were positively identified using 16S rRNA gene sequencing, recA probes specific for V. coralliilyticus, and rep-PCR fingerprinting. The antimicrobial resistance was compared between pathogenic strains of V. coralliilyticus and the Caribbean strains. First, the antimicrobial resistance of V. coralliilyticus-type strain ATCC BAA-450 was determined using an agar-overlay antimicrobial bioassay at 24°C and 27°C, temperatures which are relevant to its known temperature-dependent virulence. From 108 distinct bacteria isolated from P. americana, 12 inhibited the V. coralliilyticus-type strain at 24°C and five at 27°C. Next, the phenotypic comparison of two Caribbean phylotypes and three V. coralliilyticus reference strains against a subset of 30 bacteria demonstrated a similar resistance trend. At both temperatures, the reference strains were inhibited by three bacteria isolates, while the Caribbean strains were inhibited by four to nine bacteria. Additionally, V. coralliilyticus-type strain ATCC BAA-450 and one of the Caribbean strains were inhibited by a higher number of bacteria at 24°C compared with 27°C. Together, these results highlight that V. coralliilyticus strains have antimicrobial resistance to the majority of coral-associated bacteria tested, which may be temperature-dependent in some strains. Furthermore, all V. coralliilyticus strains tested showed multi-drug resistance to a range of 11–16 (out of 26) commercial antibiotics. This study establishes V. coralliilyticus in association with a Caribbean octocoral and demonstrates its resistance to the antimicrobial activity of coral-associated bacteria and to commercial antibiotics.