Untitled

International Journal of Systematic and Evolutionary Microbiology (2009), 59, 2605–2609 Marinobacter szutsaonensis sp. nov., isolated froma solar saltern Chung-Yi Wang, Chang-Chai Ng, Wen-Sheng Tzeng and Yuan-Tay Shyu Department of Horticulture, National Taiwan University, 140, Keelung Road, Section 4, Taipei 106, A Gram-negative, aerobic, non-spore-forming, halophilic bacterial strain, NTU-104T, was isolatedfrom the Szutsao saltern in southern Taiwan, which was previously used as salt production field.
The novel isolate grew optimally at 35–40 6C, at pH 7.5–8.0 and in the presence of 5 % (w/v)NaCl. The major fatty acids were C16 : 0, C18 : 1v9c, C16 : 1v9c, C12 : 0 3-OH and C12 : 0. Thepredominant quinone was Q-9. Phosphatidylglycerol, diphosphatidylglycerol andphosphatidylethanolamine were the predominant polar lipids. The DNA G+C content was56.5 mol%. Phylogenetic analyses based on 16S rRNA gene sequences revealed the affiliation ofthe novel isolate to the genus Marinobacter. DNA–DNA hybridization results between strainNTU-104T and the type strains of the most closely related species, Marinobacter pelagius andMarinobacter koreensis, were 36.4 % and 33.2 %, respectively. On the basis of phenotypic,phylogenetic and genetic analyses, strain NTU-104T is considered to represent a novel species ofthe genus Marinobacter. The name Marinobacter szutsaonensis sp. nov. is proposed, with strainNTU-104T (5BCRC 17809T5CGMCC 1.7011T5JCM 15751T) as the type strain.
chemotaxonomic, genetic and phylogenetic characteriza- Gammaproteobacteria, was first proposed by Gauthier tion of a halophilic Marinobacter-like strain, NTU-104T, et al. (1992). The type species of the genus, Marinobacter hydrocarbonoclasticus, was isolated from sediments col- A single isolate of strain NTU-104T was obtained from soil lected in the Gulf of Fos (French Mediterranean coast) at sediment that was collected from Szutsao saltern, southern the mouth of a petroleum refinery outlet chronically Taiwan. The isolate was cultured by the dilution method polluted by hydrocarbons (Gauthier et al., 1992). Recently, (Wang et al., 2008). Briefly, soil sediment was dissolved in many further species, for example Marinobacter gudaonen- distilled water, diluted 1 : 1 with solutions of 5, 10, 15 and sis (Gu et al., 2007), Marinobacter salsuginis (Antunes et al., 20 % (w/v) NaCl and plated on basal medium agar plates.
2007), Marinobacter segnicrescens (Guo et al., 2007), The pH of the agar plate was adjusted to 7.0 by the Marinobacter salicampi (Yoon et al., 2007), Marinobacter addition of 1 M NaOH. The plates were then incubated at pelagius (Xu et al., 2008), Marinobacter guineae (Montes 37 uC for five days. Single colonies with various morphol- et al., 2008), Marinobacter psychrophilus (Zhang et al., ogies were selected for the growth condition tests. The salt 2008), Marinobacter mobilis and Marinobacter zhejiangensis requirements of these isolates were determined using basal (Huo et al., 2008) have been described and, at the time of medium [l21: 5 g yeast extract (Difco), 5 g Casamino acid writing, the genus includes 23 recognized species. Species (Difco), 5 g MgSO4 . 7H2O] with 0–30 % (w/v) NaCl of this genus are Gram-negative, aerobic, motile, rod- content. The optimal conditions for growth were deter- shaped bacteria. All previously described members of this mined on basal medium with various temperatures (10– genus have been found to contain C16 : 0v9c, C16 : 1v9c and 70 uC) and pH values (4–10). The growth rate was C18 : 1 as the predominant fatty acids and to have DNA monitored using a spectrophotometer at OD660. The G+C contents ranging from 53 to 59.6 mol% (Liebgott Gram-reaction was monitored by Gram staining using et al., 2006). In this study, a morphological, biochemical, Gram Stain kits (BD), according to the manufacturer’sinstructions. The ability of the novel strain to utilize Abbreviations: EPS, exopolysaccharides; PHA, poly-b-hydroxyalkanoate.
various carbohydrates and amino acids was tested by usingthe GN2 MicroPlate Identification Test Panel (Biolog). The The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene results were read with a MicroPlate reader, using MicroLog sequence of strain NTU-104T is EU164778.
3.59 software to perform automated reading and iden- A table detailing the fatty acid contents of strain NTU-104T and some tification. Tests for the hydrolysis of aesculin, gelatin, related species of the genus Marinobacter and figures showing the polarlipid analysis and additional phylogenetic trees are available with the starch, Tween 20 and Tween 80, for oxidase and catalase reactions and for H2S production were performed as described by Mata et al. (2002). Enzyme activity was formed fluorometrically in triplicate using the method of determined using the API ZYM system (bioMe´rieux).
Sensitivity to antimicrobial agents was determined in basal Strain NTU-104T was found to be a Gram-negative, motile medium that contained 50 mg l–1 of each antimicrobial rod that grew optimally in medium that contained agent for at least three days. The antimicrobial agents used approximately 5 % (w/v) salt. This bacterium exhibited were ampicillin, bacitracin, carbenicillin, cefotaxime, extreme halotolerance since it was able to grow in a medium chloramphenicol, erythromycin, kanamycin, nalidixic acid, containing 0–20 % (w/v) NaCl. M. hydrocarbonoclasticus is neomycin, nitrofurantoin, novobiocin, nystatin, penicillin, also an extremely halotolerant species, whereas M. pelagius is polymyxin B, rifampicin, streptomycin and tetracycline.
a moderately halophilic bacterium with optimal growth at Cell morphology was observed by scanning electron 5 % NaCl. Strain NTU-104T grew at 10–50 uC and at pH 6– microscopy. The sample was fixed and processed following 8.5; optimal growth occurred at 35–40 uC and at pH 7.5– the recommended procedure for preparing specimens 8.0. The new isolate was aerobic and catalase- and oxidase- (Anto´n et al., 2002). Gold was used to coat the samples positive. EPS and PHA were not produced. Detailed results to an approximate thickness of 5 nm. The samples were are given in the species description.
subsequently observed under a scanning electron micro-scope (Topcon Co.). Exopolysaccharides (EPS) were The major components of the fatty acids detected in strain observed by the method of Azeredo & Oliveira (1996).
NTU-104T were C16 : 0 (37.8 %), C18 : 1v9c (23.7 %), Poly-b-hydroxyalkanoate (PHA) detection was performedusing GC as described by Mas-Castella` & Guerrero (1995).
Table 1. Differential phenotypic characteristics of strain The G+C content of DNA was determined using the NTU-104T and other species of the genus Marinobacter method of Mesbah et al. (1989). The nucleotide mixtureswere separated by HPLC (JASCO) using a Phenomenex Species: 1, NTU-104T (data from the present study); 2, M. pelagius C18 column. The set conditions were a flow rate of 1.0 ml HS225T (data from the present study and Xu et al., 2008); 3, M.
min21 at a temperature of 37 uC, quantified by measure- koreensis DD-M3T (data from the present study and Kim et al., 2006); 4, M. gudaonensis SL014B61AT (data from the present study and Gu et al., 2007). All strains are positive in tests for motility, catalase- and (Sigma) was used as the calibration reference. The oxidase-activities and for the utilization of acetate and pyruvate. All composition of the fatty acid methyl esters (FAME) was strains exhibit cream coloured colonies. All strains are negative for the analysed using the standard procedure of the Microbial Identification software (MIDI). The extraction of the fattyacids was performed as described by Heyrman et al. (1999).
Grown cultures were transferred on to trypticase soy agar(TSA) plates, which contained 3 % (w/v) trypticase soy broth, 1.5 % (w/v) Bacto-Agar (Difco) and 7 % (w/v) NaCl for 24 h at 37 uC. Single colonies were removed using a platinum inoculating loop and transferred to 10 ml Teflon centrifuge tubes with Teflon screw caps (Nalge Nunc International). FAME profiles were obtained by GLC using a model 6890N GC (HP) as described by Descheemaeker & Swings (1995). The identity of the quinones was deter- mined by HPLC (Shin et al., 1996). Polar lipids were analysed by two-dimensional TLC, as described by The nucleic acids of the novel isolates were extracted using a FastDNA Spin kit (Bio 101), following the manufac- turer’s instructions. Two universal primers (9F and 1492R) were used to amplify the 16S rRNA gene (Stackebrandt & Liesack, 1993). The amplification was performed using a Amplicons were later sequenced (Mission Biotech) and aligned with representatives from the genus Marinobacter and related taxa using multiple sequence alignment software (CLUSTAL W 1.82; Thompson et al., 1994). A phylogenetic tree was constructed with the neighbour- joining, maximum-likelihood and maximum-parsimonyalgorithms with a bootstrap robustness of 1000 using *Data from: a, Xu et al. (2008); b, Kim et al. (2006); c, Gu et al.
PHYLIP package 3.6b. DNA–DNA hybridization was per- International Journal of Systematic and Evolutionary Microbiology 59 C16 : 1v9c (10.2 %), C12 : 0 3-OH (9.3 %) and C12 : 0 (7.8 %).
structed by means of the maximum-likelihood and The fatty acid profile was similar to those of other species maximum-parsimony algorithms. Although the tree topol- of the genus Marinobacter, in particular to that of M.
ogies were slightly different, the relationships between the pelagius (Gu et al., 2007; Guo et al., 2007; Xu et al., 2008) species of the genus Marinobacter were similar (see (see Supplementary Table S1, available in IJSEM Online).
Supplementary Figs S3 and S4 in IJSEM Online). Strain The major isoprenoid quinone of strain NTU-104T was Q9.
NTU-104T exhibited high 16S rRNA gene sequence Phosphatidylglycerol, diphosphatidylglycerol and phospha- similarity to M. pelagius JCM 14804T (98.7 %), M. koreensis tidylethanolamine were the major polar lipids (see DD-M3T (97.2 %) and M. gudaonensis SL014B61AT Supplementary Fig. S2 in IJSEM Online). The DNA (97.0 %). The DNA–DNA relatedness of strain NTU-104T G+C content of strain NTU-104T was 56.5 mol%, which to M. pelagius HS225T, M. koreensis DD-M3T and M.
was within the range found previously for recognized gudaonensis SL014B61AT was 36.4 % (SD53.8 %), 33.2 % species of the genus Marinobacter (Table 1).
(SD54.1 %) and 30.7 % (SD54.6 %), respectively. All ofthese values were clearly below the 70 % threshold that is The 16S rRNA gene sequence of strain NTU-104T as generally used to delineate species. Thus, the 16S rRNA determined in this study comprised 1435 nt. Comparative gene sequence data comparisons and DNA–DNA hybrid- 16S rRNA gene sequence analyses showed that strain NTU- ization results demonstrated that strain NTU-104T was 104T was most closely related to species of the genus most closely related to the genus Marinobacter but that the Marinobacter (Fig. 1). Phylogenetic analysis based on 16S novel strain was not a member of any recognized species of rRNA gene sequence comparisons showed that strain this genus. In conclusion, these data, when combined with NTU-104T formed a coherent cluster with M. pelagius with the differences revealed by biochemical, physiological and high bootstrap resampling value (98.7 % by the neighbour- phylogenetic analyses, are sufficient to suggest that strain joining method). Other phylogenetic trees were con- NTU-104T represents a novel species of the genus Fig. 1. Neighbour-joining tree showing theposition of strain NTU-104T with other speciesof the genus Marinobacter and related taxabased on 16S rRNA gene sequences. Thebootstrap values from 1000 resamplings areindicated at nodes. Bar, 0.01 substitutions persite.
Marinobacter, for which the name Marinobacter szutsao- Anto´n, J., Oren, A., Benlloch, S., Rodrı´guez-Valera, F., Amann, R. &Rossello´-Mora, R. (2002). Salinibacter ruber gen. nov., sp. nov., a Description of Marinobacter szutsaonensis novel, extremely halophilic member of the Bacteria from saltern crystallizer ponds. Int J Syst Evol Microbiol 52, 485–491.
Marinobacter szutsaonensis (szu.tsao.nen9sis. N.L. masc.
Antunes, A., Franc¸a, L., Rainey, F. A., Huber, R., Nobre, M. F., adj. szutsaonensis of szutsao, a former salt field located in Edwards, K. J. & da Costa, M. S. (2007). Marinobacter salsuginis sp.
nov., isolated from the brine–seawater interface of the Shaban Deep,Red Sea. Int J Syst Evol Microbiol 57, 1035–1040.
Azeredo, J. & Oliveira, R. (1996). A new method for precipitating 2.2 mm) and motile with a single flagellum. Colonies are bacterial exopolysaccharides. Biotechnol Tech 10, 341–344.
irregular to regular, flat and light translucent and milky Descheemaeker, P. & Swings, J. (1995). The application of fatty acid cream colour after 48 h at 37 uC. Growth occurs at NaCl methyl ester analysis (FAME) for the identification of heterotrophic concentrations of 0–20 % (w/v), with optimum growth at bacteria present in decaying Lede-stone of the St. Bavo Cathedral in 5 % NaCl. The optimum growth temperature is 35–40 uC; Ghent. Sci Total Environ 167, 241–247.
growth is observed at 10–50 uC. The pH range for growth is Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric 6–8.5, with an optimum at pH 7.5–8.0. Growth occurs deoxyribonucleic acid-deoxyribonucleic acid hybridization in micro- under aerobic conditions. Nitrate reduction is positive, dilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among production is negative. Aesculin, acetate, casein, bacterial strains. Int J Syst Bacteriol 39, 224–229.
pyruvate, urea and gelatin are hydrolysed, but starch, L-tyrosine, Tween 20, Tween 80, DNA and xanthine are not Gauthier, M. J., Lafay, B., Christen, R., Fernandez, L., Acquaviva, M.,Bonin, P. & Bertrand, J. C. (1992). Marinobacter hydrocarbonoclasticus hydrolysed. Oxidase- and catalase-positive. Acid is pro- gen. nov., sp. nov., a new, extremely halotolerant, hydrocarbon- duced from D-glucose, D-mannose, maltose, D-mannitol, degrading marine bacterium. Int J Syst Bacteriol 42, 568–576.
sucrose and D-galactose. Acid is not produced from Gu, J., Cai, H., Yu, S.-L., Qu, R., Yin, B., Guo, Y.-F., Zhao, J.-Y. & Wu, cellobiose, D-fructose, melibiose, D-ribose, L-arabinose, X.-L. (2007). Marinobacter gudaonensis sp. nov., isolated from an oil- melezitose, D-sorbitol, raffinose, trehalose or D-xylose.
polluted saline soil in a Chinese oilfield. Int J Syst Evol Microbiol 57, The following substrates are used as sole carbon sources: L-alanine, fumarate, D-glucose, malate, succinate, D-man- Guo, B., Gu, J., Ye, Y.-G., Tang, Y.-Q., Kida, K. & Wu, X.-L. (2007).
nose, maltose and sucrose. Melezitose, trehalose, D-salicin, Marinobacter segnicrescens sp. nov., a moderate halophile isolated D-sorbitol, D-xylose, benzoate, sorbose, lactose, D-fructose, from benthic sediment of the South China Sea. Int J Syst Evol L-tryptophan, formate, L-rhamnose, L-glutamate, glycerol, gluconate, glutamate, succinate and citrate are not used.
Heyrman, J., Mergaert, J., Denys, R. & Swings, J. (1999). The use of When assayed with the API ZYM system, alkaline fatty acid methyl ester analysis (FAME) for the identification of phosphatase, esterase (C4), leucine arylamidase, valine heterotrophic bacteria present on three mural paintings showingsevere damage by microorganisms. FEMS Microbiol Lett 181, 55–62.
arylamidase, trypsin, N-acetyl-b-glucosaminidase and a-mannosidase are present, but esterase (C4), esterase lipase Huo, Y. Y., Wang, C. S., Yang, J. Y., Wu, M. & Xu, X. W. (2008).
Marinobacter mobilis sp. nov. and Marinobacter zhejiangensis sp. nov., halophilic bacteria isolated from the East China Sea. Int J Syst Evol phosphohydrolase, b-galactosidase, a-glucosidase, cystine arylamidase and a-fucosidase are absent. The type strain is Kim, B.-Y., Weon, H.-Y., Yoo, S.-H., Kim, J.-S., Kwon, S.-W., susceptible to ampicillin, bacitracin, carbenicillin, cefotax- Stackebrandt, E. & Go, S.-J. (2006). Marinobacter koreensis sp.
ime, chloramphenicol, erythromycin, kanamycin, nalidixic nov., isolated from sea sand in Korea. Int J Syst Evol Microbiol 56, acid, nitrofurantoin, nystatin, penicillin, polymyxin B and tetracycline, but is resistant to neomycin, novobiocin, Komagata, K. & Suzuki, K. (1987). Lipid and cell-wall analysis in rifampicin and streptomycin. The predominant isoprenoid bacterial systematics. Methods Microbiol 19, 161–207.
quinone is Q-9. Phosphatidylglycerol, diphosphatidylgly- Liebgott, P.-P., Casalot, L., Paillard, S., Lorquin, J. & Labat, M. (2006).
cerol and phosphatidylethanolamine are the predominant Marinobacter vinifirmus sp. nov., a moderately halophilic bacterium polar lipids. The major fatty acids are C16 : 0, C18 : 1v9c, isolated from a wine-barrel-decalcification wastewater. Int J Syst Evol 16 : 1v9c, C12 : 0 3-OH and C12 : 0.
Mas-Castella`, J. & Guerrero, R. (1995). Poly-b-hydroxyalkanoate The type strain, NTU-104T (5BCRC 17809T5CGMCC determination in bacteria from aquatic samples. J Microbiol Methods 1.7011T5JCM 15751T), was isolated from Szutsao, a discarded salt field located in southern Taiwan. The DNA Mata, J. A., Martinez-Ca´novas, J., Quesada, E. & Be´jar, V. (2002). A G+C content of the type strain is 56.5 mol%.
detailed phenotypic characterisation of the type strains of Halomonasspecies. Syst Appl Microbiol 25, 360–375.
Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precisemeasurement of the G+C content of deoxyribonucleic acid by This work was supported by Grants from Council of Agriculture and high-performance liquid chromatography. Int J Syst Bacteriol 39, International Journal of Systematic and Evolutionary Microbiology 59 Montes, M. J., Bozal, N. & Mercade´, E. (2008). Marinobacter guineae Wang, C. Y., Chang, C. C., Ng, C. C., Chen, T. W. & Shyu, Y. T. (2008).
sp. nov., a novel moderately halophilic bacterium from an Antarctic Virgibacillus chiguensis sp. nov., a novel halophilic bacterium, isolated environment. Int J Syst Evol Microbiol 58, 1346–1349.
from Chigu, a previously commercial saltern located in southern Shin, Y. K., Lee, J.-S., Chun, C. O., Kim, H.-J. & Park, Y.-H. (1996).
Taiwan. Int J Syst Evol Microbiol 58, 341–345.
Isoprenoid quinone profiles of the Leclercia adecarboxylata KCTC Xu, X. W., Wu, Y. H., Wang, C. S., Yang, J. Y., Oren, A. & Wu, M. (2008).
1036T. J Microbiol Biotechnol 6, 68–69.
Marinobacter pelagius sp. nov., a moderately halophilic bacterium. Int Stackebrandt, E. & Liesack, W. (1993). Nucleic acids and classifica- J Syst Evol Microbiol 58, 637–640.
tion. In Handbook of New Bacterial Systematics, pp. 152–189. Edited Yoon, J.-H., Lee, M.-H., Kang, S.-H. & Oh, T.-K. (2007). Marinobacter by M. Goodfellow & A. G. O’Donnell. London: Academic Press.
salicampi sp. now. isolated from a marine solar saltern in Korea. Int J Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994).
Syst Evol Microbiol 57, 2102–2105.
improving the sensitivity of progressive multiple sequence alignment Zhang, D. C., Li, H. R., Xin, Y. H., Chi, Z. M., Zhou, P. J. & Yu, Y. (2008).
through sequence weighting, position-specific gap penalties and Marinobacter psychrophilus sp. nov., a psychrophilic bacterium weight matrix choice. Nucleic Acids Res 22, 4673–4680.
isolated from the Arctic. Int J Syst Evol Microbiol 58, 1463–1466.

Source: http://www.cbrc.kaust.edu.sa/mediawiki/images/9/95/Wang_2009_2605.pdf