"Quorum sensing" regulation of lux gene expression and the structure of lux operon in marine bacteria Alivibrio logei

A group of luminescent strains of marine bacteria Alivibrio logei has been isolated (basins of the Okhotsk, White and Bering Seas). Strains A. logei were shown to be psycrophiic bacteria with an optimal growth temperature of approximately 15 degrees C. Biolumiscent characteristics of strains were studied, and the expression of lux genes was shown to be regulated by the "quorum sensing" system. The A. logei lux operon was cloned in Escherichia coli cells and the structure of this operon and its nucleotide sequence were determined. The structure of A. logei lux operon differs markedly from that in the closely related species of luminescent marine bacteria A. fischeri. In the structure of the A. logei lux operon, the the luxI gene is absent in front of luxC, and a fragment containing luxR2-luxI genes is located immediately after luxG gene. Luminescent psycrophiic marine bacteria of A. logei are assumed to be widely distributed in cold waters of northern seas.     

<Emphasis Type="Italic">Aliivibrio logei</Emphasis> KCh1 (Kamchatka isolate): Biochemical and bioluminescence characteristics and cloning of the <Emphasis Type="Italic">lux</Emphasis> operon

A new strain of luminescent marine bacteria of the genus Aliivibrio (formerly Vibrio) was isolated from the intestines of a goby Cottida sp. (Sea of Okhotsk basin; the strain was marked as KCh1). The basic conditions of growth on laboratory media were determined for the strain. Strain KCh1 was shown to be a psychrophilic bacterium with an optimal growth temperature of approximately 15°C. The nucleotide sequence of the 16S rRNA gene was determined and shown to be almost identical to the 16S rRNA gene sequence of Aliivibrio logei and A. salmonicida but considerably different from that of A. fischeri. The biochemical characteristics (nitrate reduction, lysine decarboxylation, and D-galactose fermentation) of strain KCh1 matched those of A. logei and A. fischeri strains. An antibioticogram for strain KCh1 was determined. The lux operon of the strain was cloned in Escherichia coli cells and partially sequenced, the results show a high homology with the lux operon of A. salmonicida.     

“Quorum sensing” regulation and the structure of <Emphasis Type="Italic">lux</Emphasis> the operon in marine bacteria <Emphasis Type="Italic">Aliivibrio logei</Emphasis>

A group of luminescent strains of marine bacteria Aliivibrio logei has been isolated (basins of the Okhotsk, White and Bering Seas). Strains A. logei were shown to be psycrophilic bacteria with an optimal growth temperature of approximately 15°C. Bioluminescent characteristics of strains were studied, and the expression of lux genes was shown to be regulated by the “quorum sensing” system. The A. logei lux operon was cloned in Escherichia coli cells and the structure of this operon and its nucleotide sequence were determined. The structure of A. logei lux operon differs markedly from that in the closely related species of luminescent marine bacteria A. fischeri. In the structure of the A. logei lux operon, the luxI gene is absent in front of luxC, and a fragment containing luxR2-luxI genes is located immediately after luxG gene. Luminescent psycrophilic marine bacteria of A. logei are assumed to be widely distributed in cold waters of northern seas.     

Identification and cloning of immunogenic Aliivibrio salmonicida Pal-like protein present in profiled outer membrane and secreted subproteome

Aliivibrio salmonicida is the aetiological agent of cold water vibriosis affecting farmed fish species, a disease that today is fully controlled by vaccination. However, the molecular mechanisms behind the successful vaccine are largely unknown. In order to gain insight into the possible mechanisms of A. salmonicida vaccines, we report here the profiles of both the outer membrane and secreted subproteomes of A. salmonicida LFI315. The 2 subproteomes were resolved by 2-dimensional electrophoresis that identified a total of 82 protein entries. Monoclonal antibodies specific to an unidentified protein antigen were utilized in the immunoproteomic analysis of both outer membrane proteins and extracellular proteins. The immunogenic protein was located in both subproteomes and identified as a 20 kDa peptidoglycan-associated lipoprotein (Pal). The identity of the antigen was verified by heterologous expression of the cloned A. salmonicida pal gene (VSAL_I1899). It is likely that the immunogenic Pal-like protein is among the constituents that act as a protective antigen in the successful vaccine used today. In view of this, it may be considered a potentially useful component in future vaccine development and pathogenicity studies.     

Global responses of Aliivibrio salmonicida to hydrogen peroxide as revealed by microarray analysis

Aliivibrio salmonicida causes "cold-water vibriosis" (or "Hitra disease") in fish, including marine-reared Atlantic salmon. During development of the disease the bacterium will encounter macrophages with antibacterial activities such as production of damaging reactive oxygen species (ROS). To defend itself the bacterium will presumably start producing detoxifying enzymes, reducing agents, and proteins involved in DNA and protein repair systems. Even though responses to oxidative stress are well studied for a few model bacteria, little work has been done in general to explain how important groups of pathogens, like members of the Vibrionaceae family, can survive at high levels of ROS. We have used bioinformatic tools and microarray to study how A. salmonicida responds to hydrogen peroxide (H(2)O(2)). First, we used the recently published genome sequence to predict potential binding sites for OxyR (H(2)O(2) response regulator). The computer-based search identified OxyR sites associated with 20 single genes and 8 operons, and these predictions were compared to experimental data from Northern blot analysis and microarray analysis. In general, OxyR binding site predictions and experimental results are in agreement. Up- and down regulated genes are distributed among all functional gene categories, but a striking number of ≥2 fold up regulated genes encode proteins involved in detoxification and DNA repair, are part of reduction systems, or are involved in carbon metabolism and regeneration of NADPH. Our predictions and -omics data corroborates well with findings from other model bacteria, but also suggest species-specific gene regulation.     

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.     

Evidence for a type III secretion system in Aeromonas salmonicida subsp. salmonicida

Aeromonas salmonicida subsp. salmonicida, the etiological agent of furunculosis, is an important fish pathogen. We have screened this bacterium with a broad-host-range probe directed against yscV, the gene that encodes the archetype of a highly conserved family of inner membrane proteins found in every known type III secretion system. This has led to the identification of seven open reading frames that encode homologues to proteins functioning within the type III secretion systems of Yersinia species. Six of these proteins are encoded by genes comprising a virA operon. The A. salmonicida subsp. salmonicida yscV homologue, ascV, was inactivated by marker replacement mutagenesis and used to generate an isogenic ascV mutant. Comparison of the extracellular protein profiles from the ascV mutant and the wild-type strain indicates that A. salmonicida subsp. salmonicida secretes proteins via a type III secretion system. The recently identified ADP-ribosylating toxin AexT was identified as one such protein. Finally, we have compared the toxicities of the wild-type A. salmonicida subsp. salmonicida strain and the ascV mutant against RTG-2 rainbow trout gonad cells. While infection with the wild-type strain results in significant morphological changes, including cell rounding, infection with the ascV mutant has no toxic effect, indicating that the type III secretion system we have identified plays an important role in the virulence of this pathogen.     

Freshwater bioluminescence in Vibrio albensis (Vibrio cholerae biovar albensis) NCIMB 41 is caused by a two-nucleotide deletion in luxO

We previously proposed that the function of the lux operon is to produce a halotolerant flavodoxin, FP390 or P-flavin binding protein, and not to produce light. A crucial basis of this hypothesis is that almost all species of luminous bacteria emit light in culture media containing over 2% NaCl. However, Vibrio albensis (Vibrio cholerae biovar albensis) NCIMB 41 emits light in freshwater and this appears to be in direct conflict with our hypothesis. To determine why this exceptional freshwater bioluminescence is emitted, we studied the lux operon and the regulatory system of the operon in this strain, and found that expression of the operon is regulated by a system involving a derivative of 4,5-dihydroxy 2,3-pentanedione, DPD, as an inducer, and the repressor gene for the lux operon, luxO, is damaged by deletion of two nucleotides. Furthermore, to study the effect of damage to the luxO gene, pUC18 derivatives containing the damaged and repaired luxO sequences were prepared. Cells transfected with the damaged luxO sequence emitted light like the parental strain, whereas ones transfected with the repaired one did so only sparingly. Here we show that the light emission in freshwater by this strain is not in conflict with our hypothesis.