Pseudovibrio Denitrificans Research Articles

Initial analysis of profiles from Pseudovibrio denitrificans and Vibrio parahaemolyticus antagonism by LC-MS-based metabolomics

Research on bioprospecting bioactive secondary metabolites from marine bacteria has rapidly expanded in the past few decades. The complexity of the environment in which bacterial communities live involves complex interactions with other communities competing for resources for survival. Previously, the ability of Pseudovibrio denitrificans has been demonstrated to inhibit the growth and virulence of different patho-gens such as Vibrio parahaemolyticus. The antagonism of these bacteria has been studied, considering the latter's importance in producing farmed shrimp. The molecules produced by P. denitrificans may be fun-damental for its antibacterial effect. In the present work, we examine LC/MS profiles of the interaction under two different circumstances: direct and delayed antagonism. The results show marked differences in the chemical composition between both interactions and the tentative identification of antibacterial and antibiofilm compounds. The observed profile could be part of P. denitrificans chemical arsenal to halt V. parahaemolyticus growing. The presented data is relevant to designing future studies on bacterial antago-nism with relevant implications for the aquaculture industry. Keywords: vibriosis, probiotics, chemometrics, white shrimp.

Initial analysis of profiles from Pseudovibrio denitrificans and Vibrio parahaemolyticus antagonism by LC-MS-based metabolomics

Research on bioprospecting bioactive secondary metabolites from marine bacteria has rapidly expanded in the past few decades. The complexity of the environment in which bacterial communities live involves complex interactions with other communities competing for resources for survival. Previously, the ability of Pseudovibrio denitrificans has been demonstrated to inhibit the growth and virulence of different pathogens such as Vibrio parahaemolyticus. The antagonism of these bacteria has been studied, considering the latter's importance in producing farmed shrimp. The molecules produced by P. denitrificans may be fundamental for its antibacterial effect. In the present work, we examine LC/MS profiles of the interaction under two different circumstances: direct and delayed antagonism. The results show marked differences in the chemical composition between both interactions and the tentative identification of antibacterial and antibiofilm compounds. The observed profile could be part of P. denitrificans chemical arsenal to halt V. parahaemolyticus growing. The presented data is relevant to designing future studies on bacterial antagonism with relevant implications for the aquaculture industry. Keywords: vibriosis, probiotics, chemometrics, white shrimp.

An exploratory study of sponges, sponge-associated, and planktonic prokaryotic communities in an artificial coral reef environment

Abstract In the present study, we assessed the sponge fauna, sponge-associated, and planktonic prokaryotic communities residing in Burgers' Zoo Ocean aquarium, Arnhem, the Netherlands. The Ocean aquarium consisted of separate displays and life support systems, and included fish-only systems in addition to a large, 750,000 L tank containing a living, tropical coral reef ecosystem. Sponges were observed throughout the aquarium system and were identified as belonging to the genera Chalinula, Chondrilla, Chondrosia, Cinachyrella, Stylissa, Suberites and Tethya. There was a highly significant difference in composition between sponge-associated and planktonic prokaryotic communities. The tanks in which the sponges were sampled appeared to have a secondary structural effect on prokaryotic composition with sponges and water from the same tanks sharing several microorganisms. Both sponge-associated and planktonic prokaryotic communities housed prokaryotic taxa, which were highly similar to microorganisms previously recorded in sponges or coral reef environments, including taxa potentially involved in nitrification, denitrification, sulphur oxidation, and antibiotic biosynthesis. Several abundant microorganisms were only recorded in sponges and these may play a role in maintaining water quality in the aquarium system. Potential pathogens, e.g. related to Photobacterium damselae, and beneficial organisms, e.g. related to Pseudovibrio denitrificans, were also detected. The present study showed that Burgers' Zoo Ocean aquarium housed diverse free-living and host-associated prokaryotic communities. Future research should focus on identifying conditions and microbial communities conducive to a healthy aquarium environment.

Linking differences in microbial network structure with changes in coral larval settlement

Coral cover and recruitment have decreased on reefs worldwide due to climate change-related disturbances. Achieving reliable coral larval settlement under aquaculture conditions is critical for reef restoration programmes; however, this can be challenging due to the lack of reliable and universal larval settlement cues. To investigate the role of microorganisms in coral larval settlement, we undertook a settlement choice experiment with larvae of the coral Acropora tenuis and microbial biofilms grown for different periods on the reef and in aquaria. Biofilm community composition across conditioning types and time was profiled using 16S and 18S rRNA gene sequencing. Co-occurrence networks revealed that strong larval settlement correlated with diverse biofilm communities, with specific nodes in the network facilitating connections between modules comprised of low- vs high-settlement communities. Taxa associated with high-settlement communities were identified as Myxoccales sp., Granulosicoccus sp., Alcanivoraceae sp., unassigned JTB23 sp. (Gammaproteobacteria), and Pseudovibrio denitrificans. Meanwhile, taxa closely related to Reichenbachiella agariperforans, Pleurocapsa sp., Alcanivorax sp., Sneathiella limmimaris, as well as several diatom and brown algae were associated with low settlement. Our results characterise high-settlement biofilm communities and identify transitionary taxa that may develop settlement-inducing biofilms to improve coral larval settlement in aquaculture.

Natural Indoles From the BacteriumPseudovibrio denitrificansP81 Isolated From a Marine Sponge,AaptosSpecies

A new natural indole, vibrindole B (1), together with known analogs, vibrindole A (2), trisindoline (3), norharmane (4), and 3-(hydroxyacetyl)indole (5), produced by the bacterium Pseudovibrio denitrificans P81, were isolated from a sponge, Aaptos species. The structures of indoles 1 to 5 were established by spectroscopic methods. The proposed biosynthetic pathway of 1 to 5 is also discussed, starting from tryptophan. Moreover, indoles 1 to 3 were found to exhibit cytotoxicity toward T24 tumor cells with IC50values of 1.71 ± 0.11, 4.53 ± 0.14, and 2.26 ± 0.26 µM, respectively.

Arsenate reducing bacteria isolated from the marine sponge Theonella swinhoei: Bioremediation potential

Arsenic (As) contamination of freshwater resources constitutes a major environmental issue affecting over 200 million people worldwide. Although the use of microorganisms for the bioremediation of As has been well studied, only very few candidates have been identified to date. Here, we investigated bacteria associated with the Red Sea sponge Theonella swinhoei and their potential to reduce As in a low-salinity liquid medium. This Indo-Pacific common sponge has been shown to hyper-accumulate As, at an average concentration of 8600 mg/g-1 in an environment uncontaminated by arsenic or barium. Four isolated strains of bacteria exhibited arsenic reduction potential by transforming inorganic As in the form of arsenate (iAsV) to arsenite (iAsIII). Two of these isolates were identified as Alteromonas macleodii and Pseudovibrio ascidisceicola, and the other two isolates, both belonging to the same species, were identified as Pseudovibrio denitrificans. The four isolates were then cultured in a low-salinity iAsV-rich medium (5 mM) and As concentration was measured over time using a specifically designed high-performance liquid chromatograph coupled to a mass spectrometer (HPLC-MS). Out of the four isolates, A. macleodii and P. ascidisceicola grew successfully in a low-salinity liquid medium and reduced AsV to AsIII at an average rate of 0.094 and 0.083 mM/h, respectively, thereby demonstrating great potential for the bioremediation of As-contaminated groundwater.

Mono- and multispecies biofilms from a crustose coralline alga induce settlement in the scleractinian coral Leptastrea purpurea

Microorganisms have been reported to induce settlement in various marine invertebrate larvae but their specificity of inductive capacities for the settlement of coral larvae remains poorly understood. In this study, we isolated 56 microbial strains from the crustose coralline alga (CCA) Hydrolithon reinboldii using five different media either with or without additional antibiotics and/or spiked CCA extract. We tested the isolates for their potential to induce settlement behavior in larvae of the brooding scleractinian coral Leptastrea purpurea. From these 56 CCA-associated microbial strains, we identified six bacterial classes and 18 families. The culturable bacterial community associated with H. reinboldii was dominated by Gammaproteobacteria, Actinobacteria, and Alphaproteobacteria while the Illumina MiSeq analysis showed that the culture-independent bacterial community was dominated by Gammaproteobacteria, Alphaproteobacteria, and Flavobacteria. Furthermore, we found no correlation between inductive settlement capacities and phylogenetic relationships. Instead, settlement behavior of L. purpurea larvae was induced by specific isolated species. Strains #1792 (Pseudovibrio denitrificans), #1678 (Acinetobacter pittii), #1633 (Pseudoalteromonas phenolica), #1772 (Marine bacterium LMG1), #1721 (Microbulbifer variabilis), and #1783 (Pseudoalteromonas rubra) induced settlement behavior in coral larvae at mostly high and significant levels (≥ 40%) but the remaining isolates strongly varied in their activity. Multispecies biofilms consisting of four strains (#1792, #1678, #1633, and #1721) were observed to synergistically increase settlement behavior levels (> 90%); however, the addition of #1772 to the multispecies biofilms negatively affected coral larvae and resulted in a total loss of inducing activity. The findings provide new insights into the role of bacteria in the settlement process of scleractinian corals and may help to identify the true nature of bacteria-derived morphogenic cues.

Phylogenomic Analyses of Members of the Widespread Marine Heterotrophic Genus Pseudovibrio Suggest Distinct Evolutionary Trajectories and a Novel Genus, Polycladidibacter gen. nov.

Bacteria belonging to the Pseudovibrio genus are widespread, metabolically versatile, and able to thrive as both free-living and host-associated organisms. Although more than 50 genomes are available, a comprehensive comparative genomics study to resolve taxonomic inconsistencies is currently missing. We analyzed all available genomes and used 552 core genes to perform a robust phylogenomic reconstruction. This in-depth analysis revealed the divergence of two monophyletic basal lineages of strains isolated from polyclad flatworm hosts, namely, Pseudovibrio hongkongensis and Pseudovibrio stylochi These strains have reduced genomes and lack sulfur-related metabolisms and major biosynthetic gene clusters, and their environmental distribution appears to be tightly associated with invertebrate hosts. We showed experimentally that the divergent strains are unable to utilize various sulfur compounds that, in contrast, can be utilized by the type strain Pseudovibrio denitrificans Our analyses suggest that the lineage leading to these two strains has been subject to relaxed purifying selection resulting in great gene loss. Overall genome relatedness indices (OGRI) indicate substantial differences between the divergent strains and the rest of the genus. While 16S rRNA gene analyses do not support the establishment of a different genus for the divergent strains, their substantial genomic, phylogenomic, and physiological differences strongly suggest a divergent evolutionary trajectory and the need for their reclassification. Therefore, we propose the novel genus Polycladidibacter gen. nov.IMPORTANCE The genus Pseudovibrio is commonly associated with marine invertebrates, which are essential for ocean health and marine nutrient cycling. Traditionally, the phylogeny of the genus has been based on 16S rRNA gene analysis. The use of the 16S rRNA gene or any other single marker gene for robust phylogenetic placement has recently been questioned. We used a large set of marker genes from all available Pseudovibrio genomes for in-depth phylogenomic analyses. We identified divergent monophyletic basal lineages within the Pseudovibrio genus, including two strains isolated from polyclad flatworms. These strains showed reduced sulfur metabolism and biosynthesis capacities. The phylogenomic analyses revealed distinct evolutionary trajectories and ecological adaptations that differentiate the divergent strains from the other Pseudovibrio members and suggest that they fall into a novel genus. Our data show the importance of widening the use of phylogenomics for better understanding bacterial physiology, phylogeny, and evolution.

The marine symbiont Pseudovibrio denitrificans, is effective to control pathogenic Vibrio spp. in shrimp aquaculture

In the search for probiotic bacteria from marine environments to control pathogenic Vibrio of cultivable marine species, this study focused on members of the genus Pseudovibrio. Based on its symbiotic features and antibacterial capacity, the genus Pseudovibrio could be used as probiotic to control vibriosis in shrimp culture. Forty-three isolates of Pseudovibrio were isolated from sponges Aplysina gerardogreeni, and in vitro competitive exclusion assays were performed against shrimp pathogenic Vibrio spp. (Vibrio harveyi, Vibrio campbellii, Vibrio vulnificus and two strains of Vibrio parahaemolyticus, one positive to PirA/PirB toxins). The most bioactive isolates (named Ps11, Ps17 and Ps18) against the pathogenic Vibrio spp. were validated by in vivo trials in the laboratory and in shrimp ponds. A significant increase in the survival of larvae and juveniles challenged. Additionally, the trial performed in PL3, which was naturally infected with V. harveyi, strongly indicates the P. denitrificans capabilities to compete and displace pathogenic Vibrio spp. present in shrimp postlarvae cultures enhancing their survival. Two isolates of P. denitrificans, coded as Ps17 and Ps18, were chosen due to the excellent results obtained in vitro and in the challenge tests and were assessed in experimental culture ponds of Penaeus vannamei. Ps17 and Ps18 were applied in the food once/day, during a complete culture cycle. Survival and production performances were significantly improved in ponds treated with Ps17. These results indicate the advantage of using P. denitrificans as probiotic for shrimp production.

Genomic Attributes of Novel Symbiont Pseudovibrio brasiliensis sp. nov. Isolated From the Sponge Arenosclera brasiliensis

Sponge holobionts are defined as the host animals and their associated microbiomes. Both host and microbiome produce extracellular products that facilitate interaction within the holobiont. For example, microbes may provide nutrition for the animal host and protection against pathogens. The genomic study of bacterial cultures may shed light on the properties of novel symbiotic bacteria isolated from marine holobionts. In this study, we performed a genome-based analysis of Pseudovibrio brasiliensis Ab134T isolated from the sponge Arenosclera brasiliensis. This novel strain is phylogenetically related to Pseudovibrio denitrificans. In silico DNA-DNA hybridization and calculation of the average amino acid identity between the strain Ab134T and P. denitrificans JCM 12308T showed <70% similarity and <95% identity, respectively. This novel bacterial species possesses genomic features that hint at several possible roles in symbiosis (e.g., production of secondary metabolites, including bromotyrosine-derived alkaloids) in sponge and coral holobionts. We also detected gene clusters encoding type III, type IV, and type VI secretion systems and 26 toxin-like proteins, including probable paralogs. Our results demonstrate the genome versatility of P. brasiliensis Ab134T and the potential to attach to host cells, which may play a role in its symbiotic lifestyle.

Cytotoxic indole alkaloids from Pseudovibrio denitrificans BBCC725

In this study, we investigated a culture supernatant of a Pseudovibrio denitrificans strain isolated from sea water. Five indole alkaloids were isolated from this culture. These include four known compounds, arundine (1), vibrindole A (2), 1,1,1-tris(indol-3-yl)methane (3), 7,7-bis(indol-3-yl)-p-cresol (4) and the new tetra(indol-3-yl)ethanone (5). Compound 5 showed interesting cytotoxic effect against L929 cells (EC50=7µM) and A549 cells (EC50=8µM).

Cultures of the Marine Bacterium Pseudovibrio denitrificans Ab134 Produce Bromotyrosine-Derived Alkaloids Previously Only Isolated from Marine Sponges.

Herein we report the isolation and spectroscopic identification of fistularin-3 (1), 11-hydroxyaerothionin (2), and verongidoic acid (3), as well as the UPLC-HRMS detection of aerothionin (4), homopurpuroceratic acid B (5), purealidin L (6), and aplysinamisine II (7), from cultures of the marine bacterium Pseudovibrio denitrificans Ab134, isolated from tissues of the marine sponge Arenosclera brasiliensis. These results unambiguously demonstrate for the first time that bromotyrosine-derived alkaloids that were previously isolated only from Verongida sponges can be biosynthesized by a marine bacterium.

Pseudovibrio stylochi sp. nov., isolated from a marine flatworm.

A Gram-stain-negative, motile, rod-shaped bacterial strain, designated UST20140214-052T, was isolated from a marine flatworm (polyclad). The bacterium was found to be catalase-positive and weakly oxidase-positive, and motile by means of several peritrichous or subpolar flagella. Strain UST20140214-052T grew optimally at 28 °C, at pH 7.0 and in the presence of 3 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain UST20140214-052T belongs to the genus Pseudovibrio, with highest sequence similarity to Pseudovibrio hongkongensis UST20140214-015BT (98.8 %), followed by Pseudovibrio japonicus WSF2T (96.2 %), Pseudovibrio ascidiaceicola F423T (96.2 %), Pseudovibrio denitrificans DN34T (95.9 %), Pseudovibrio axinellae Ad2T (95.9 %). All the other species shared < 95.5 % sequence similarity. The DNA-DNA hybridization estimate value between strain UST20140214-052T and P. hongkongensis UST20140214-015BT was 24.7 ± 2.4 %. The major fatty acid was summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c, as defined by the MIDI system; 62.6 %). The DNA G+C content was 47.0 mol%. The combined genotypic and phenotypic data show that strain UST20140214-052T represents a novel species within the genus Pseudovibrio, for which the name Pseudovibrio stylochi sp. nov. is proposed, with the type strain UST20140214-052T ( = KCTC 42384T = MCCC 1K00452T).

Low-Toxicity Diindol-3-ylmethanes as Potent Antifouling Compounds.

In the present study, eight natural products that belonged to di(1H-indol-3-yl)methane (DIM) family were isolated from Pseudovibrio denitrificans UST4-50 and tested for their antifouling activity against larval settlement (including both attachment and metamorphosis) of the barnacle Balanus (=Amphibalanus) amphitrite and the bryozoan Bugula neritina. All diindol-3-ylmethanes (DIMs) showed moderate to strong inhibitory effects against larval settlement of B. amphitrite with EC50 values ranging from 18.57 to 1.86 μM and could be considered as low-toxicity antifouling compounds since their LC50/EC50 ratios were larger than 15. Furthermore, the DIM- and 4-(di(1H-indol-3-yl)methyl)phenol (DIM-Ph-4-OH)-treated larvae completed normal settlement when they were transferred to clean seawater after being exposed to those compounds for 24 h. DIM also showed comparable antifouling performance to the commercial antifouling biocide Sea-Nine 211(™) in the field test over a period of 5 months, which further confirmed that DIMs can be considered as promising candidates of environmentally friendly antifouling compounds.

Synergistic in vitro cytotoxicity of adociaquinone B and heptyl prodigiosin against MCF-7 breast cancer cell line

A major limitation of cancer chemotherapy is the inability to escalate doses of an anticancer drug due to intolerable cytotoxic side effects. This limitation can be overcome by using a combination of drugs administered at lower doses. In this study, we investigated the effects of combining two compounds isolated from marine organisms for in vitro cytotoxicity to the MCF-7 breast cancer cell line by the MTT assay. Adociaquinone B, from the marine sponge Xestospongia sp., and heptyl prodigiosin, from the marine bacterium Pseudovibrio denitrificans strain Z143-1, were combined in three molar proportions and the combinatorial effect was determined by isobologram analysis and the fractional inhibitory concentration (FIC) of each compound. To determine if these compounds individually caused apoptosis in MCF-7 cells, we performed brightfield microscopy to investigate cell morphology and Hoechst 33342 staining to check for chromatin condensation. Apoptosis is the preferred mode of cell death in cancer therapy because it causes minimal inflammation. Initial findings of this study suggest a synergistic cytotoxic effect of A combinations of these two compounds against MCF-7 breast cancer cells, and their ability to induce apoptosis individually.

Isolation, characterization and phylogeny of sponge-associated bacteria with antimicrobial activities from Brazil

Bacteria associated with marine sponges represent a rich source of bioactive metabolites. The aim of this study was to isolate and characterize bacteria with antimicrobial activities from Brazilian sponges. A total of 158 colony-forming units were isolated from nine sponge species. Among these, 12 isolates presented antimicrobial activities against pathogenic bacteria. Based on comparative sequence analysis of their 16S rRNA genes, the sponge-associated bacterial strains could be subdivided into three phylogenetically different clusters. Five strains were affiliated with Firmicutes (genera Bacillus and Virgibacillus), three with α-Proteobacteria ( Pseudovibrio sp.) and four with γ-Proteobacteria (genera Pseudomonas and Stenotrophomonas). The sponge-associated bacterial strains Pseudomonas fluorescens H40 and H41 and Pseudomonas aeruginosa H51 exhibited antimicrobial activity against both Gram-negative and Gram-positive bacteria, including strains such as vancomycin-resistant Enterococcus faecium and multiresistant Klebsiella pneumoniae. Bacillus pumilus Pc31 and Pc32, Pseudovibrio ascidiaceicola Pm31 and Ca31 and Pseudovibrio denitrificans Mm37 strains were more effective against Gram-positive bacteria. These findings suggest that the identified strains may contribute to the search for new sources of antimicrobial substances, an important strategy for developing alternative therapies to treat infections caused by multidrug-resistant bacteria.

Pseudovibrio denitrificansstrain Z143-1, a heptylprodigiosin-producing bacterium isolated from a Philippine tunicate

Microbial isolate Z143-1 found to be associated with an unidentified tunicate was characterized due to its significant antimicrobial activity. Z143-1 is similar to Pseudovibrio ascidiaceicola and Pseudovibrio denitrificans in morphological, physiological and biochemical characteristics, except for its ability to ferment glucose and produce a characteristic red pigment. Fatty acid methyl ester analysis revealed a predominance of the fatty acid 18:1 omega7c at 80.55%, at levels slightly lower than the Pseudovibrio denitrificans type strain DN34(T) (87.7%). The mol% G+C of Z143-1 is 54.02, relatively higher than the Pseudovibrio denitrificans type strain DN34(T) and Pseudovibrio ascidiaceicola with mol% G+C of 51.7 and 51.4, respectively. However, phylogenetic analysis of the 16S rRNA gene sequence of Z143-1 showed 100% similarity with the Pseudovibrio denitrificans type strain DN34(T). In this study, the bacterium Z143-1 is reported as a new strain of Pseudovibrio denitrificans. While there is no report of a secondary metabolite for Pseudovibrio denitrificans, Z143-1 produces the red pigment heptylprodigiosin, also known as 16-methyl-15-heptyl-prodiginine, which shows anti-Staphylococcus aureus activity.

Abundance and Bioactivity of Cultured Sponge-Associated Bacteria from the Mediterranean Sea

In this study, the search for new antibiotics was combined with quantitative ecological studies. The cultured fraction of the associated bacterial communities from ten different Mediterranean sponge species was investigated. To obtain quantitative and qualitative data of sponge-associated bacterial communities and to expand the cultured diversity, different media were used. The largest morphological diversity and highest yield of isolates was obtained by using oligotrophic media, which consisted of natural habitat seawater amended with (1% additional carbon sources. The dominant bacterial morphotypes were determined and bacterial isolates were tested for antimicrobial activity and identified using 16S rDNA sequencing. The sponge-associated most abundant morphotypes were all affiliated to the Alphaproteobacteria and showed antimicrobial activity against at least one of the tested strains. In contrast, the ambient seawater was dominated by Gammaproteobacteria. One single alphaproteobacterium, which was related to Pseudovibrio denitrificans, was shown to dominate the cultured community of at least six of the sponges. This designated MBIC3368-like alphaproteobacterium has been isolated from sponges before and seems to be restricted to associations with members of the phylum Porifera. It displays a weak and unstable antimicrobial activity, which gets easily lost during cultivation. However, this bioactive bacterium was present in the sponges by up to 10(6) cells per gram wet-weight sponge tissue and dominated the cultured fraction with up to 74%. The association of this alphaproteobacterium with sponges is probably evolutionary young and facultative and possibly involves biologically active secondary metabolites. Besides a demonstrated vertical transfer, additional horizontal transfer between the sponges is assumed. Members of the genus Bacillus displaying antimicrobial activity were found regularly, too. However, actinomycetes, which are known for their production of bioactive substances, were present in very low abundance.

Pseudovibrio denitrificans gen. nov., sp. nov., a marine, facultatively anaerobic, fermentative bacterium capable of denitrification

Two denitrifying strains of heterotrophic, facultatively anaerobic, marine bacteria, designated DN34(T) and DN33, were isolated from sea-water samples collected in Nanwan Bay, Kenting National Park, Taiwan. They were Gram-negative. Cells in late exponential to early stationary phase of growth were predominantly straight or curved rods, but Y- or V-shaped forms were also observed. They were motile by means of one to several lateral or subpolar flagella. Both strains required NaCl for growth and exhibited optimal growth at about 30 degrees C, pH 8 and 3 % NaCl. They were capable of anaerobic growth by carrying out denitrifying metabolism using nitrate, nitrite or nitrous oxide as terminal electron acceptors or, alternatively, by fermenting glucose, mannose, sucrose or trehalose as substrates. Anaerobic fermentative growth on glucose resulted in formation of various organic acids, including formate, lactate, acetate, pyruvate and fumarate. The major cellular fatty acids were 2-OH-14 : 0, 3-OH-14 : 0 and 16 : 0. DN34(T) and DN33 had DNA G+C contents of 51.7 and 51.6 mol%, respectively. Physiological characterization, together with phylogenetic analysis based on 16S rRNA gene sequence analysis, revealed that the two denitrifying strains could be accommodated in a novel genus, for which the name Pseudovibrio gen. nov. is proposed. Pseudovibrio denitrificans sp. nov. is the type species, with DN34(T) (=BCRC 17323(T)=JCM 12308(T)) as the type strain.