| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Departamento de Microbiología y Genética, Lab. 209, Edificio Departamental de Biología, Campus Miguel de Unamuno, Universidad de Salamanca, 37007 Salamanca, Spain
Correspondence
Raúl Rivas
raul{at}wwwedu-micro.usal.es
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
-galactosidase and hydrolysed gelatin, but did not produce arginine dihydrolase, indole or acetoin. Strain MACL01T used several carbohydrates and fermented glucose, L-arabinose and sucrose. The most abundant fatty acids were summed feature 3 (32.6 %; comprising C16 : 1
7c and/or C15 : 0 iso 2-OH), C16 : 0 (21.2 %) and C18 : 17c (19.9 %). The G+C content of the genomic DNA was 49.8 mol%. On the basis of genotypic, phenotypic, chemotaxonomic and phylogenetic results, strain MACL01T (=LMG 22194T=CECT 5860T) should be classified as the type strain of a novel species of the genus Photobacterium, for which the name Photobacterium halotolerans sp. nov. is proposed.
An electron micrograph, neighbour-joining trees based on partial recA and rpoA gene sequences and a table of the differential phenotypic characteristics of strain MACL01T are available as supplementary material in IJSEM Online.
Present address: Laboratorium voor Microbiologie, Vakgroep Biochemie, Fysiologie en Microbiologie, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium. 
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
). Strain MACL01T was isolated under aseptic conditions from water samples taken from Lake Martel at a depth of 10 cm. A 200 ml sample was filtered, under vacuum in sterile conditions, through a membrane filter (Millipore) with a pore diameter of 45 µm. The membrane was placed on a plate containing YED medium (yeast extract, 0.5 %; glucose, 0.7 %; agar, 2 %) supplemented with 1.5 % (w/v) NaCl and was incubated at 28 °C. Colony morphology was examined in cultures grown on TSA medium (BD Difco) supplemented with 1.5 % (w/v) NaCl. To test the ability to grow in TCBS, strain MACL01T was tested for growth on thiosulphate/citrate/bile salts/sucrose agar (TCBS; BD Difco) plates by incubating them 48 h at 28 °C.
Isolate MACL01T was observed under a phase-contrast microscope after 48 h growth in YED medium at 22 °C to check for cell shape and motility. The cells were also stained according to the classic Gram procedure described by Doetsch (1981). For electron microscopy, cells were grown on nutrient agar for 2 days at 22 °C. Cells were gently suspended in sterile water and then stained with 0.2 % uranyl acetate and examined at 80 kV with a Zeiss EM 209 transmission electron microscope (Peix et al., 2003).
Extraction and amplification of genomic DNA for 16S rRNA sequence analysis were carried out as described previously (Rivas et al., 2003) and the recA and rpoA genes were amplified and sequenced as described by Thompson et al. (2005a, b). The sequences of these genes were compared against the sequences available from GenBank using the BLASTN programme (Altschul et al., 1990) and were aligned using CLUSTAL X software (Thompson et al., 1997). Distances were calculated according to Kimura's method (Kimura, 1980). Phylogenetic trees were inferred using the neighbour-joining method (Saitou & Nei, 1987). Bootstrap analysis was based on 1000 resamplings. The MEGA2 package (Kumar et al., 2001) was used for all analyses.
DNA for the determination of G+C content was prepared according to Chun & Goodfellow (1995); the value was obtained by using the thermal denaturation method (Mandel & Marmur, 1968).
For fatty acids analyses, strain MACL01T was cultivated for 24 h at 28 °C in TSB (BD Difco) amended with 1.5 % agar. The cellular fatty acids of strain MACL01T were analysed as methyl esters by GLC analysis at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany) according to the instructions of the Microbial Identification System (MIDI). Physiological and biochemical tests were performed using various API strips (bioMérieux). The API 20NE strip (Seo et al., 2005) was inoculated according to the manufacturer's instructions, without salt addition, with a suspension of strain MACL01T in sterile water containing 1.5 % NaCl. For the inoculation of API ZYM strips (Thompson et al., 2005b), suspensions of strain MACL01T incubated for 48 h in TSA plus 1.5 % NaCl were obtained according to the manufacturer's instructions. Finally, for the inoculation of the API 50CH strip, a suspension in AUX medium plus 1.5 % NaCl was used. The API 20NE, API 20E and API 50CH strips were incubated for 48 and 72 h: the results were identical for both of these incubation times. The temperature range for growth was determined by incubating cultures in YED medium between 4 and 45 °C. The pH range was determined in YED medium, with a final pH between 4 and 10. Salt tolerance was studied in YED medium containing 0–12 % (w/v) NaCl. Luminescence was observed in the dark on MA (BD Difco), in agreement with Macián et al. (2001). Standard phenotypic tests were performed as described previously (Baumann & Schubert, 1984; Farmer & Hickman-Brenner, 1992; Thompson et al., 2002a, b, 2005b). Resistance to the vibriostatic agents O/129 (10 and 150 µg; Oxoid) and novobiocin (5 µg; Oxoid) was tested on MA medium.
Strain MACL01T comprised cells that were Gram-negative, rod-shaped (1.7x0.7 µm) and motile by means of two polar flagella (see Supplementary Fig. S1 available in IJSEM Online).
According to the 16S rRNA gene sequence analysis, the organism under study belongs to the genus Photobacterium and forms a branch within this genus together with Photobacterium rosenbergii and Photobacterium ganghwense (Fig. 1). Strain MACL01T shows 96.9 and 96.2 % 16S rRNA gene sequence similarity with P. rosenbergii LMG 22223T and P. ganghwense FR1311T, respectively. These values are close to the cut-off value of 97 % for bacterial species definition (Stackebrandt & Goebel, 1994) and, hence, even though they suggested that the novel isolate could belong to a novel species of the genus Photobacterium, we analysed other housekeeping genes useful in the differentiation of Vibrio and related species (Thompson et al., 2004, 2005a, b).
|
; Thompson et al., 2005b). In the bacterial genomes sequenced to date, the rpoA gene is a ubiquitous single-copy gene that seems to be resistant to lateral gene transfer (Lerat et al., 2003; Zeigler, 2003; Gevers et al., 2004). Accordingly, it has recently been proposed as an alternative marker for bacterial classification and as a chronometer in Vibrio-like species (Thompson et al., 2005a).
The recA gene has been also proposed as an alternative marker in the family Vibrionaceae, and its sequences seem to be more discriminatory than those of the 16S rRNA gene in the genus Vibrio (Thompson et al., 2004). A pairwise analysis of the recA sequence of strain MACL01T also revealed low levels of similarity between this strain and several species from the genera Vibrio and Photobacterium. For example, the analysis pointed to 83.7 % similarity with Vibrio proteolyticus LMG 3772T, 83.4 % similarity with Photobacterium leiognathii LMG 4228T and 82.4 % similarity with P. rosenbergii LMG 22223T. Nevertheless, neighbour-joining phylogenetic analysis of the recA gene from strain MACL01T showed that this strain clustered with several species of the genus Photobacterium (Supplementary Fig. S3). The results of the recA analysis confirm those obtained by other workers (Thompson et al., 2004, 2005a, b), and show that Photobacterium and Vibrio are two different phylogenetic groups, although Photobacterium species did not form a homogeneous group.
According to Thompson et al. (2005a), there is variation of about 0.4 and 5.5 % among strains from P. rosenbergii species in rpoA and recA sequences, respectively. The lower similarity values found between the sequences of these two genes of P. rosenbergii and MACL01T confirm that this strain does not belong to P. rosenbergii. Therefore, analysis of the 16S rRNA, rpoA and recA genes indicates that strain MACL01T belongs to a novel species of the genus Photobacterium.
The DNA G+C content of strain MACL01T is 49.8 mol%, which is slightly higher than those reported for the remaining species of the genus Photobacterium.
The results of the fatty acid methyl ester analysis are given in the species description below. The fatty acid composition of strain MACL01T displayed qualitative and quantitative differences with respect to the species to which it is most closely related phylogenetically, namely P. rosenbergii and P. ganghwense. The main differences between strain MACL01T and its closest relatives were as follows. Some fatty acids detected in P. rosenbergii were not detected in strain MACL01T, e.g. C15 : 0, C15 : 0 iso, C15 : 0 iso 3-OH and C17 : 19c iso. The fatty acid C16 : 0 was detected in larger amounts in strain MACL01T and P. ganghwense (21 %) than in P. rosenbergii (10–15 %). In contrast, the amount of fatty acids detected from summed feature 3 (comprising C16 : 17c and/or C15 : 0 iso 2-OH) was larger in P. rosenbergii (41–44 %) than in strain MACL01T (32.6 %) and P. ganghwense (27.8 %). The amount of fatty acid C18 : 17c (29.6 %) was larger in P. ganghwense than in P. rosenbergii (23 %) and strain MACL01T (20 %). Finally, some fatty acids that were detected in amounts greater than 1 % in strain MACL01T, such as C16 : 19c (2.5 %), C16 : 0 iso (1 %) and an unknown fatty acid at 12.484 (1.2 %), were not found for P. rosenbergii or P. ganghwense.
The phenotypic characteristics of strain MACL01T are given in the species description below. The strain differed from P. rosenbergii and P. ganghwense in several physiological and biochemical respects. Colonies of strain MACL01T on TCBS plates were blue, those of P. rosenbergii were yellow and those of P. ganghwense were green. In contrast with P. rosenbergii, strain MACL01T grew at 4 °C and was able to grow at 8 % NaCl. Strain MACL01T produced gelatinase and fermented L-arabinose whereas P. rosenbergii did not. Strain MACL01T did not produce arginine dihydrolase, esterase, esterase lipase, valine arylamidase or naphthol-AS-BI-phosphohydrolase, whereas these compounds were produced by P. rosenbergii. Strain MACL01T did not ferment melibiose, L-rhamnose or amygdalin and did not assimilate cellobiose, galactose, mannose, melibiose, raffinose or methyl 
-D-glucoside. These tests were positive for P. rosenbergii. Strain MACL01T differed from P. ganghwense in terms of growth at 4 °C. Indole and arginine dihydrolase were not produced by strain MACL01T but were assimilated or produced by P. ganghwense. Strain MACL01T produced -galactosidase and fermented sucrose and L-arabinose, unlike P. ganghwense. Several differential phenotypic characteristics of strain MACL01T and the remaining Photobacterium species are shown in Supplementary Table S1.
In conclusion, the overall results of the present study indicate that isolate MACL01T should be classified within a novel species, for which the name Photobacterium halotolerans sp. nov. is proposed.
Description of Photobacterium halotolerans sp. nov.
Photobacterium halotolerans (ha.lo.to'le.rans. Gr. n. hals salt; L. part. adj. tolerans tolerating; N.L. part. adj. halotolerans referring to the ability to tolerate high salt concentrations).
Cells are rod-shaped, Gram-negative and motile by means of polar flagella. Colonies on TSA supplemented with 1.5 % (w/v) NaCl are circular, smooth, white to cream in colour, opaque and usually 2–4 mm in diameter within 2 days at 28 °C. The isolate grows on TCBS medium, producing blue colonies. Oxidase- and catalase-positive. The DNA G+C content of the type strain is 49.8 mol%. Aerobic or facultatively anaerobic, chemo-organotrophic, mesophilic and halotolerant. Able to ferment carbohydrates. Negative Voges–Proskauer reaction and negative for indole production and H2S production. Does not produce arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, caseinase, urease, esterase, esterase lipase, valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, phosphohydrolase or glucuronidase. Gelatin is hydrolysed and the hydrolysis of aesculin is weak. The strain produces amylases, -galactosidase, acid and alkaline phosphatase, leucine arylamidase and -glucosaminidase. Positive for reduction of nitrate to nitrite; tryptophan deaminase production is weak. Negative for luminescence on marine agar. Grows in the presence of NaCl concentrations up to 8 % (w/v) and optimally in the presence of 1.5 % (w/v) NaCl at 28 °C, although salt is not essential for growth. Growth occurs at 4–37 °C (optimal growth occurs at 28 °C), at pH 5–8.5 (optimal growth occurs at pH 7). Shows sensitivity to the vibriostatic agent O/129 and is novobiocin-positive. The most abundant fatty acids are summed feature 3 (32.6 %; comprising C16 : 17c and/or C15 : 0 iso 2-OH), C16 : 0 (21.2 %), C18 : 17c (19.9 %), C12 : 0 3-OH (6.6 %) and C12 : 0 (5.9 %). The following fatty acids were detected in small amounts: summed feature 2 (3.0 %; comprising C14 : 0 3-OH, C16 : 1 iso I, an unidentified fatty acid with an equivalent chain length of 10.928 and/or C12 : 0 ALDE), C16 : 19c (2.5 %), an unidentified fatty acid with an equivalent chain length of 12.484 (1 %), C16 : 0 iso (1 %) and C14 : 0 (1 %). Ferments glucose, mannitol, sucrose and L-arabinose. Uses N-acetylglucosamine, citrate, adipate, glucose, malate, maltose, mannitol, phenylacetate, sucrose, fructose and trehalose as sole carbon sources. Utilization of L-arabinose, D-ribose, D-xylose, gentiobiose, starch and glycogen is weak. Does not use amygdalin, caproate, D-arabinose, L-xylose, L-rhamnose, galactose, sorbose, D-mannose, melibiose, cellobiose, lactose, melezitose, raffinose, turanose, lyxose, tagatose, D-fucose, L-fucose, methyl -D-xyloside, methyl -D-mannoside, methyl -D-glucoside, arbutin, salicin, inulin, adonitol, inositol, sorbitol, dulcitol, erythritol, xylitol, arabinitol, glycerol and 2-ketogluconate or 5-ketogluconate as carbon sources.
The type strain, MACL01T (=LMG 22194T=CECT 5860T), was isolated from Lake Martel, Mallorca, Spain.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Baumann, P. & Schubert, R. H. W. (1984). Family II. Vibrionaceae Veron 1965, 5245AL. In Bergey's Manual for Systematic Bacteriology, vol. 1, pp. 517–545. Edited by N. R. Krieg & J. G. Holt. Baltimore: Williams & Wilkins.
Chun, J. & Goodfellow, M. (1995). A phylogenetic analysis of the genus Nocardia with 16S rRNA sequences. Int J Syst Bacteriol 45, 240–245.
Doetsch, R. N. (1981). Determinative methods of light microscopy. In Manual of Methods for General Bacteriology, pp. 21–33. Edited by P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg & G. B. Phillips. Washington, DC: American Society for Microbiology.
Farmer, J. J., III & Hickman-Brenner, F. W. (1992). The genera Vibrio and Photobacterium. In The Prokaryotes. A Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, and Applications, 2nd edn, pp. 2952–3011. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K. H. Schleifer. New York: Springer.
Gevers, D., Vandepoele, K., Simillion, C. & van de Peer, Y. (2004). Gene duplication and biased functional retention of paralogs in bacterial genomes. Trends Microbiol 12, 148–154.[CrossRef][Medline]
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef][Medline]
Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2001). Molecular Evolutionary Genetics Analysis software. Tempe, AZ: Arizona State University.
Lerat, E., Daubin, V. & Moran, N. A. (2003). From gene trees to organismal phylogeny in prokaryotes: the case of the 
-Proteobacteria. PLoS Biol 1, E19.[Medline]
Macián, M. C., Ludwig, W., Aznar, R., Grimont, P. A. D., Schleifer, K. H., Garay, E. & Pujalte, M. J. (2001). Vibrio lentus sp. nov., isolated from Mediterranean oysters. Int J Syst Evol Microbiol 51, 1449–1456.[Abstract]
Mandel, M. & Marmur, J. (1968). Use of ultraviolet absorbance temperature profile for determining the guanine plus cytosine content of DNA. Methods Enzymol 12B, 195–206.[CrossRef]
Peix, A., Rivas, R., Mateos, P. F., Martínez-Molina, E., Rodríguez-Barrueco, C. & Velázquez, E. (2003). Pseudomonas rhizosphaerae sp. nov., a novel species that actively solubilizes phosphate in vitro. Int J Syst Evol Microbiol 53, 2067–2072.
Rivas, R., Sánchez, M., Trujillo, M. E., Zurdo-Piñeiro, J. L., Mateos, P. F., Martínez-Molina, E. & Velázquez, E. (2003). Xylanimonas cellulosilytica gen. nov., sp. nov., a xylanolytic bacterium isolated from a decayed tree (Ulmus nigra). Int J Syst Evol Microbiol 53, 99–103.
Rivas, R., Sánchez-Márquez, S., Mateos, P. F., Martínez-Molina, E. & Velázquez, E. (2005). Martelella mediterranea gen. nov., sp. nov., a novel -proteobacterium isolated from a subterranean saline lake. Int J Syst Evol Microbiol 55, 955–959.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Seo, H. J., Bae, S. S., Lee, J. Y. & Kim, S. J. (2005). Photobacterium frigidiphilum sp. nov., a psychrophilic, lipolytic bacterium isolated from deep-sea sediments of Edison Seamount. Int J Syst Evol Microbiol 55, 1661–1666.
Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.
Thompson, F. L., Hoste, B., Vandemeulbroecke, K., Engelbeen, K., Denys, R. & Swings, J. (2002a). Vibrio trachuri Iwamoto et al. 1995 is a junior synonym of Vibrio harveyi (Johnson and Shunk 1936) Baumann et al. 1981. Int J Syst Evol Microbiol 52, 973–976.[Abstract]
Thompson, F. L., Hoste, B., Thompson, C. C., Goris, J., Gomez-Gil, B., Huys, L., De Vos, P. & Swings, J. (2002b). Enterovibrio norvegicus gen. nov., sp. nov., isolated from the gut of turbot (Scophthalmus maximus) larvae: a new member of the family Vibrionaceae. Int J Syst Evol Microbiol 52, 2015–2022.[Abstract]
Thompson, F. L., Thompson, C. C., Vandemeulebroecke, S., Hoste, K., Dawyndt, P. & Swings, J. (2004). Use of recA as an alternative phylogenetic marker in the family Vibrionaceae. Int J Syst Evol Microbiol 54, 919–924.
Thompson, F. L., Gevers, D., Thompson, C. C., Dawyndt, P., Naser, S., Hoste, B., Munn, C. B. & Swings, J. (2005a). Phylogeny and molecular identification of vibrios on the basis of multilocus sequence analysis. Appl Environ Microbiol 71, 5107–5115.
Thompson, F. L., Thompson, C. C., Naser, S., Hoste, B., Vandemeulebroecke, K., Munn, C., Bourne, D. & Swings, J. (2005b). Photobacterium rosenbergii sp. nov. and Enterovibrio coralii sp. nov., vibrios associated with coral bleaching. Int J Syst Evol Microbiol 55, 913–1917.
Zeigler, D. R. (2003). Gene sequences useful for predicting relatedness of whole genomes in bacteria. Int J Syst Evol Microbiol 53, 1893–1900.
This article has been cited by other articles:
|
|
 |
|
  D. M. Adin, K. L. Visick, and E. V. Stabb Identification of a Cellobiose Utilization Gene Cluster with Cryptic {beta}-Galactosidase Activity in Vibrio fischeri Appl. Envir. Microbiol., July 1, 2008; 74(13): 4059 - 4069. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-Y. Jung, Y.-T. Jung, T.-K. Oh, and J.-H. Yoon Photobacterium lutimaris sp. nov., isolated from a tidal flat sediment in Korea Int J Syst Evol Microbiol, February 1, 2007; 57(2): 332 - 336. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
