PhD Thesis 2011 Chapter 3 Polyphasic taxonomy: Idiomarina and Pseudoidiomarina species Jay Siddharth Page 82 PhD Thesis 2011 Chapter 3 Polyphasic taxonomy: Idiomarina and Pseudoidiomarina species. Chapter synopsis. An attempt to cultivate the extremophiles from Lonar lake was made using standard microbiological approaches. This employed an array of media combinations for the initial screening process. This was followed by 16S rRNA based identification of the isolates. The isolates that had very low similarity was subjected to polyphasic taxonomic investigation. This resulted in the isolation of a new species whose proposed names are Idiomarina lonarii and Pseudoidiomarina lonarii. Jay Siddharth Page 83 PhD Thesis 2011 Introduction: Genus Idiomarina Two strains, KMM 227T and KMM 231T, were isolated from seawater sampled at a depths of 4000 and 5000 m, respectively, in the northwestern part of the Pacific Ocean. The new isolates were similar in their phenotypic properties to other marine aerobic proteobacteria, including Alteromonas, Pseudoalteromonas and Marinomonas, but they were characterized by several salient differences from known genera: morphological peculiarities, inability to use carbohydrates as the sole source of carbon and energy, and unusual cellular fatty acid composition. On the basis of phenotypic, chemotaxonomic and phylogenetic data, these marine deep-sea bacteria were classified as Idiomarina abyssalis and Idiomarina zobellii (177). Phylogeny Comparative 16S rDNA sequence analysis of Idiomarina strains KMM 227 and KMM 231 showed both strains to be similar (sequence similarity 96.9%). They formed a separate monophyletic branch within the γ-subclass of Proteobacteria, and are distantly related to Alteromonas, Colwellia and Pseudoalteromonas. Colwellia psychrerythraea was found to be the nearest relative to Idiomarina KMM 227T and KMM 231 (90.5 and 90.1% 16S rDNA sequence similarities, respectively; Taxonomy The genus Idiomarina was proposed to accommodate strictly aerobic, Gram-negative, psychrotolerant heterotrophic halophilic, marine deep sea microorganisms with a respiratory type of metabolism. The bacteria require NaCl for growth. The genus includes the type species Idiomarina abyssalis KMM 227T and Idiomarina zobellii KMM 231T (177). Idiomarina abyssalis forms an outer sheath-like structures covering one of the cell poles. It grows in 0.6– 15% NaCl, produces proteinase, and oxidizes cyclodextran, dextran, glycogen, methylpyruvate, monomethylsuccinate, acetate, glycyl-L-glutamic acid, L-proline, glycerol, and glucose-6- phosphate. The G+C content in the DNA is 50 mol%. Idiomarina zobellii is characterized by growth in 1–10% NaCl, ability to form long fimbria along with a polar flagellum, production of chitinase, and monomethylsuccinate, glycyl-L-glutamic acid, and L-ornithine utilization. It does Jay Siddharth Page 84 PhD Thesis 2011 not utilize cyclodextran, dextran, glycogen, L-proline, acetate, glycerol, and glucose-6-phosphate and produce proteinase. The G+C content in the DNA is 48 mol%. Comparative characterization of I. abyssalis and I. zobellii species are shown in table below. Habitat At present, the only known habitat of Idiomarina bacteria is deep-sea water (depths of 4000– 5000 m), in the Pacific Ocean. Genomic Relatedness The DNA-DNA reassociation levels between the strains were determined to be as follows: I. abyssalis KMM 227T and I. zobellii KMM 231T— 27%; I. abyssalis KMM 227T and other species: Alteromonas macleodii—7%; Vibrio cholerae— 7%; Aeromonas jandai—3%; Marinomonas communis—7%; Pseudoalteromonas haloplanktis— 6%; and Escherichia coli— 3%. Physiology The genus Idiomarina comprises Gram-negative, strictly aerobic, chemoorganotrophic, oxidase- positive, asporogenic, motile by one polar flagellum, rod-shaped, single, and sometimes paired Jay Siddharth Page 85 PhD Thesis 2011 cells of 0.7–0.9 μm in diameter. Bacteria may have fimbria and are able to form external sheath- like structures on the one pole of the cells. They do not accumulate poly-β-hydroxybutyrate as a reserve product and have no arginine dihydrolase. The bacteria do not need organic growth factors but require seawater or 0.6–15% NaCl for growth. They grow at 4–35°C, with an optimum at 20–25°C, but do not grow at 40°C. Growth occurs at pH 5.5–9.5, with the optimum pH being 7.5–8.5. They produce gelatinases, lipases, and DNAses. Idiomarina abyssalis produces proteinase. Strains do not hydrolyze starch and agar. The following substrates are utilized: D-arginine, L-tyrosine, L-alanine, alaninamide, L-alanine-glycine, α-ketobutyrate, and α-ketovalerate. Bacteria do not utilize D-arabinose, D-rhamnose, D-mannose, sucrose, maltose, lactose, melibiose, glycerol, mannitol, L-lysine, and L-phenylalanine. Bacteria are sensitive to erythromycin, and tolerant to kanamycin, ampicillin, benzylpenicillin, oleandomycin, lincomycin, tetracycline, oxacillin, vancomycin, and vibriostatic substance O/129. Iso-branched fatty acids with the odd number of carbon atoms predominate (up to 70%), and saturated and monounsaturated fatty acids are detected in cellular fatty acid composition of Idiomarina strains. Idiomarina strains are similar in their properties to members of Alteromonas, Pseudoalteromonas, Marinomonas, Halomonas and Oceanospirillum. Representatives of related Alteromonas and Pseudoalteromonas genera differ from Idiomarina strains by lower DNA G+C content values, ability to utilize a broad spectrum of carbon compounds, predominance of saturated and monounsaturated cellular fatty acids (177). Marinomonas species have specific physiological and biochemical properties and a bipolar type of flagellation (178). Halomonas species are characterized by tolerance to high concentration of sodium ions, a DNA G+C content of 57–68 mol%, peritrichous flagella and a specific fatty acid pattern (179). Distinctive features of Oceanospirillum are specific cell morphology and phenotypic properties (180). The main characteristics differentiating Idiomarina species from related genera are shown in table below Jay Siddharth Page 86 PhD Thesis 2011 Jay Siddharth Page 87 PhD Thesis 2011 Materials and methods: The materials and methods employed for the polyphasic taxonomy of the four isolates described in this thesis has been added as a section at the begening as a seperate chapter titled materials and methods. Results. (1) Isolation of bacteria from water: The isolate QA8 was obtained from the Lonar water filtrate on Zobell marine broth supplemented with 40% Lonar water. The isolate was subcultured to test for purity this was followed by morphology characterization. The isolate exhibited aerobic growth and did not require any supplements when grown on Zobell marine agar. (2) Morphology characterization The colonies of QA8 were creamish white and formed round opaque colonies on agar plates. Upon longer growth the colonies were seen to spread and grow dark (after more than 72hrs). Cell morphology Primary microscopic observation showed that the isolate was rod shaped. (3) Gram reaction Gram staining of the isolate showed that the isolate was gram negative. (4) Motility by microscopy. The isolate showed motility in the hanging drop preparation. The motility could be observed until the slide dried out. To show the images for documentation purposes the culture was grown on soft agar plate. (5) Motility assay on soft agar. Motility assay on soft agar revealed that the isolate was aerobic as most of the growth was near the surface of the agar in the tube. (6) Determination of NaCl optima The isolate was found to have a NaCl optima of 10% NaCl, it however showed growth only at high salt conditions. Jay Siddharth Page 88 PhD Thesis 2011 (7) Determination of pH optima The isolate exhibited a pH optima between 9 and 10, thus showing an alkaliphillic nature. (8) Determination of Temp optima. The isolate was able to survive at mesophillic temperatures with an optimum temperature range of 30-37 C (9) BIOLOG (Oxidation of carbon sources). The isolate showed metabolic versatility as it was able to degrade few of the test compounds during the BIOLOG assay. (10) Whole cell fatty acid profile. The fatty acid profile showed the most abundant lipid was 15:0 iso which accounted for about 32% of the total fatty acids this wa s followed by iso 17:1 w9c and 17:0 fatty acids that accounted for approximately 16% and 14 % of the the total fatty acids. Jay Siddharth Page 89 PhD Thesis 2011 Jay Siddharth Page 90 PhD Thesis 2011 (11) Mol % G+C determination of genomic DNA The isolate QA8 exhibited a 50.5 % of G+C, this values are close to other type strain isolates of the same genus. (12) PCR amplification of 16S rRNA gene and phylogenetic analysis. The amplification of the near complete gene of the isolate resulted in an approximately 1.5 kb fragment of DNA this was completely sequenced and showed less than 95% sequence homology to the type strains of the genus Idiomarina and Pseudoidiomarina. Jay Siddharth Page 91 PhD Thesis 2011 (13) DNA-DNA hybridization studies The DNA-DNA hybridization studies of strain strain QA8 (ID 06-304) against Idiomarina baltica DSM 15154T (ID 06-299) and against Pseudoidiomarina taiwanensis (ID 06-300)., yielded the following results. (V % DNA-DNA similarity (in 2 X SSC at 69 °C) QA8 (ID 06-304) alu es T 12.3 (4.3) Idiomarina baltica DSM 15154 (ID 06-299) in par Pseudoidiomarina taiwanensis (ID 06-300) 22.6 (24.7) ent heses are results of measurements in duplicate) Jay Siddharth Page 92 PhD Thesis 2011 Discussion: When the isolate QA8 was subjected to polyphasic taxonomic it showed affiliation to the genus Idiomarina and Pseudoidiomarina. Further investigation showed that is exhibited a marked differences in terms of Major fatty acids and carbon source metabolism. The mol % G+C does not match with either of the genus namely Idiomarina or Pseudoidiomarina. The 16S rRNA data in terms of % similarity of the 16S rRNA gene reveals that there is a high degree of difference with the known type strains. A whole genome DNA-DNA hybridization has revealed that the isolate has very less homology with the type strains that it showed maximum homology at the 16S rRNA level.