Kaihao Tang

MomL, a novel marine-derived N-acyl homoserine lactonase from Muricauda olearia

ABSTRACT Gram-negative bacteria use N-acyl homoserine lactones (AHLs) as quorum sensing (QS) signaling molecules for interspecies communication, and AHL-dependent QS is related with virulence factor production in many bacterial pathogens. Quorum quenching, the enzymatic degradation of the signaling molecule, would attenuate virulence rather than kill the pathogens, and thereby reduce the potential for evolution of drug resistance. In a previous study, we showed that Muricauda olearia Th120, belonging to the class Flavobacteriia, has strong AHL degradative activity. In this study, an AHL lactonase (designated MomL), which could degrade both short- and long-chain AHLs with or without a substitution of oxo-group at the C-3 position, was identified from Th120. Liquid chromatography-mass spectrometry analysis demonstrated that MomL functions as an AHL lactonase catalyzing AHL degradation through lactone hydrolysis. MomL is an AHL lactonase belonging to the metallo-β-lactamase superfamily that harbors an N-terminal signal peptide. The overall catalytic efficiency of MomL for C6-HSL is ∼2.9 × 105 s−1 M−1. Metal analysis and site-directed mutagenesis showed that, compared to AiiA, MomL has a different metal-binding capability and requires the histidine and aspartic acid residues for activity, while it shares the “HXHXDH” motif with other AHL lactonases belonging to the metallo-β-lactamase superfamily. This suggests that MomL is a representative of a novel type of secretory AHL lactonase. Furthermore, MomL significantly attenuated the virulence of Pseudomonas aeruginosa in a Caenorhabditis elegans infection model, which suggests that MomL has the potential to be used as a therapeutic agent.

Evaluation of a new high-throughput method for identifying quorum quenching bacteria

Quorum sensing (QS) is a population-dependent mechanism for bacteria to synchronize social behaviors such as secretion of virulence factors. The enzymatic interruption of QS, termed quorum quenching (QQ), has been suggested as a promising alternative anti-virulence approach. In order to efficiently identify QQ bacteria, we developed a simple, sensitive and high-throughput method based on the biosensor Agrobacterium tumefaciens A136. This method effectively eliminates false positives caused by inhibition of growth of biosensor A136 and alkaline hydrolysis of N-acylhomoserine lactones (AHLs), through normalization of β-galactosidase activities and addition of PIPES buffer, respectively. Our novel approach was successfully applied in identifying QQ bacteria among 366 strains and 25 QQ strains belonging to 14 species were obtained. Further experiments revealed that the QQ strains differed widely in terms of the type of QQ enzyme, substrate specificity and heat resistance. The QQ bacteria identified could possibly be used to control disease in aquaculture.

Quorum sensing in marine snow and its possible influence on production of extracellular hydrolytic enzymes in marine snow bacterium Pantoea ananatis B9

Marine snow is a continuous shower of organic and inorganic detritus, and plays a crucial role in transporting materials from the sea surface to the deep ocean. The aims of the current study were to identify N-acyl homoserine lactone (AHL)-based quorum sensing (QS) signaling molecules directly from marine snow particles and to investigate the possible regulatory link between QS signals and extracellular hydrolytic enzymes produced by marine snow bacteria. The marine snow samples were collected from the surface water of China marginal seas. Two AHLs, i.e. 3OC6-HSL and C8-HSL, were identified directly from marine snow particles, while six different AHL signals, i.e. C4-HSL, 3OC6-HSL, C6-HSL, C10-HSL, C12-HSL and C14-HSL were produced by Pantoea ananatis B9 inhabiting natural marine snow particles. Of the extracellular hydrolytic enzymes produced by P. ananatis B9, alkaline phosphatase activity was highly enhanced in growth medium supplemented with exogenous AHL (C10-HSL), while quorum quenching enzyme (AiiA) drastically reduced the enzyme activity. To our knowledge, this is the first report revealing six different AHL signals produced by P. ananatis B9 and AHL-based QS system enhanced the extracellular hydrolytic enzyme in P. ananatis B9. Furthermore, this study first time revealing 3OC6-HSL production by Paracoccus carotinifaciens affiliated with Alphaproteobacteria.

Description of Thalassotalea piscium gen. nov., sp. nov., isolated from flounder (Paralichthys olivaceus), reclassification of four species of the genus Thalassomonas as members of the genus Thalassotalea gen. nov. and emended description of the genus Thalassomonas.

A Gram-staining-negative, aerobic, rod-shaped bacterium, designated strain T202(T), was isolated from the gill of a cultured flounder (Paralichthys olivaceus). Based on 16S rRNA gene sequence similarity, strain T202(T) was a member of the family Colwelliaceae and shared 93.32-96.58 % similarity with type strains of all members of the most closely related genus Thalassomonas. Phylogenetically, the isolate shared a root with the type strains of four marine species, Thalassomonas agariperforans M-M1(T), Thalassomonas agarivorans TMA1(T), Thalassomonas loyana CBMAI 722(T) and Thalassomonas ganghwensis JC2041(T). Optimal growth occurred in the presence of 2-4 % (w/v) NaCl, at pH 7.0-8.0 and at 28 °C. Ubiquinone 8 (Q-8) was the predominant respiratory quinone. The major fatty acids were C16 : 0, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 1ω9c and C17 : 1ω8c. The major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content of strain T202(T) was 37 mol%. On the basis of polyphasic analysis, especially the phylogenetic relationships and the lower DNA G+C content, strain T202(T) is considered to represent a novel species in a new genus, for which the name Thalassotalea piscium gen. nov., sp. nov. is proposed. The type strain of Thalassotalea piscium is T202(T) ( = JCM 18590(T) = DSM 26287(T) = KCTC 32144(T)). Because Thalassomonas agariperforans M-M1(T), Thalassomonas agarivorans TMA1(T), Thalassomonas loyana CBMAI 722(T) and Thalassomonas ganghwensis JC2041(T) formed a phylogenetic group together with strain T202(T) that was clearly separated from other known strains of Thalassomonas, these four species are reclassified as members of the genus Thalassotalea as Thalassotalea agariperforans comb. nov. (type strain M-M1(T) = KCTC 23343(T) = CCUG 60020(T)), Thalassotalea agarivorans comb. nov. (type strain TMA1(T) = BCRC 17492(T) = JCM 13379(T) = DSM 19706(T)), Thalassotalea loyana comb. nov. (type strain CBMAI 722(T) = LMG 22536(T)) and Thalassotalea ganghwensis comb. nov. (type strain JC2041(T) = IMSNU 14005(T) = KCTC 12041(T) = DSM 15355(T)). The type species of the genus Thalassotalea is Thalassotalea ganghwensis gen. nov., comb. nov. An emended description of the genus Thalassomonas is also proposed.

Genome analysis of Pseudoalteromonas flavipulchra JG1 reveals various survival advantages in marine environment

BackgroundCompetition between bacteria for habitat and resources is very common in the natural environment and is considered to be a selective force for survival. Many strains of the genus Pseudoalteromonas were confirmed to produce bioactive compounds that provide those advantages over their competitors. In our previous study, P. flavipulchra JG1 was found to synthesize a Pseudoalteromonas flavipulchra antibacterial Protein (PfaP) with L-amino acid oxidase activity and five small chemical compounds, which were the main competitive agents of the strain. In addition, the genome of this bacterium has been previously sequenced as Whole Genome Shotgun project (PMID: 22740664). In this study, more extensive genomic analysis was performed to identify specific genes or gene clusters which related to its competitive feature, and further experiments were carried out to confirm the physiological roles of these genes when competing with other microorganisms in marine environment.ResultsThe antibacterial protein PfaP may also participate in the biosynthesis of 6-bromoindolyl-3-acetic acid, indicating a synergistic effect between the antibacterial macromolecule and small molecules. Chitinases and quorum quenching enzymes present in P. flavipulchra, which coincide with great chitinase and acyl homoserine lactones degrading activities of strain JG1, suggest other potential mechanisms contribute to antibacterial/antifungal activities. Moreover, movability and rapid response mechanisms to phosphorus starvation and other stresses, such as antibiotic, oxidative and heavy metal stress, enable JG1 to adapt to deleterious, fluctuating and oligotrophic marine environments.ConclusionsThe genome of P. flavipulchra JG1 exhibits significant genetic advantages against other microorganisms, encoding antimicrobial agents as well as abilities to adapt to various adverse environments. Genes involved in synthesis of various antimicrobial substances enriches the antagonistic mechanisms of P. flavipulchra JG1 and affords several admissible biocontrol procedures in aquaculture. Furthermore, JG1 also evolves a range of mechanisms adapting the adverse marine environment or multidrug rearing conditions. The analysis of the genome of P. flavipulchra JG1 provides a better understanding of its competitive properties and also an extensive application prospect.

Flaviramulus ichthyoenteri sp. nov., an N-acylhomoserine lactone-degrading bacterium isolated from the intestine of a flounder (Paralichthys olivaceus), and emended descriptions of the genus Flaviramulus and Flaviramulus basaltis.

A Gram-stain-negative, strictly aerobic, yellow-pigmented, rod-shaped and N-acylhomoserine lactone-degrading bacterium, designated strain Th78(T), was isolated from the intestine of a cultured flounder (Paralichthys olivaceus). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain Th78(T) belonged to the genus Flaviramulus (family Flavobacteriaceae) and showed the highest 16S rRNA gene sequence similarity to Flaviramulus basaltis H35(T) (96.70 %). Optimal growth occurred in the presence of 2-3 % (w/v) NaCl, at pH 7.0-8.0 and at 28 °C. The major fatty acids were iso-C15 : 0 3-OH, iso-C15 : 1 G and iso-C17 : 0 3-OH. The major polar lipids were phosphatidylethanolamine, one unidentified aminolipid and three unidentified polar lipids. Menaquinone 6 (MK-6) was the only respiratory quinone. The DNA G+C content of strain Th78(T) was 31.5 mol%. On the basis of polyphasic analysis, strain Th78(T) is considered to represent a novel species of the genus Flaviramulus, for which the name Flaviramulus ichthyoenteri sp. nov. is proposed. The type strain is Th78(T) ( = JCM 18634(T) = KCTC 32142(T) = DSM 26285(T)). Emended descriptions of the genus Flaviramulus and Flaviramulus basaltis are also proposed.

Genome sequence of Pseudoalteromonas flavipulchra JG1, a marine antagonistic bacterium with abundant antimicrobial metabolites.

The marine bacterium Pseudoalteromonas flavipulchra JG1 can synthesize various antibacterial metabolites, including protein and small molecules. The draft genome of JG1 is about 5.36 Mb and harbors approximate 4,913 genes, which will provide further insight into the synthesis of antimicrobial agents and antagonistic mechanisms of P. flavipulchra against pathogens.

Genome analysis of Flaviramulus ichthyoenteri Th78T in the family Flavobacteriaceae: insights into its quorum quenching property and potential roles in fish intestine

BackgroundIntestinal microbes play significant roles in fish and can be possibly used as probiotics in aquaculture. In our previous study, Flaviramulus ichthyoenteri Th78T, a novel species in the family Flavobacteriaceae, was isolated from fish intestine and showed strong quorum quenching (QQ) ability. To identify the QQ enzymes in Th78T and explore the potential roles of Th78T in fish intestine, we sequenced the genome of Th78T and performed extensive genomic analysis.ResultsAn N-acyl homoserine lactonase FiaL belonging to the metallo-β-lactamase superfamily was identified and the QQ activity of heterologously expressed FiaL was confirmed in vitro. FiaL has relatively little similarity to the known lactonases (25.2 ~ 27.9% identity in amino acid sequence). Various digestive enzymes including alginate lyases and lipases can be produced by Th78T, and enzymes essential for production of B vitamins such as biotin, riboflavin and folate are predicted. Genes encoding sialic acid lyases, sialidases, sulfatases and fucosidases, which contribute to utilization of mucus, are present in the genome. In addition, genes related to response to different stresses and gliding motility were also identified. Comparative genome analysis shows that Th78T has more specific genes involved in carbohydrate transport and metabolism compared to other two isolates in Flavobacteriaceae, both isolated from sediments.ConclusionsThe genome of Th78T exhibits evident advantages for this bacterium to survive in the fish intestine, including production of QQ enzyme, utilization of various nutrients available in the intestine as well as the ability to produce digestive enzymes and vitamins, which also provides an application prospect of Th78T to be used as a probiotic in aquaculture.

Flavirhabdus iliipiscaria gen. nov., sp. nov., isolated from intestine of flounder (Paralichthys olivaceus) and emended descriptions of the genera Flavivirga, Algibacter, Bizionia and Formosa.

A Gram-stain-negative, orange-coloured, rod-shaped bacterium, designated strain Th68(T), was isolated from the intestine of flounder (Paralichthys olivaceus). The isolate required sea salts for growth. Gliding motility was not observed. Flexirubin-type pigments were present. 16S rRNA gene sequence analysis indicated that strain Th68(T) represented a distinct phyletic line within the family Flavobacteriaceae with less than 96.1% similarity to members of the recognized genera of the family. The DNA G+C content was 33.0 mol%. The major fatty acids were iso-C(15 : 0), iso-C(15 : 1) G, iso-C(17 : 0) 3-OH and iso-C(15 : 0) 3-OH. The major polar lipids were phosphatidylethanolamine, two unidentified aminolipids and two unidentified polar lipids. Menaquinone 6 (MK-6) was the only respiratory quinone. On the basis of the phenotypic, chemotaxonomic and phylogenetic data, strain Th68(T) represents a novel species of a new genus in the family Flavobacteriaceae , for which the name Flavirhabdus iliipiscaria gen. nov., sp. nov. is proposed. The type strain of Flavirhabdus iliipiscaria is Th68(T) ( = JCM 18637(T) = KCTC 32141(T)).

Ichthyenterobacterium magnum gen. nov., sp. nov., a member of the family Flavobacteriaceae isolated from olive flounder (Paralichthys olivaceus).

A novel marine bacterium isolated from the intestine of cultured flounder (Paralichthys olivaceus) was studied by using a polyphasic taxonomic approach. The isolate was Gram-stain-negative, pleomorphic, aerobic, yellow and oxidase- and catalase-negative. Phylogenetic analysis of 16S rRNA gene sequences indicated that isolate Th6(T) formed a distinct branch within the family Flavobacteriaceae and showed 96.6% similarity to its closest relative, Bizionia hallyeonensis T-y7(T). The DNA G+C content was 29 mol%. The major respiratory quinone was MK-6. The predominant fatty acids were iso-C(15 : 1) G, iso-C(15 : 0), iso-C(15 : 0) 3-OH, iso-C(17 : 0) 3-OH and summed feature 3 (C(15 : 1)ω6c and/or C(16 : 1)ω7c). On the basis of the phenotypic, chemotaxonomic and phylogenetic characteristics, the novel bacterium has been assigned to a novel species of a new genus for which the name Ichthyenterobacterium magnum gen. nov., sp. nov. is proposed. The type strain is Th6(T) ( = JCM 18636(T) = KCTC 32140(T)).