Development of a haemolysin gene‐based multiplex PCR for simultaneous detection of Vibrio campbellii,Vibrio harveyi and Vibrio parahaemolyticus

Aim: To develop a haemolysin (hly) gene‐based species‐specific multiplex PCR for simple and rapid detection of Vibrio campbellii, V. harveyi and V. parahaemolyticus. Methods and Results: The complete hly genes of three V. campbellii strains isolated from diseased shrimps were sequenced and species‐specific PCR primers were designed based on these sequences and the registered hly gene sequences of Vibrio harveyi and Vibrio parahaemolyticus. Specificity and sensitivity of the multiplex PCR was validated with 27 V. campbellii, 16 V. harveyi, and 69 V. parahaemolyticus, 18 other Vibrio species, one Photobacterium damselae and nine other bacterial species. The detection limits of all the three target species were in between 10 and 100 cells per PCR tube. Conclusions: Specificity and sensitivity of the multiplex PCR is 100% each and sufficient to be considered as an effective tool in a prediction system to prevent potential disease outbreak by these Vibrio species. Significance and Impact of the Study: Because there is lack of simple, rapid and cost‐effective method to differentiate these closely related V. campbellii, V. harveyi and V. parahaemolyticus species, the multiplex PCR developed in this study will be very effective in epidemiological, ecological and economical points of view.     

Luminescence, virulence and quorum sensing signal production by pathogenic Vibrio campbellii and Vibrio harveyi isolates

Aims:  To study the relationship between luminescence, autoinducer production and virulence of pathogenic vibrios. Methods and Results:  Luminescence, quorum sensing signal production and virulence towards brine shrimp nauplii of 13 Vibrio campbellii and Vibrio harveyi strains were studied. Although only two of the tested strains were brightly luminescent, all of them were shown to produce the three different types of quorum sensing signals known to be produced by Vibrio harveyi. Cell-free culture fluids of all strains significantly induced bioluminescence in the cholerae autoinducer 1, autoinducer 2 and harveyi autoinducer 1 reporter strains JAF375, JMH597 and JMH612, respectively. There was no relation between luminescence and signal production and virulence towards brine shrimp. Conclusions:  There is a large difference between different strains of Vibrio campbellii and Vibrio harveyi with respect to bioluminescence. However, this is not reflected in signal production and virulence towards gnotobiotic brine shrimp. Moreover, there seems to be no relation between quorum sensing signal production and virulence towards brine shrimp. Significance and Impact of the Study:  The results presented here indicate that strains that are most brightly luminescent are not necessarily the most virulent ones and that the lower virulence of some of the strains is not due to a lack of autoinducer production.     

Analysis of a gene (vch) encoding hemolysin isolated and sequenced from Vibrio campbellii

This study describes the amplification, localization, and sequence analysis of a hemolysin gene from type strain V. campbellii NBRC 15631--the first report of a full-length hemolysin gene for the species. An amplicon ( approximately 600 bp) of polymerase chain reaction performed using V. campbellii DNA template and primers previously designed to target a fragment of V. harveyi hemolysin gene (vhh) was shotgun-cloned and sequenced, generating 576 bp nucleotide sequences of the V. campbellii hemolysin gene. PCR primers designed based on these initial sequences were used to amplify a 551-bp V. campbellii hemolysin gene fragment that was used as probe in Southern hybridization, which localized the complete hemolysin gene within a 3.5-kb HindIII restriction fragment of the V. campbellii genomic DNA. To obtain the remaining DNA sequences upstream and downstream of the 576-bp hemolysin gene sequences, inverse PCR was performed using a self-ligated (circularized) V. campbellii HindIII restriction fragment as the template and PCR primers designed to amplify flanking regions of the 576-bp gene fragment. Nucleotide sequences from the terminal regions of the 3.1-kb product of inverse PCR provided the flanking sequences, resulting in the complete sequence for the V. campbellii hemolysin gene. A VCH PCR primer set was designed to amplify a 1.3-kb region containing the entire hemolysin gene even from other V. campbellii strains, which was sequenced to confirm the V. campbellii hemolysin gene sequence. An open reading frame (ORF) of 1,254 bp (designated as vch) was identified, sharing 79% sequence identity with V. harveyi hemolysin gene vhh, representing 262 base substitutions between V. campbellii and V. harveyi. The deduced amino acid sequence of V. campbellii hemolysin (VCH) shows homologies to the V. harveyi hemolysin (VHH), thermolabile hemolysin of V. parahaemolyticus, proteins such as phospholipase of V. vulnificus and lecithinases of V. mimicus and V. cholerae. The VCH primer set did not produce any amplicon in PCR using V. harveyi DNA, and may therefore be used to distinguish environmental strains of V. campbellii from V. harveyi.     

Increased susceptibility of white spot syndrome virus-infected Litopenaeus vannamei to Vibrio campbellii

The concept of polymicrobial disease is well accepted in human and veterinary medicine but has received very little attention in the field of aquaculture. This study was conducted to investigate the synergistic effect of white spot syndrome virus (WSSV) and Vibrio campbellii on development of disease in specific pathogen-free (SPF) shrimp Litopenaeus vannamei. The juvenile shrimp were first injected with WSSV at a dose of 30 SID(50) shrimp(-1) (SID(50) = shrimp infectious dose with 50% endpoint) and 24 h later with 10(6) colony-forming units (cfu) of V. campbellii shrimp(-1). Controls receiving just one of the pathogens or negative inocula were included. In the treatment with WSSV only, shrimp started to die at 48-108 h post injection (hpi) and cumulative mortality reached 100% at 268-336 hpi. In the treatment with only V. campbellii injection (10(6) cfu shrimp(-1)), cumulative mortality reached 16.7%. Shrimp in the dual treatment died very quickly after V. campbellii injection and 100% cumulative mortality was obtained at 72-96 hpi. When WSSV-injected shrimp were given sonicated V. campbellii instead of live V. campbellii, no synergistic effect was observed. Density of V. campbellii in the haemolymph of co-infected moribund shrimp collected 10 h after V. campbellii injection was significantly higher than in shrimp injected with V. campbellii only (P < 0.01). However, there was no difference in WSSV replication between shrimp inoculated with WSSV only compared with dually inoculated ones. This study revealed that prior infection with WSSV enhances the multiplication and disease inducing capacity of V. campbellii in shrimp.     

The probabilistic identification of Vibrio spp. isolated from surface seawater with special reference to Vibrio campbellii

A subset (n = 20) of 143 putative Vibrio spp. isolated from surface seawater samples collected along a transect from Barbados to Puerto Rico to Bermuda were included in a numerical taxonomy study. Fourteen strains were identified as Vibrio campbellii . It was concluded that V. campbellii occurs in the open ocean with high frequency, and probably represents an autochthonous species in surface waters of the Atlantic Ocean.     

Quorum Sensing-Disrupting Brominated Furanones Protect the Gnotobiotic Brine Shrimp Artemia franciscana from Pathogenic Vibrio harveyi, Vibrio campbellii, and Vibrio parahaemolyticus Isolates

Autoinducer 2 (AI-2) quorum sensing was shown before to regulate the virulence of Vibrio harveyi towards the brine shrimp Artemia franciscana. In this study, several different pathogenic V. harveyi, Vibrio campbellii, and Vibrio parahaemolyticus isolates were shown to produce AI-2. Furthermore, disruption of AI-2 quorum sensing by a natural and a synthetic brominated furanone protected gnotobiotic Artemia from the pathogenic isolates in in vivo challenge tests.     

Quorum sensing-disrupting brominated furanones protect the gnotobiotic brine shrimp Artemia franciscana from pathogenic Vibrio harveyi, Vibrio campbellii, and Vibrio parahaemolyticus isolates

Autoinducer 2 (AI-2) quorum sensing was shown before to regulate the virulence of Vibrio harveyi towards the brine shrimp Artemia franciscana. In this study, several different pathogenic V. harveyi, Vibrio campbellii, and Vibrio parahaemolyticus isolates were shown to produce AI-2. Furthermore, disruption of AI-2 quorum sensing by a natural and a synthetic brominated furanone protected gnotobiotic Artemia from the pathogenic isolates in in vivo challenge tests.     

Multilocus sequence analysis reveals that Vibrio harveyi and V. campbellii are distinct species

Identification and classification of Vibrio species have relied upon band pattern methods (e.g., amplified fragment length polymorphism) and DNA-DNA hybridization. However, data generated by these methods cannot be used to build an online electronic taxonomy. In order to overcome these limitations, we developed the first standard multilocus sequence scheme focused on the ubiquitous and pathogenic Vibrio harveyi species group (i.e., V. harveyi, V. campbellii, V. rotiferianus, and a new as yet unnamed species). We examined a collection of 104 isolates from different geographical regions and hosts using segments of seven housekeeping genes. These two species formed separated clusters on the basis of topA, pyrH, ftsZ, and mreB gene sequences. The phylogenetic picture obtained by the other three loci, i.e., gyrB, recA, and gapA, was more complex though. V. campbellii appeared nested within V. harveyi in the recA trees, whereas V. harveyi formed a tight nested cluster within V. campbellii by gapA. The gyrB gene had no taxonomic resolution and grouped the two species together. The fuzziness observed in these three genes seems not be related to recombination but to low divergence due to the accumulation of only a few substitutions. In spite of this, the concatenated sequences provided evidence that the two species form two separated clusters. These clusters did not arise by recombination but by accumulation of point mutations. V. harveyi and V. campbellii isolates can be readily identified through the open database resource developed in this study (http://www.taxvibrio.lncc.br/). We argue that the species should be defined by evolutionary criteria. Strains of the same species will share at least 95% concatenated sequence similarity using the seven loci, and, most importantly, cospecific strains will form cohesive readily recognizable phylogenetic clades.     

The C2 entity of chitosugars is crucial in molecular selectivity of the Vibrio campbellii chitoporin

The marine bacterium Vibrio campbellii expresses a chitooligosaccharide-specific outer-membrane channel (chitoporin) for the efficient uptake of nutritional chitosugars that are externally produced through enzymic degradation of environmental host shell chitin. However, the principles behind the distinct substrate selectivity of chitoporins are unclear. Here, we employed black lipid membrane (BLM) electrophysiology, which handles the measurement of the flow of ionic current through porins in phospholipid bilayers for the assessment of porin conductivities, to investigate the pH dependency of chitosugar–chitoporin interactions for the bacterium’s natural substrate chitohexaose and its deacetylated form, chitosan hexaose. We show that efficient passage of the N-acetylated chitohexaose through the chitoporin is facilitated by its strong affinity for the pore. In contrast, the deacetylated chitosan hexaose is impermeant; however, protonation of the C2 amino entities of chitosan hexaose allows it to be pulled through the channel in the presence of a transmembrane electric field. We concluded from this the crucial role of C2-substitution as the determining factor for chitoporin entry. A change from N-acetylamino- to amino-substitution effectively abolished the ability of approaching molecules to enter the chitoporin, with deacetylation leading to loss of the distinctive structural features of nanopore opening and pore access of chitosugars. These findings provide further understanding of the multistep pathway of chitin utilization by marine Vibrio bacteria and may guide the development of solid-state or genetically engineered biological nanopores for relevant technological applications.     

Acquired phototrophy in ciliates: a review of cellular interactions and structural adaptations

Many ciliates acquire the capacity for photosynthesis through stealing plastids or harboring intact endosymbiotic algae. Both phenomena are a form of mixotrophy and are widespread among ciliates. Mixotrophic ciliates may be abundant in freshwater and marine ecosystems, sometimes making substantial contributions toward community primary productivity. While mixotrophic ciliates utilize phagotrophy to capture algal cells, their endomembrane system has evolved to partially bypass typical heterotrophic digestion pathways, enabling metabolic interaction with foreign cells or organelles. Unique adaptations may also be found in certain algal endosymbionts, facilitating establishment of symbiosis and nutritional interactions, while reducing their fitness for survival as free-living cells. Plastid retaining oligotrich ciliates possess little selectivity from which algae they sequester plastids, resulting in unstable kleptoplastids that require frequent ingestion of algal cells to replace them. Mesodinium rubrum (=Myrionecta rubra) possesses cryptophyte organelles that resemble a reduced endosymbont, and is the only ciliate capable of functional phototrophy and plastid division. Certain strains of M. rubrum may have a stable association with their cryptophyte organelles, while others need to acquire a cryptophyte nucleus through feeding. This process of stealing a nucleus, termed karyoklepty, was first described in M. rubrum and may be an evolutionary precursor to a stable, reduced endosymbiont, and perhaps eventually a tertiary plastid. The newly described Mesodinium"chamaeleon," however, is less selective of which cryptophyte species it will retain organelles, and appears less capable of sustained phototrophy. Ciliates likely stem from a phototrophic ancestry, which may explain their propensity to practice acquired phototrophy.     

Luciferase from Vibrio campbellii is more thermostable and binds reduced FMN better than its homologues

A new luciferase from V. campbellii (Lux_Vc) was cloned and expressed in Escherichia coli and purified to homogeneity. Although the amino acid sequences and the catalytic reactions of Lux_Vc are highly similar to those of the luciferase from V. harveyi (Lux_Vh), the two enzymes have different affinities toward reduced FMN (FMNH(-)). The catalytic reactions of Lux_Vc and Lux Vh were monitored by stopped-flow absorbance and luminescence spectroscopy at 4 degrees C and pH 8. The measured Kd at 4 degrees C for the binding of FMNH(-) to Lux_Vc was 1.8 microM whereas to Lux_Vh, it was 11 microM. Another difference between the two enzymes is that Lux_Vc is more stable than Lux_Vh over a range of temperatures; Lux_Vc has t1/2 of 1020 min while Lux_Vh has t1/2 of 201 min at 37 degrees C. The superior thermostability and tighter binding of FMNH(-) make Lux_Vc a more tractable luciferase than Lux_Vh for further structural and functional studies, as well as a more suitable enzyme for some applications. The kinetics results reported here reveal transient states in the reaction of luciferase that have not been documented before.     

Effects of hypercapnic hypoxia on inactivation and elimination of Vibrio campbellii in the Eastern oyster, Crassostrea virginica

The Eastern oyster, Crassostrea virginica, inhabits shallow coastal waters that frequently experience periods of low dissolved oxygen (hypoxia) and elevated CO(2) (hypercapnia) levels. Bacteria are extremely abundant in these environments and accumulate in large numbers in filter-feeding oysters, which can act as passive carriers of human pathogens. Although hypercapnic hypoxia (HH) can affect certain specific immune mechanisms, its direct effect on the inactivation, degradation and elimination of bacteria in oysters is unknown. This research was conducted to determine whether exposure to HH reduces the ability of C. virginica to inactivate and eliminate Vibrio campbellii following its injection into the adductor muscle. Oysters were held in fully air-saturated (normoxic; partial O(2) pressure [P(O2)] = 20.7 kPa, CO(2) < 0.06 kPa, pH 7.8 to 8.0) or HH (P(O2) = 4 kPa, CO(2) = 1.8 kPa, pH 6.5 to 6.8) seawater at 25 degrees C for 4 h before being injected in the adductor muscle with 10(5) live Vibrio campbellii bacteria and remained under these conditions for the remainder of the experiment (up to 24 h postinjection). Real-time PCR was used to quantify the number of intact V. campbellii bacteria, while selective plating was used to quantify the number of injected bacteria remaining culturable in whole-oyster tissues, seawater, and feces/pseudofeces at 0, 1, 4, and 24 h postinjection. We found that oysters maintained under normoxic conditions were very efficient at inactivating and degrading large numbers of injected bacteria within their tissues. Moreover, a small percentage ( approximately 10%) of injected bacteria were passed into the surrounding seawater, while less than 1% were recovered in the feces/pseudofeces. In contrast, HH increased the percentage of culturable bacteria recovered from the tissues of oysters, suggesting an overall decrease in bacteriostasis. We suggest that poor water quality may increase the risk that oysters will harbor and transmit bacterial pathogens hazardous to human and ecosystem health.     

Temperature‐dependent phagotrophy and phototrophy in a mixotrophic chrysophyte

The roles of temperature and light on grazing and photosynthesis were examined for Dinobryon sociale, a common freshwater mixotrophic alga. Photosynthetic rate was determined for D. sociale adapted to temperatures of 8, 12, 16, and 20°C under photosynthetically active radiation light irradiances of 25, 66, and 130 μmol photons · m^?2 · s^?1, with concurrent measurement of bacterial ingestion at all temperatures under medium and high light (66 and 130 μmol photons · m^?2 · s^?1). Rates of ingestion and photosynthesis increased with temperature to a maximum at 16°C under the two higher light regimes, and declined at 20°C. Although both light and temperature had a marked effect on photosynthesis, there was no significant difference in bacterivory at medium and high irradiances at any given temperature. At the lowest light condition (25 μmol photons · m^?2 · s^?1), photosynthesis remained low and relatively stable at all temperatures. D. sociale acquired the majority of carbon from photosynthesis, although the low photosynthetic rate without a concurrent decline in feeding rate at 8°C suggested 20%–30% of the carbon budget could be attributed to bacterivory at low temperatures. Grazing experiments in nutrient‐modified media revealed that this mixotroph had increased ingestion rates when either dissolved nitrogen or phosphorus was decreased. This work increases our understanding of environmental effects on mixotrophic nutrition. Although the influence of abiotic factors on phagotrophy and phototrophy in pure heterotrophs and phototrophs has been well studied, much less is known for mixotrophic organisms.     

Spectroscopic and photochemical characterization of a deep ocean proteorhodopsin

A second group of proteorhodopsin-encoding genes (blue-absorbing proteorhodopsin, BPR) differing by 20-30% in predicted primary structure from the first-discovered green-absorbing (GPR) group has been detected in picoplankton from Hawaiian deep sea water. Here we compare BPR and GPR absorption spectra, photochemical reactions, and proton transport activity. The photochemical reaction cycle of Hawaiian deep ocean BPR in cells is 10-fold slower than that of GPR with very low accumulation of a deprotonated Schiff base intermediate in cells and exhibits mechanistic differences, some of which are due to its glutamine residue rather than leucine at position 105. In contrast to GPR and other characterized microbial rhodopsins, spectral titrations of BPR indicate that a second titratable group, in addition to the retinylidene Schiff base counterion Asp-97, modulates the absorption spectrum near neutral pH. Mutant analysis confirms that Asp-97 and Glu-108 are proton acceptor and proton donor, respectively, in retinylidene Schiff base proton transfer reactions during the BPR photocycle as previously shown for GPR, but BPR contains an alternative acceptor evident in its D97N mutant, possibly the same as the second titratable group modulating the absorption spectrum. BPR, similar to GPR, carries out outward light-driven proton transport in Escherichia coli vesicles but with a reduced translocation rate attributable to its slower photocycle. In energized E. coli cells at physiological pH, the net effect of BPR photocycling is to generate proton currents dominated by a triggered proton influx, rather than efflux as observed with GPR-containing cells. Reversal of the proton current with the K+-ionophore valinomycin supports that the influx is because of voltage-gated channels in the E. coli cell membrane. These observations demonstrate diversity in photochemistry and mechanism among proteorhodopsins. Calculations of photon fluence rates at different ocean depths show that the difference in photocycle rates between GPR and BPR as well as their different absorption maxima may be explained as an adaptation to the different light intensities available in their respective marine environments. Finally, the results raise the possibility of regulatory (i.e. sensory) rather than energy harvesting functions of some members of the proteorhodopsin family.     

Priming the prophenoloxidase system of Artemia franciscana by heat shock proteins protects against Vibrio campbellii challenge

Like other invertebrates, the brine shrimp Artemia franciscana relies solely on innate immunity, which by definition lacks adaptive characteristics, to combat against invading pathogens. One of the innate mechanisms is melanisation of bacteria mediated by the activation of the prophenoloxidase (proPO) system. The 70 kDa heat shock proteins (Hsp70) derived from either prokaryote (Escherichia coli) or eukaryote (Artemia), well conserved and immune-dominant molecules, protect Artemia against Vibrio campbellii. However, the molecular mechanisms by which these proteins protect Artemia against Vibrio campbellii infection are unknown. Here we demonstrated that feeding gnotobiotically grown Artemia with either Artemia Hsp70 or the E. coli Hsp70 equivalent DnaK, each overproduced in E. coli, followed by V. campbellii challenge enhanced the proPO system, at both mRNA and protein activity levels. Additionally, the Artemia fed with these proteins survived well in a Vibrio challenge assay. These results indicated that Hsp70s derived from either prokaryotic or eukaryotic sources generate protective immunity in the crustacean Artemia against V. campbellii infection by priming the proPO system. This is apparently the first in vivo report on priming activity of Hsp70 in an invertebrate.     

Folding and assembly of proteorhodopsin

Proteorhodopsins (PRs), the recently discovered light-driven proton pumps, play a major role in supplying energy for microbial organisms of oceans. In contrast to PR, rhodopsins found in Archaea and Eukarya are structurally well characterized. Using single-molecule microscopy and spectroscopy, we observed the oligomeric assembly of native PR molecules and detected their folding in the membrane. PR showed unfolding patterns identical with those of bacteriorhodopsin and halorhodopsin, indicating that PR folds similarly to archaeal rhodopsins. Surprisingly, PR predominantly assembles into hexameric oligomers, with a smaller fraction assembling into pentamers. Within these oligomers, PR arranged into radial assemblies. We suggest that this structural assembly of PR may have functional implications.