Potential antitumor therapeutic application of Grimontia hollisae thermostable direct hemolysin mutants.

We report on the preparation of a new type of immunotoxin by conjugation of an epidermal growth factor receptor (EGFR)-binding peptide and an R46E mutation of thermostable direct hemolysin from Grimontia hollisae, (Gh-TDH(R) (46E) /EB). The hybrid immunotoxin was purified to homogeneity and showed a single band with slight slower mobility than that of Gh-TDH(R) (46E) . Cytotoxicity assay of Gh-TDH(R) (46E) /EB on EGFR highly, moderately, low, and non-expressed cells, A431, MDA-MB-231, HeLa, and HEK293 cells, respectively, showed apparent cytotoxicity on A431 and MDA-MB-231 cells but not on HeLa or HEK293 cells. In contrast, no cytotoxicity was observed for these cells treated with either Gh-TDH(R) (46E) or EB alone, indicating enhanced cytotoxic efficacy of Gh-TDH(R) (46E) by the EGFR binding moiety. Further antitumor activity assay of Gh-TDH(R) (46E) /EB in a xenograft model of athymic nude mice showed obvious shrinkage of tumor size and degeneration, necrosis, and lesions of tumor tissues compared to the normal tissues. Therefore, the combination of Gh-TDH(R) (46E) with target affinity agents opens new possibilities for pharmacological treatment of cancers and potentiates the anticancer drug's effect.

Production of bioactive secondary metabolites by marine vibrionaceae.

Bacteria belonging to the Vibrionaceae family are widespread in the marine environment. Today, 128 species of vibrios are known. Several of them are infamous for their pathogenicity or symbiotic relationships. Despite their ability to interact with eukaryotes, the vibrios are greatly underexplored for their ability to produce bioactive secondary metabolites and studies have been limited to only a few species. Most of the compounds isolated from vibrios so far are non-ribosomal peptides or hybrids thereof, with examples of N-containing compounds produced independent of nonribosomal peptide synthetases (NRPS). Though covering a limited chemical space, vibrios produce compounds with attractive biological activities, including antibacterial, anticancer, and antivirulence activities. This review highlights some of the most interesting structures from this group of bacteria. Many compounds found in vibrios have also been isolated from other distantly related bacteria. This cosmopolitan occurrence of metabolites indicates a high incidence of horizontal gene transfer, which raises interesting questions concerning the ecological function of some of these molecules. This account underlines the pending potential for exploring new bacterial sources of bioactive compounds and the challenges related to their investigation.

Salinivibrio sharmensis sp. nov., a novel haloalkaliphilic bacterium from a saline lake in Ras Mohammed Park (Egypt).

A novel haloalkaliphilic, facultative anaerobic and Gram-negative Salinivibrio-like microorganism (designated strain BAG(T)) was recovered from a saline lake in Ras Mohammed Park (Egypt). Cells were motile, curved rods, not spore-forming and occurred singly. Strain BAG(T) grew optimally at 35°C (temperature growth range 25-40°C) with 10.0% (w/v) NaCl [NaCl growth range 6.0-16.0% (w/v)] and at pH 9.0 (pH growth range 6.0-10.0). Strain BAG(T) had phosphatidylethanolamine (PEA) and phosphatidylglycerol (PG) as the main polar lipids, C16:0 (54.0%) and C16:1 (26.0%) as the predominant cellular fatty acids and Q-8 as the major respiratory quinone. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain BAG(T) was a member of Salinivibrio genus, with the highest sequence similarities of 99.1, 98.4 and 98.1% to Salinivibrio siamensis JCM 14472(T), Salinivibrio proteolyticus DSM 19052(T) and Salinivibrio costicola subsp. alcaliphilus DSM 16359(T), respectively. DNA-DNA hybridization values of strain BAG(T) with members of Salinivibrio genus were lower than 55.0%. DNA G + C content was 51.0 mol%. On the basis of the polyphasic taxonomic results revealed in this study, strain BAG(T) should be classified as a novel species of Salinivibrio genus, for which the name Salinivibrio sharmensis sp. nov. is proposed, with the type strain BAG(T) (=ATCC BAA-1319(T) = DSM 18182(T)).

Accumulation and role of compatible solutes in fast-growing Salinivibrio costicola subsp. yaniae.

The moderately halophilic bacterium Salinivibrio costicola subsp. yaniae showed an extremely fast growth rate. Optimal growth was observed in artificial seawater containing 1.4 mol/L NaCl and in MM63 media containing 0.6 mol/L NaCl. We analyzed a variety of compatible solutes that had accumulated in this strain grown in the media. The supplementation effect of the compatible solutes glycine betaine, glutamate, and ectoine to the growth of S. costicola subsp. yaniae was examined. Glycine betaine and glutamate had no supplementation effect on the fast growth rate. Growth of salt-sensitive mutants MU1 and MU2, both of which were defective in the ability to synthesize ectoine, was not observed in MM63 medium in the presence of more than 1.0 mol/L NaCl. From these data, we conclude that ectoine was the predominant compatible solute synthesized in this bacterium that effected an extremely fast growth rate.

Description of Enterovibrio nigricans sp. nov., reclassification of Vibrio calviensis as Enterovibrio calviensis comb. nov. and emended description of the genus Enterovibrio Thompson et al. 2002.

Eleven strains of halophilic, facultative anaerobes isolated from healthy and diseased Dentex dentex and Sparus aurata (bony fishes) cultured in Spanish Mediterranean fisheries have been studied by a polyphasic approach that included a wide phenotypic characterization, DNA-DNA hybridization and phylogenetic analysis using 16S rRNA, recA and rpoD gene sequences. All strains were phylogenetically related to Enterovibrio species and Vibrio calviensis. On the basis of sequence analysis and DNA-DNA hybridization data, eight of the strains were identified as Enterovibrio coralii. The remaining three strains formed a tight, independent clade in all sequence analyses and showed less than 70 % DNA-DNA hybridization with strains of the closest Enterovibrio species, from which they could be differentiated by several phenotypic traits. We conclude that these three strains represent a novel species in the genus Enterovibrio and we thus propose the name Enterovibrio nigricans sp. nov., with strain DAl 1-1-5(T) (=CECT 7320(T) =CAIM 661(T)) as the type strain. In addition, we propose the reclassification of Vibrio calviensis Denner et al. 2002 as Enterovibrio calviensis comb. nov. (type strain RE35F/12(T) [corrected] =CIP 107077(T) =DSM 14347(T) =CECT 7414(T)) and we provide an emended description of the genus Enterovibrio.

Phylogeny and molecular identification of vibrios on the basis of multilocus sequence analysis.

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.

Photobacterium rosenbergii sp. nov. and Enterovibrio coralii sp. nov., vibrios associated with coral bleaching.

Six new Vibrio-like isolates originating from different species of bleached and healthy corals around Magnetic Island (Australia) were investigated using a polyphasic approach. Phylogenetic analyses based on 16S rRNA, recA and rpoA gene sequences split the isolates in two new groups. Strains LMG 22223(T), LMG 22224, LMG 22225, LMG 22226 and LMG 22227 were phylogenetic neighbours of Photobacterium leiognathi LMG 4228(T) (95.6 % 16S rRNA gene sequence similarity), whereas strain LMG 22228(T) was related to Enterovibrio norvegicus LMG 19839(T) (95.5 % 16S rRNA gene sequence similarity). The two new groups can be distinguished from closely related species on the basis of several phenotypic features, including fermentation of d-mannitol, melibiose and sucrose, and utilization of different compounds as carbon sources, arginine dihydrolase activity, nitrate reduction, resistance to the vibriostatic agent O/129 and the presence of fatty acids 15 : 0 iso and 17 : 0 iso. The names Photobacterium rosenbergii sp. nov. (type strain LMG 22223(T)=CBMAI 622(T)=CC1(T)) and Enterovibrio coralii sp. nov. (type strain LMG 22228(T)=CBMAI 623(T)=CC17(T)) are proposed to accommodate these new isolates. The G+C contents of the DNA of the two type strains are respectively 47.6 and 48.2 mol%.

Use of recA as an alternative phylogenetic marker in the family Vibrionaceae.

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 %.

Genetic organization of the Vibrio harveyi DnaA gene region and analysis of the function of the V. harveyi DnaA protein in Escherichia coli.

The Vibrionaceae family is distantly related to Enterobacteriaceae within the group of bacteria possessing the Dam methylase system. We have cloned, sequenced, and analyzed the dnaA gene region of Vibrio harveyi and found that although the organization of the V. harveyi dnaA region differs from that of Escherichia coli, the expression of both genes is autoregulated and ATP-DnaA binds cooperatively to ATP-DnaA boxes in the dnaA promoter region. The DnaA proteins of V. harveyi and E. coli are interchangeable and function nearly identically in controlling dnaA transcription and the initiation of chromosomal DNA replication despite the evolutionary distance between these bacteria.

Enterovibrio norvegicus gen. nov., sp. nov., isolated from the gut of turbot (Scophthalmus maximus) larvae: a new member of the family Vibrionaceae.

Twenty-two isolates originating from the gut of healthy cultured turbot larvae in Norway were investigated using a polyphasic approach. Amplified fragment length polymorphism fingerprinting analysis showed that the isolates have typical patterns and form two main groups. Phylogenetic analysis revealed that the isolates belong to the gamma-Proteobacteria, with Vibrio hollisae as their closest neighbour. DNA-DNA hybridization, chemotaxonomic and phenotypic analyses further proved that these isolates represent a tight novel taxon that differs from currently described species in the family Vibrionaceae. It is proposed that these novel isolates be accommodated in a new genus, Enterovibrio gen. nov., with Enterovibrio norvegicus sp. nov. as the type species. Isolates were motile by a polar flagellum, positive for oxidase, catalase, arginine dihydrolase and beta-galactosidase, but negative for the Voges-Proskauer reaction. They produced indole, did not reduce nitrate and were resistant to the vibriostatic agent O/129. The DNA G+C content of E. norvegicus was 47.1-47.9 mol%. The type strain is E. norvegicus LMG 19839(T) (= CAIM 430(T)).

Polyphasic approach to the characterisation of marine luminous bacteria.

Fifteen luminous bacterial strains were isolated from the Tyrrhenian Sea coastal waters off northeastern Sicily and characterised by a combination of phenotypic and molecular tests in order to identify them and to determine their intraspecific genetic variability. Five luminous type strains, Vibrio splendidus NCIMB 1, V. harveyi NCIMB 1280, V. fischeri NCIMB 1281, V. orientalis NCIMB 2195 and Photobacterium leiognathi NCIMB 2193, were used as reference. On the basis of their phenotypic characters, the isolates were assigned to the family Vibrionaceae and all were related to the V. harveyi reference strain. Amplified 16S ribosomal DNA restriction analysis (ARDRA) enabled the strains to be subdivided into three groups, two of which exhibited the same restriction pattern as the two reference strains, V. harveyi and V. splendidus. Comparison of the full 16S rDNA sequence and of a 100-bp highly variable 16S rDNA region (selected as a 'signature' sequence for the luminous bacteria) confirmed ARDRA data and suggested that the strains of the third group could be considered a subspecies of V. harveyi or a tyrrhenian biovar, different from the other reference strains whose 16S rDNAs have already been sequenced. Random amplified polymorphic DNA (RAPD) fingerprinting and analysis of plasmid content suggested a high degree of intraspecific genetic variability within the largest ARDRA group. Data obtained suggest that the ARDRA method and the sequencing of the 16S rDNA signature region could be a powerful tool for a rapid identification of marine luminous bacteria.

Characterization of hapR, a positive regulator of the Vibrio cholerae HA/protease gene hap, and its identification as a functional homologue of the Vibrio harveyi luxR gene.

The Vibrio cholerae HA/protease gene (hap) promoter is inactive in Escherichia coli. We cloned and sequenced the 0.7kb hap promoter fragment from strain 3083-2 and showed that hap is located immediately 3' of ompW, encoding a minor outer membrane protein. A clone from a genomic library of strain 3083-2 was isolated, which was required for activation of the hap promoter in E. coli. Expression from the hap promoter only occurred late in the growth phase. A single complete open reading frame (ORF) designated HapR was identified on a 1.7 kb DNA fragment that was required for activation. Allelic replacements showed that hapR was also essential for hap expression in V. cholerae. In El Tor, but not in classical biotypes of V. cholerae, hapR mutations also produced a rugose colonial phenotype. HapR was shown to encode a 203-amino-acid polypeptide with 71% identity to LuxR of V. harveyi, an essential positive regulator of the lux operon that has no previously identified homologues. The amino-terminal domain (residues 21-68) showed significant homology to the TetR family of helix-turn-helix DNA-binding domains and was 95% identical to the same domain of LuxR. HapR and LuxR activated both the hap and the lux promoters at near wild-type levels, despite only limited homology in the promoter sequences (46% identity with 12 gaps over 420bp). DNA sequences and ORFs 5' (but not 3') of the hapR and luxR loci were homologous, suggesting a common origin for these loci, and hapR-hybridizing sequences were found in other vibrios. We conclude that HapR is absolutely required for hap expression and that HapR and LuxR form a new family of transcriptional activator proteins.

Iron uptake in Plesiomonas shigelloides: cloning of the genes for the heme-iron uptake system.

The iron uptake systems of Plesiomonas shigelloides strains were determined. Siderophore production was not detected by chemical or biological assays, and the strains tested were unable to use enterobactin, aerobactin, or vibriobactin for growth in low-iron media. Both hemin and hemoglobin supported full growth of the bacteria in media lacking other iron sources, but neither transferrin nor lactoferrin served as a source of iron. Hemolysin was detected, and the production of hemolysin was iron repressible. DNA sequences encoding hemolysin production and DNA sequences encoding the ability to use heme or hemoglobin as a sole source of iron were cloned from P. shigelloides and expressed in Escherichia coli. The abilities to use heme and hemoglobin as iron sources were closely linked, and the cloned sequences encoded the ability to transport the porphyrin, as well as iron, into the cells.

Plasmid screening amongst Aeromonas species and Plesiomonas shigelloides isolated from subjects with diarrhoea in Lagos, Nigeria.

Fifty-three Aeromonas strains and 16 Plesiomonas shigelloides isolated from subjects with diarrhoea in Lagos were screened for the presence of plasmids. Nine (17%) of the Aeromonas strains and one (6.3%) of the P. shigelloides harboured one or more plasmids, ranging in size from 2.4 to 16.8 MDa. As has been documented in other enteropathogens, the possibilities are that these plasmids code for some factors to enhance the virulence of their hosts.