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Laboratori de Microbiologia, Facultat de Farmacia, Universitat de Barcelona, Av. Joan XXIII s/n, 08028 Barcelona, Spain
Correspondence
Elena Mercadé
mmercade{at}ub.edu
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ABSTRACT |
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An electron micrograph of a cell of strain M8T, an extended phylogenetic tree for strains M8T and M6 within the genus Pseudomonas and a table showing cellular fatty acid compositions are available with the online version of this paper.
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). Antarctica has become a great source of novel psychrophilic and psychrotolerant strains, some of which belong to the genus Pseudomonas (Kriss et al., 1976; Shivaji et al., 1989; Ma et al., 2006; Maugeri et al., 1996; Bruni et al., 1999; Reddy et al., 2004). The genus Pseudomonas once comprised more than 100 species, but, over the last decade, many of these have been reclassified into different genera (Kersters et al., 1996; Anzai et al., 2000). During a taxonomic investigation of cold-adapted bacteria from soil samples collected in the Antarctic area of the South Shetland Islands, two strains, M6 and M8T, able to grow at –4 °C and capable of forming swarming colonies on trypticase soy agar (TSA) were isolated. In this study, the taxonomic status of these two strains was investigated by using a combination of phenotypic characterization, 16S rRNA gene sequencing, DNA G+C content determination, DNA–DNA hybridization and cellular fatty acid analysis. The data obtained show that strains M6 and M8T belong to a novel species of the genus Pseudomonas, for which the name Pseudomonas guineae sp. nov. is proposed, with M8Tas the type strain. Strains M8T and M6 were isolated from a soil sample collected from Deception Island (South Shetland Islands, Antarctica). Sample aliquots were removed with a platinum loop and diluted in a saline solution (pH 7) containing the following salts (g l–1): NaCl, 0.56; KCl, 0.027; CaCl2, 0.03; NaHCO3, 0.01. TSA plates were inoculated with loopfuls of several sample dilutions by using the streak-plate method to obtain isolated colonies. Plates were incubated for 4 days at 15 °C. Isolates were maintained aerobically on TSA slopes at 4 °C and also at –80 °C on cryo-beads.
The morphology, cell size and shape of cells grown on TSA at 15 °C were determined by means of negative staining and transmission electron microscopy. Motility was determined by phase-contrast microscopy. Oxidase, catalase and urease activities, nitrate reduction and hydrolysis of casein, lecithin, gelatin, DNA, starch and Tween 80 were determined according to Cowan & Steel (1993). The presence of fluorescent pigments was tested under UV light after 8 days on King's B medium (King et al., 1954). Acid production from carbohydrates, enzyme production and additional characteristics were determined by using API 50 CH, API ZYM and API 20NE strips (bioMérieux). Tolerance of NaCl was measured on nutrient agar containing 0.5–7.5 % (w/v) NaCl; plates were incubated at 15 °C for 30 days. The temperature range for growth was determined on TSA incubated for 14 days at temperatures from –4 to 37 °C. Anaerobic growth was determined on trypticase soy broth (TSB) plus 1.5 % agar-agar and on Marine agar (Difco) after incubation in an anaerobic chamber at 15 °C for 14 days.
The cells were Gram-negative, rod-shaped (0.4–0.5 µm wide and 1.5–2.0 µm long) and motile by means of polar flagella (see Supplementary Fig. S1, available in IJSEM Online). Colonies of the isolates grown on TSA at 15 °C for 72 h were non-pigmented, round with irregular edges, slightly convex, 1.5–2.0 mm in diameter and did not produce fluorescent pigment on King's B medium. After 1 week, colonies had swarmed over the plate, merging together and becoming more mucous. The isolates grew at temperatures ranging from –4 to 30 °C and tolerated NaCl concentrations up to 4 % (w/v) on TSA. The isolates were positive for the hydrolysis of lecithin and negative for the hydrolysis of casein, starch, Tween 80 and DNA. Other phenotypic characteristics of the Antarctic isolates and their closest phylogenetic relatives are shown in Table 1. These phenotypic studies showed that the isolates displayed characteristics consistent with those for the genus Pseudomonas.
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). The mean fatty acid compositions of strains M8T and M6, together with those of type strains of the closest phylogenetic neighbours, are shown in Supplementary Table S1 (available in IJSEM Online). The most abundant fatty acids were C16 : 0, C18 : 1
7c and summed feature 3 (C16 : 17c and/or iso-C15 : 0 2-OH). The isolates had cellular fatty acid profiles similar to that of Pseudomonas peli LMG 23201T, containing the same percentages of C16 : 0 and C18 : 17c, whereas the summed feature 3 content was lower for P. peli. Pseudomonas anguilliseptica LMG 21629T contained a significantly higher proportion of C16 : 0 and also had a smaller proportion of summed feature 3.
Total DNA for complete 16S rRNA gene sequence analysis was prepared according to the protocol of Niemann et al. (1997). Phylogenetic analyses were carried out by using the neighbour-joining method as described previously by Bozal et al. (2002), with the software package BioNumerics (Applied Maths). For DNA–DNA hybridizations and determination of the G+C content, total DNA was prepared according to a modification of the procedure of Wilson (1987). The G+C content was determined by using the HPLC technique, as described by Mesbah et al. (1989). The DNA–DNA hybridizations were performed at 47 °C according to a modification (Goris et al., 1998; Cleenwerck et al., 2002) of the method described by Ezaki et al. (1989).
16S rRNA phylogenetic studies confirmed that the Antarctic isolates (strains M8T and M6) were members of the genus Pseudomonas. The highest level of 16S rRNA gene sequence similarity (99.1 %) was found with P. peli LMG 23201T; lower levels of similarity occurred with other Pseudomonas species with validly published names (Fig. 1; Supplementary Fig. S2, available in IJSEM Online, shows the complete phylogenetic tree). Strain M6 showed 100.0 % 16S rRNA gene sequence similarity to M8T, indicating that these strains probably belong to the same species. To verify the taxonomic position of strain M8T, DNA–DNA hybridizations were performed with P. peli LMG 23201T and P. anguilliseptica LMG 21629T. The low DNA–DNA reassociation values (33 % with P. peli LMG 23201T and 37 % with P. anguilliseptica LMG 21629T) and the 16S rRNA gene sequence data indeed showed that strain M8T occupies a distinct position within the genus Pseudomonas (Wayne et al., 1987). Strain M6 showed 99 % DNA similarity to M8T and it can be concluded that they belong to the same genospecies. The DNA G+C contents of M8T and M6 (58.5 and 58.4 mol%, respectively) lie within the range described for members of the genus Pseudomonas.
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) and P. anguilliseptica (Wakabayashi & Egusa, 1972). On the basis of the data from this polyphasic study, therefore, it is proposed that strains M8T and M6 represent a novel species of the genus Pseudomonas, for which the name Pseudomonas guineae sp. nov. is proposed.
Description of Pseudomonas guineae sp. nov.
Pseudomonas guineae (gui.ne'ae. N.L. gen. masc. n. guineae of Guinea, in honour of the late Professor Jesús Guinea, a prominent Spanish microbiologist, who isolated this strain).
Cells are rod-shaped (0.4–0.5 µm wide and 1.5–2.0 µm long), Gram-negative, non-spore-forming and do not produce fluorescent pigment on King's B medium. Cells are motile by means of polar flagella. After 72 h incubation at 15 °C on TSA, colonies are 1.5–2.0 mm in diameter, smooth and round with irregular edges. Growth occurs at temperatures between –4 and 30 °C, but not at 37 °C. NaCl is tolerated at concentrations up to 4 % (w/v). Growth is very poor under anaerobic conditions. Enzyme activities and details of the carbon sources utilized are given in Table 1
. The DNA G+C content is 58.5 mol%.
The type strain, M8T (=LMG 24016T=CECT 7231T), was isolated from a soil sample collected from Deception Island (South Shetland Islands, Antarctica).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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