Biochemical and Molecular Characterization of Carotenogenic Flavobacterial Isolates from Marine Waters.

Carotenoids are known to possess immense nutraceutical properties and microorganisms are continuously being explored as natural source for production of carotenoids. In this study, pigmented bacteria belonging to Flavobacteriaceae family were isolated using kanamycin-containing marine agar and identified using the molecular techniques and their phenotypic characteristics were studied along with their potential to produce carotenoids. Analysis of random amplification of polymorphic DNA (RAPD) banding patterns and the fragment size of the bands indicated that the 10 isolates fall under two major groups. Based on 16S rRNA gene sequence analysis the isolates were identified as Vitellibacter sp. (3 isolates), Formosa sp. (2 isolates) and Arenibacter sp. (5 isolates). Phenotypically, the isolates showed slight variation from the reported species of these three genera of Flavobacteriaceae. Only the isolates belonging to Vitellibacter and Formosa produced flexirubin, a typical yellow orange pigment produced by most of the organisms of the family Flavobacteriaceae. Vitellibacter sp. and Formosa sp. were found to produce higher amount of carotenoids compared to Arenibacter sp. and zeaxanthin was found to be the major carotenoid produced by these two species. The study indicated that Vitellibacter sp. and Formosa sp. can be exploited for production of carotenoids, particularly zeaxanthin.

Algoriella xinjiangensis gen. nov., sp. nov., a new psychrotolerant bacterium of the family Flavobacteriaceae.

An aerobic, Gram-stain negative, non-spore-forming and psychrotolerant bacterium, designated strain XJ109(T), was isolated from a sewage water sample collected from Xinjiang Uigur Autonomous Region, China. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain XJ109(T) represents a novel member of the family Flavobacteriaceae. The strain showed 95.5 % similarity with the 16S rRNA gene sequence of Empedobacter brevis LMG 4011(T), 95.4% with Chishuiella changwenlii BY4(T), 95.3% with Empedobacter falsenii NF 993(T) and 92.3% with Weeksella virosa DSM 16922(T). Strain XJ109(T) showed the common phenotypic and chemotaxonomic characteristics of the family Flavobacteriaceae, containing menaquinone-6 (MK-6) as the predominant respiratory quinone and iso-C17:0 3OH and iso-C15:0 as the major fatty acids. The polar lipid profile consisted of phosphatidylethanolamine, one unidentified phospholipid and two unidentified lipids. The genomic DNA G+C content was 38.0 mol%. Strain XJ109(T) was positive for catalase and oxidase activities, and it was observed to grow at 4-30 °C (optimal 16-20 °C), pH 6.5-10.0 (optimal 7.0-7.5) and in media containing 0-2.0% (w/v) NaCl (optimal 0.5 %). On the basis of the polyphasic evidence presented, strain XJ109(T) is considered to represent a novel genus and species of the family Flavobacteriaceae, for which the name Algoriella xinjiangensis gen. nov., sp. nov. is proposed. The type strain is XJ109(T) (=CGMCC 1.10229(T)=JCM 16590(T)).

Mesonia sediminis sp. nov., isolated from a sea cucumber culture pond.

A yellow-pigmented, Gram-stain negative and facultatively anaerobic bacterium, designated MF326(T), was isolated from a sample of sediment collected from a sea cucumber culture pond in Rongcheng, China (122°14'34″E 36°54'36″N). Cells of strain MF326(T) were found to be catalase negative and oxidase positive. Optimal growth was found to occur at 30 °C and pH 7.0-7.5 in the presence of 2.0-3.0% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain MF326(T) is a member of the genus Mesonia and exhibits the high sequence similarity (94.3%) with the type strain of Mesonia ostreae, followed by Mesonia algae (93.9%). The dominant fatty acids of strain MF326(T) were identified as iso-C(15:0), an unidentified fatty acid with an equivalent chain-length of 13.565 and anteiso-C(15:0). The major polar lipids were found to be two unidentified lipids and phosphatidylethanolamine. The major respiratory quinone was found to be MK-6 and the genomic DNA G+C content was determined to be 40.7 mol%. On the basis of the phylogenetic analysis and differential phenotypic characteristics, it is concluded that strain MF326(T) (=KCTC 42255(T) =MCCC 1H00125(T)) should be assigned to the genus Mesonia as the type strain of a novel species, for which the name Mesonia sediminis sp. nov. is proposed.

Aureimonas jatrophae sp. nov. and Aureimonas phyllosphaerae sp. nov., leaf-associated bacteria isolated from Jatropha curcas L.

Four orange-pigmented isolates, L7-456, L7-484(T), L9-479 and L9-753(T), originating from surface-sterilized leaf tissues of Jatropha curcas L. cultivars were characterized using a polyphasic taxonomic approach. Phylogenetic analyses based on 16S rRNA gene sequences indicated that all four isolates belong to the genus Aureimonas. In these analyses, strain L7-484(T) appeared to be most closely related to Aureimonas ureilytica 5715S-12(T) (95.7 % sequence identity). The 16S rRNA gene sequences of strains L7-456, L9-479 and L9-753(T) were found to be identical and also shared the highest similarity with A. ureilytica 5715S-12(T) (97.5 %). Both L7-484(T) and L9-753(T) contained Q-10 and Q-9 as predominant ubiquinones and diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, phosphatidyldimethylethanolamine, sulfoquinovosyldiacylglycerol and an aminophospholipid as the major polar lipids. C18 : 1ω7c and C16 : 0 were the major fatty acids. Similar to other species in the genus Aureimonas, hydroxylated fatty acids (e.g. C18 : 1 2-OH) and cyclic fatty acids (C19 : 0 cyclo ω8c) were also present. The DNA G+C contents of L7-484(T) and L9-753(T) were 66.1 and 69.4 mol%, respectively. Strains L7-484(T) and L9-753(T) exhibited less than 40 % DNA-DNA hybridization both between themselves and to A. ureilytica KACC 11607(T). Our results support the proposal that strain L7-484(T) represents a novel species within the genus Aureimonas, for which the name Aureimonas jatrophae sp. nov. is proposed, and that strains L9-753(T), L7-456 ( = KACC 16229  = DSM 25023) and L9-479 ( = KACC 16228  = DSM 25024) represent a second novel species within the genus, for which the name Aureimonas phyllosphaerae sp. nov. is proposed. The type strains of Aureimonas jatrophae sp. nov. and Aureimonas phyllosphaerae sp. nov. are respectively L7-484(T) ( = KACC 16230(T)  = DSM 25025(T)) and L9-753(T) ( = KACC 16231(T)  = DSM 25026(T)).

Pontibacillus sp. ALD_SL1 and Psychroflexus sp. ALD_RP9, two novel moderately halophilic bacteria isolated from sediment and water from the Aldabra Atoll, Seychelles.

Pontibacillus sp. ALD_SL1 and Psychroflexus sp. ALD_RP9 are two novel bacterial isolates from mangrove sediment and a moderately hypersaline pool on the Aldabra Atoll, Seychelles. The isolates represent two novel species were characterised physiologically and genomically. Pontibacillus sp. ALD_SL1 is a facultatively anaerobic yellow, motile, rod-shaped Gram-positive, which grows optimally at a NaCl concentration of 11%, pH 7 and 28°C. It is the third facultatively anaerobic member of the genus Pontibacillus. The organism gains energy through the fermentation of pyruvate to acetate and ethanol under anaerobic conditions. The genome is the first among Pontibacillus that harbours a megaplasmid. Psychroflexus sp. ALD_RP9 is an aerobic heterotroph, which can generate energy by employing bacteriorhodopsins. It forms Gram-negative, orange, non-motile rods. The strain grows optimally at NaCl concentrations of 10%, pH 6.5-8 and 20°C. The Psychroflexus isolate tolerated pH conditions up to 10.5, which is the highest pH tolerance currently recorded for the genus. Psychroflexus sp. ALD_RP9 taxonomically belongs to the clade with the smallest genomes. Both isolates show extensive adaptations to their saline environments yet utilise different mechanisms to ensure survival.

Huge symbiotic organs in giant scale insects of the genus Drosicha (Coccoidea: Monophlebidae) harbor flavobacterial and enterobacterial endosymbionts.

Giant scale insects (Drosicha: Coccoldea: Monophlebidae) were investigated for their symbiotic organs and bacterial endosymbionts. Two types of bacterial 16S rRNA gene sequences, flavobacterial and enterobacterial, were consistently detected in D. corpulenta and D. pinicola. The former sequences formed a compact clade in the Bacteroidetes, allied to the symbionts of cushion and armored scales. The latter sequences formed a robust clade in the gamma-Proteobacteria, allied to enteric bacteria like Enterobacter aerogenes and Escherichia coli. Another type of 16S sequence derived from Wolbachia was also detected in D. pinicola. In-situ hybridization demonstrated that the flavobacterial and enterobacterial symbionts were localized in a pair of huge bacteriomes in the abdomen, the former in uninucleated peripheral bacteriocytes and the latter in syncytial central bacteriocytes. Electron microscopy confirmed the endocellular locations of the pleomorphic flavobacterial symbiont and the rod-shaped enterobacterial symbiont, and also revealed the location and fine structure of the Wolbachia symbiont in D. pinicola. Infection frequencies of the flavobacterial and enterobacterial symbionts were consistently 100% in populations of D. corpulenta and D. pinicola, while the Wolbachia symbiont exhibited 0% and 100% infection frequencies in D. corpulente and D. pinicola, respectively. Neither the flavobacterial symbiont nor the enterobacterial symbiont exhibited AT-biased nucleotide composition or accelerated molecular evolution. The huge bacteriomes of Drosicha giant scales would provide a useful system for investigating biochemical, physiological, and genomic aspects of the host-symbiont and symbiont-symbiont interactions.

Antibody-mediated bacterial killing of Ichthyobacterium seriolicida in Japanese amberjack.

Ichthyobacterium seriolicida is the causative agent of bacterial hemolytic jaundice (BHJ) in Japanese amberjack, Seriola quinqueradiata. Fish recovering from BHJ acquire protective immunity against reinfection. In this study, fish were passively immunized to determine whether serum antibody is involved in protection against BHJ. The susceptibility of I. seriolicida to the bactericidal activity of Japanese amberjack serum was also investigated. In passive immunization tests, significantly lower mortality was noted in fish that received convalescent serum. Bacteria were killed when exposed to convalescent serum but not serum from naïve fish. Electron microscopic analyses showed that I. seriolicida cells were morphologically altered by reaction with convalescent serum. Naïve fish serum became bactericidal upon addition of purified IgM from convalescent serum. Involvement of the classical complement pathway in the bactericidal mechanism was confirmed because bactericidal activity was lost upon heating convalescent serum or chelation treatment using EDTA. Convalescent fish serum thus protects against reinfection by I. seriolicida via humoral immunity mediated by activation of the classical complement pathway.

Genome Information of Maribacter dokdonensis DSW-8 and Comparative Analysis with Other Maribacter Genomes.

Maribacter dokdonensis DSW-8 was isolated from the seawater off Dokdo in Korea. To investigate the genomic features of this marine bacterium, we sequenced its genome and analyzed the genomic features. After de novo assembly and gene prediction, 16 contigs totaling 4,434,543 bp (35.95% G+C content) in size were generated and 3,835 protein-coding sequences, 36 transfer RNAs, and 6 ribosomal RNAs were detected. In the genome of DSW-8, genes encoding the proteins associated with gliding motility, molybdenum cofactor biosynthesis, and utilization of several kinds of carbohydrates were identified. To analyze the genomic relationships among Maribacter species, we compared publically available Maribacter genomes, including that of M. dokdonensis DSW-8. A phylogenomic tree based on 1,772 genes conserved among the eight Maribacter strains showed that Maribacter speices isolated from seawater are distinguishable from species originating from algal blooms. Comparison of the gene contents using COG and subsystem databases demonstrated that the relative abundance of genes involved in carbohydrate metabolism are higher in seawater-originating strains than those of algal blooms. These results indicate that the genomic information of Maribacter species reflects the characteristics of their habitats and provides useful information for carbon utilization of marine flavobacteria.

A unique self-organization of bacterial sub-communities creates iridescence in Cellulophaga lytica colony biofilms.

Iridescent color appearances are widespread in nature. They arise from the interaction of light with micron- and submicron-sized physical structures spatially arranged with periodic geometry and are usually associated with bright angle-dependent hues. Iridescence has been reported for many animals and marine organisms. However, iridescence has not been well studied in bacteria. Recently, we reported a brilliant "pointillistic" iridescence in colony biofilms of marine Flavobacteria that exhibit gliding motility. The mechanism of their iridescence is unknown. Here, using a multi-disciplinary approach, we show that the cause of iridescence is a unique periodicity of the cell population in the colony biofilm. Cells are arranged together to form hexagonal photonic crystals. Our model highlights a novel pattern of self-organization in a bacterial biofilm. "Pointillistic" bacterial iridescence can be considered a new light-dependent phenomenon for the field of microbiology.

Antimicrobial peptides of an anti-lipopolysaccharide factor, epinecidin-1, and hepcidin reduce the lethality of Riemerella anatipestifer sepsis in ducks.

Antimicrobial peptides (AMPs) are effective against a wide range of microbes, but still no research results have reported their use in duck disease therapy. Riemerella anatipestifer (RA) is a Gram-negative bacterium which infects ducks and causes very significant economic losses. The minimum inhibitory concentrations (MICs) of epinecidin-1 for the tested RA strains ranged 6.25-50microg/ml, those of the SALF55-76 cyclic peptide ranged 12.5-25microg/ml, those of the SALF55-76 linear peptide ranged 6.25-25microg/ml, those of hepcidin TH1-5 ranged 25-400microg/ml, and those of hepcidin TH2-3 ranged 100-400microg/ml. The antimicrobial activities of these peptides were confirmed by transmission electron microscopy which showed that RA disruption of the outer membrane brought about cell death. In addition, pretreatment, co-treatment, and post-treatment with peptides were all effective in promoting a significant decrease in duck mortality and decreasing the number of infectious bacteria. A quantitative RT-PCR was performed to survey levels of gene expressions of Mn superoxide dismutase in the brain, lipoprotein lipase in the liver, and H5 histone in the spleen induced in response to bacterial infection and an injection of the AMPs in experiments with the duck, Cairina moschata. Our results indicated that the rescue of ducks by the peptides and the behavior of the peptides, which was like an enhancer in immunology, may involve regulation of the expressions of these genes. Collectively, these peptides reduced the mortality in ducks during bacterial challenge, suggesting that AMPs have the potential to serve as therapeutic drugs for use against bacterial infectious diseases in ducks.

Characterization of biofilm formation by Riemerella anatipestifer.

Riemerella anatipestifer (RA) causes epizootics of infectious disease in poultry and results in serious economic losses, especially for the duck industry. The present study focuses on understanding the biofilm-producing ability of RA strains in attempt to explain the intriguing persistence of RA post-infection on duck farms. Four RA serotype reference strains and 39 field RA isolates were measured for the biofilm formation by crystal violet staining. Eighteen out of the 43 RA strains produced biofilms. Furthermore, RA isolate CH3 was treated with carbohydrates (sucrose; glucose), disodium EDTA (EDTA), antibiotics (ampicillin; chloramphenicol) or detergent (Triton X-100) to determine the effect of the treatments on biofilm formation. Biofilm formation by RA isolate CH3 was independent of sucrose but significantly inhibited by 5% glucose and 0.1 mmol/L EDTA. Biofilmed CH3 culture (CH3 grown with a biofilm) was 5-31 times more resistant to the treatments of ampicillin, chloramphenicol or Triton X-100 than planktonic CH3 culture on the basis of minimal inhibitory concentration and minimal bactericidal concentration. The development and architecture of the biofilm formed by CH3 were also assessed using confocal laser scanning microscopy, scanning electron microscopy and fluorescence microscopy. In addition, animal experiment was performed to determine the median lethal doses (LD(50)) of three RA isolates with different biofilm formation abilities. Despite the result that virulence is strain-dependent as a result of various factors other than biofilm-producing ability, the fact that biofilmed isolate is more resistant to antibiotic and detergent treatments than planktonic isolate suggest that biofilm formation by RA may contribute to the persistent infections on duck farms.

Lutibacter litoralis gen. nov., sp. nov., a marine bacterium of the family Flavobacteriaceae isolated from tidal flat sediment.

A rod-shaped marine bacterium, designated strain CL-TF09T, isolated from a tidal flat in Ganghwa, Korea, was characterized based on its physiological and biochemical features, fatty acid profile and phylogenetic position. 16S rRNA gene sequence analysis revealed a clear affiliation with the family Flavobacteriaceae. Strain CL-TF09T showed the closest phylogenetic relationship with the genera Tenacibaculum and Polaribacter; sequence similarities between CL-TF09T and the type strains of Tenacibaculum and Polaribacter species ranged from 90.7 to 91.8 %. Cells of strain CL-TF09T were non-motile and grew on solid media as yellow colonies. The strain grew in the presence of 1-5 % sea salts, within a temperature range of 5-30 degrees C and at pH 7-8. The strain had iso-C(15 : 0) 3-OH (17.4 %), iso-C(15 : 0) (16.7 %), anteiso-C(15 : 0) (15.1 %) and iso-C(16 : 0) 3-OH (13.4 %) as predominant fatty acids. The DNA G+C content was 33.9 mol%. Based on the physiological, fatty acid composition and phylogenetic data presented, strain CL-TF09T is considered to represent a novel genus and species of the family Flavobacteriaceae, for which the name Lutibacter litoralis gen. nov., sp. nov. is proposed. The type strain is CL-TF09T (=KCCM 42118T = JCM 13034T).

Leeuwenhoekiella blandensis sp. nov., a genome-sequenced marine member of the family Flavobacteriaceae.

Bacteria in the family Flavobacteriaceae are increasingly recognized to play important roles in the degradation of organic matter during and following algal blooms. A novel heterotrophic, rod-shaped, aerobic, yellow-pigmented and gliding bacterium was isolated from a seawater sample collected in the Bay of Blanes in the north-western Mediterranean Sea. Analysis of its 16S rRNA gene sequence, retrieved from the whole-genome sequence, showed that the bacterium was closely related to members of the genus Leeuwenhoekiella within the family Flavobacteriaceae, phylum Bacteroidetes. Phenotypic, genotypic, chemotaxonomic and phylogenetic analyses supported the creation of a novel species to accommodate this bacterium, for which the name Leeuwenhoekiella blandensis sp. nov. is proposed. The type strain is MED 217(T) (=CECT 7118(T)=CCUG 51940(T)).