Two Gram-negative, cold-adapted, aerobic bacteria, designated strains M8T and M6, were isolated from soil collected from the South Shetland Islands. The organisms were rod-shaped, catalase- and oxidase-positive and motile by means of polar flagella. These two psychrotolerant strains grew between −4 and 30 °C. 16S rRNA gene sequence analysis placed strains M8T and M6 within the genus Pseudomonas. DNA–DNA hybridization experiments between the Antarctic isolate M8T and type strains of phylogenetically related species, namely Pseudomonas peli and Pseudomonas anguilliseptica, revealed levels of relatedness of 33 and 37 %, respectively. Strain M6 showed 99 % DNA similarity to strain M8T. Several phenotypic characteristics, together with data on cellular fatty acid composition, served to differentiate strains M8T and M6 from related pseudomonads. On the basis of the polyphasic taxonomic evidence presented in this study, it can be concluded that strains M8T and M6 belong to the same genospecies, representing a novel species of the genus Pseudomonas, for which the name Pseudomonas guineae sp. nov. is proposed. The type strain is M8T (=LMG 24016T=CECT 7231T)

Bozal de Febrer, Núria

Mercadé Gil, M. Elena

Montes López, María Jesús

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY

English

Diposit Digital de la Universitat de Barcelona

Pseudomonas guineae sp. nov., a novelpsychrotolerant bacterium from an AntarcticenvironmentNúria Bozal, M. Jesús Montes and Elena MercadéCorrespondenceElena Mercadémmercade@ub.eduLaboratori de Microbiologia, Facultat de Farmacia, Universitat de Barcelona, Av. Joan XXIII s/n,08028 Barcelona, SpainTwo Gram-negative, cold-adapted, aerobic bacteria, designated strains M8T and M6, wereisolated from soil collected from the South Shetland Islands. The organisms were rod-shaped,catalase- and oxidase-positive and motile by means of polar flagella. These two psychrotolerantstrains grew between ”4 and 30 6C. 16S rRNA gene sequence analysis placed strains M8T andM6 within the genus Pseudomonas. DNA–DNA hybridization experiments between theAntarctic isolate M8T and type strains of phylogenetically related species, namely Pseudomonaspeli and Pseudomonas anguilliseptica, revealed levels of relatedness of 33 and 37 %,respectively. Strain M6 showed 99 % DNA similarity to strain M8T. Several phenotypiccharacteristics, together with data on cellular fatty acid composition, served to differentiate strainsM8T and M6 from related pseudomonads. On the basis of the polyphasic taxonomic evidencepresented in this study, it can be concluded that strains M8T and M6 belong to the samegenospecies, representing a novel species of the genus Pseudomonas, for which the namePseudomonas guineae sp. nov. is proposed. The type strain is M8T (5LMG 24016T5CECT7231T).In recent years, increasing attention has been devotedto cold-adapted micro-organisms and their enzymes(Antranikian et al., 2005). Antarctica has become a greatsource of novel psychrophilic and psychrotolerant strains,some of which belong to the genus Pseudomonas (Krisset al., 1976; Shivaji et al., 1989; Ma et al., 2006; Maugeriet al., 1996; Bruni et al., 1999; Reddy et al., 2004). Thegenus Pseudomonas once comprised more than 100 species,but, over the last decade, many of these have beenreclassified into different genera (Kersters et al., 1996;Anzai et al., 2000). During a taxonomic investigation ofcold-adapted bacteria from soil samples collected in theAntarctic area of the South Shetland Islands, two strains,M6 and M8T, able to grow at 24 uC and capable offorming swarming colonies on trypticase soy agar (TSA)were isolated. In this study, the taxonomic status of thesetwo strains was investigated by using a combination ofphenotypic characterization, 16S rRNA gene sequencing,DNA G+C content determination, DNA–DNA hybridiza-tion and cellular fatty acid analysis. The data obtainedshow that strains M6 and M8T belong to a novel species ofthe genus Pseudomonas, for which the name Pseudomonasguineae sp. nov. is proposed, with M8Tas the type strain.Strains M8T and M6 were isolated from a soil samplecollected from Deception Island (South Shetland Islands,Antarctica). Sample aliquots were removed with aplatinum loop and diluted in a saline solution (pH 7)containing the following salts (g l21): NaCl, 0.56; KCl,0.027; CaCl2, 0.03; NaHCO3, 0.01. TSA plates wereinoculated with loopfuls of several sample dilutions byusing the streak-plate method to obtain isolated colonies.Plates were incubated for 4 days at 15 uC. Isolates weremaintained aerobically on TSA slopes at 4 uC and also at280 uC on cryo-beads.The morphology, cell size and shape of cells grown on TSAat 15 uC were determined by means of negative stainingand transmission electron microscopy. Motility wasdetermined by phase-contrast microscopy. Oxidase, cata-lase and urease activities, nitrate reduction and hydrolysisof casein, lecithin, gelatin, DNA, starch and Tween 80 weredetermined according to Cowan & Steel (1993). Thepresence of fluorescent pigments was tested under UV lightafter 8 days on King’s B medium (King et al., 1954). Acidproduction from carbohydrates, enzyme production andadditional characteristics were determined by using API 50CH, API ZYM and API 20NE strips (bioMérieux).Tolerance of NaCl was measured on nutrient agarThe GenBank/EMBL/DDBJ accession numbers for the 16S rRNA genesequences of strains M8T and M6 are AM491810 and AM491811,respectively.An electron micrograph of a cell of strain M8T, an extended phylogenetictree for strains M8T and M6 within the genus Pseudomonas and a tableshowing cellular fatty acid compositions are available with the onlineversion of this paper.International Journal of Systematic and Evolutionary Microbiology (2007), 57, 2609–2612 DOI 10.1099/ijs.0.65141-065141 G 2007 IUMS Printed in Great Britain 2609containing 0.5–7.5 % (w/v) NaCl; plates were incubated at15 uC for 30 days. The temperature range for growth wasdetermined on TSA incubated for 14 days at temperaturesfrom 24 to 37 uC. Anaerobic growth was determined ontrypticase soy broth (TSB) plus 1.5 % agar-agar and onMarine agar (Difco) after incubation in an anaerobicchamber at 15 uC for 14 days.The cells were Gram-negative, rod-shaped (0.4–0.5 mmwide and 1.5–2.0 mm long) and motile by means of polarflagella (see Supplementary Fig. S1, available in IJSEMOnline). Colonies of the isolates grown on TSA at 15 uC for72 h were non-pigmented, round with irregular edges,slightly convex, 1.5–2.0 mm in diameter and did notproduce fluorescent pigment on King’s B medium. After1 week, colonies had swarmed over the plate, mergingtogether and becoming more mucous. The isolates grew attemperatures ranging from 24 to 30 uC and tolerated NaClconcentrations up to 4 % (w/v) on TSA. The isolates werepositive for the hydrolysis of lecithin and negative for thehydrolysis of casein, starch, Tween 80 and DNA. Otherphenotypic characteristics of the Antarctic isolates andtheir closest phylogenetic relatives are shown in Table 1.These phenotypic studies showed that the isolates displayedcharacteristics consistent with those for the genusPseudomonas.Fatty acids were prepared from 40 mg wet cell materialharvested from a TSB agar culture (30 g TSB l21 and 15 gagar l21) incubated for 4 days at 15 uC. The whole-cell fattyacids were determined as described previously (Bozal et al.,2002). The mean fatty acid compositions of strains M8T andM6, together with those of type strains of the closestphylogenetic neighbours, are shown in Supplementary TableS1 (available in IJSEM Online). The most abundant fattyacids were C16 : 0, C18 : 1v7c and summed feature 3 (C16 : 1v7cand/or iso-C15 : 0 2-OH). The isolates had cellular fatty acidprofiles similar to that of Pseudomonas peli LMG 23201T,containing the same percentages of C16 : 0 and C18 : 1v7c,whereas the summed feature 3 content was lower for P. peli.Pseudomonas anguilliseptica LMG 21629T contained asignificantly higher proportion of C16 : 0 and also had asmaller proportion of summed feature 3.Total DNA for complete 16S rRNA gene sequence analysiswas prepared according to the protocol of Niemann et al.(1997). Phylogenetic analyses were carried out by using theneighbour-joining method as described previously by Bozalet al. (2002), with the software package BioNumerics(Applied Maths). For DNA–DNA hybridizations anddetermination of the G+C content, total DNA wasprepared according to a modification of the procedure ofWilson (1987). The G+C content was determined by usingthe HPLC technique, as described by Mesbah et al. (1989).The DNA–DNA hybridizations were performed at 47 uCaccording to a modification (Goris et al., 1998; Cleenwercket al., 2002) of the method described by Ezaki et al. (1989).16S rRNA phylogenetic studies confirmed that theAntarctic isolates (strains M8T and M6) were members ofthe genus Pseudomonas. The highest level of 16S rRNAgene sequence similarity (99.1 %) was found with P. peliLMG 23201T; lower levels of similarity occurred with otherPseudomonas species with validly published names (Fig. 1;Supplementary Fig. S2, available in IJSEM Online, showsthe complete phylogenetic tree). Strain M6 showed 100.0 %16S rRNA gene sequence similarity to M8T, indicating thatthese strains probably belong to the same species. To verifythe taxonomic position of strain M8T, DNA–DNAhybridizations were performed with P. peli LMG 23201Tand P. anguilliseptica LMG 21629T. The low DNA–DNAreassociation values (33 % with P. peli LMG 23201T and37 % with P. anguilliseptica LMG 21629T) and the 16SrRNA gene sequence data indeed showed that strain M8Toccupies a distinct position within the genus Pseudomonas(Wayne et al., 1987). Strain M6 showed 99 % DNAsimilarity to M8T and it can be concluded that they belongto the same genospecies. The DNA G+C contents of M8TTable 1. Phenotypic characteristics of strains M8T and M6and their closest phylogenetic neighboursStrains: 1, M8T; 2, M6; 3, P. peli LMG 23201T [data from Vanparyset al. (2006)]; 4, P. anguilliseptica LMG 21629T [data from Vanparyset al. (2006)]. All strains are positive for oxidase, catalase, leucinearylamidase and naphthol-AS-BI-phosphohydrolase. All are negativefor trypsin, chymotrypsin, a-galactosidase, b-galactosidase, b-gluc-uronidase, b-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase,a-mannosidase, a-fucosidase, indole production, acidification ofglucose, arginine dihydrolase, urease, aesculin hydrolysis, gelatinhydrolysis and the assimilation of glycerol, L-arabinose, D-ribose, D-glucose, D-fructose, D-mannose, D-mannitol, N-acetylglucosamine, D-maltose, D-sucrose, D-trehalose, D-arabitol, gluconate, adipate,phenylacetate, erythritol, D-arabinose, L-xylose, D-adonitol, methylb-D-xyloside, L-sorbose, L-rhamnose, dulcitol, inositol, D-sorbitol,methyl a-D-mannoside, methyl a-D-glucoside, amygdalin, arbutin,aesculin, salicin, D-cellobiose, D-lactose, D-melibiose, inulin, D-melezitose, D-raffinose, starch, glycogen, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, D- and L-fucose, L-arabitol and2- and 5-ketogluconate. +, Positive; W, weakly positive; 2, negative.Characteristic 1 2 3 4Growth at 4 uC + + 2 +Nitrate reduction 2 2 2 +Enzyme activitiesAlkaline phosphatase + + 2 WEsterase + + 2 2Esterase lipase + + + 2Lipase + + W 2Valine arylamidase + + 2 2Cystine arylamidase + + 2 2Acid phosphatase + + 2 +Assimilation of:D-Galactose 2 2 2 +Caprate + + 2 +Malate + + + +Citrate + + 2 +N. Bozal, M. J. Montes and E. Mercadé2610 International Journal of Systematic and Evolutionary Microbiology 57and M6 (58.5 and 58.4 mol%, respectively) lie within therange described for members of the genus Pseudomonas.The morphological, physiological, chemotaxonomic andphylogenetic data showed that strains M8T and M6 belongto the genus Pseudomonas. The DNA–DNA hybridizationanalyses clearly distinguished strain M8T from P. peli(Vanparys et al., 2006) and P. anguilliseptica (Wakabayashi& Egusa, 1972). On the basis of the data from thispolyphasic study, therefore, it is proposed that strains M8Tand M6 represent a novel species of the genusPseudomonas, for which the name Pseudomonas guineaesp. nov. is proposed.Description of Pseudomonas guineae sp. nov.Pseudomonas guineae (gui.ne9ae. N.L. gen. masc. n. guineaeof Guinea, in honour of the late Professor Jesús Guinea, aprominent Spanish microbiologist, who isolated this strain).Cells are rod-shaped (0.4–0.5 mm wide and 1.5–2.0 mmlong), Gram-negative, non-spore-forming and do notproduce fluorescent pigment on King’s B medium. Cellsare motile by means of polar flagella. After 72 h incubationat 15 uC on TSA, colonies are 1.5–2.0 mm in diameter,smooth and round with irregular edges. Growth occurs attemperatures between 24 and 30 uC, but not at 37 uC.NaCl is tolerated at concentrations up to 4 % (w/v).Growth is very poor under anaerobic conditions. Enzymeactivities and details of the carbon sources utilized aregiven in Table 1. The DNA G+C content is 58.5 mol%.The type strain, M8T (5LMG 24016T5CECT 7231T), wasisolated from a soil sample collected from Deception Island(South Shetland Islands, Antarctica).AcknowledgementsThis paper is dedicated, with sorrow, respect and gratitude, to thememory of Jesús Guinea Sánchez, who died on 29 September 2006.We would like to thank Josefina Castellvı́ for providing Antarcticsamples. We gratefully acknowledge the assistance of F. Garcia(Departament d’Agricultura, Ramaderia i Pesca, Generalitat deCatalunya, Spain) with the fatty acid analysis. We thank theBCCM/LMG Identification Service (BCCM/LMG BacteriaCollection, Laboratorium voor Microbiologie, University of Ghent,Ghent, Belgium) for performing hybridization analyses and 16S rRNAgene sequence analysis. We would also like to thank Dr H. G. Trüperfor advice on Latin nomenclature. This research was supportedby the Autonomous Government of Catalonia, Spain (grant2005SGR00066).ReferencesAntranikian, G., Vorgias, C. E. & Bertoldo, C. (2005). Extremeenvironments as a source for microorganisms and novel biocatalysts.Adv Biochem Eng Biotechnol 96, 219–262.Anzai, Y., Kim, H., Park, J. Y., Wakabayashi, H. & Oyaizu, H. (2000).Phylogenetic affiliation of the pseudomonads based on 16S rRNAsequence. Int J Syst Evol Microbiol 50, 1563–1589.Bozal, N., Montes, M. J., Tudela, E., Jiménez, F. & Guinea, J. (2002).Shewanella frigidimarina and Shewanella livingstonensis sp. nov.isolated from Antarctic coastal areas. Int J Syst Evol Microbiol 52,195–205.Bruni, V., Gugliandolo, C., Maugeri, T. & Allegra, A. (1999).Psychrotrophic bacteria from a coastal station in the Ross Sea(Terra Nova Bay, Antarctica). New Microbiol 22, 357–363.Cleenwerck, I., Vandemeulebroecke, K., Janssens, D. & Swings, J.(2002). Re-examination of the genus Acetobacter, with descriptions ofAcetobacter cerevisiae sp. nov. and Acetobacter malorum sp. nov. Int JSyst Evol Microbiol 52, 1551–1588.Cowan, S. T. & Steel, K. J. (1993). Manual for the Identification ofMedical Bacteria, 3rd edn. Edited by G. I. Barrow & R. K. A. Feltham.Cambridge: Cambridge University Press.Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometricdeoxyribonucleic acid-deoxyribonucleic acid hybridization in micro-dilution wells as an alternative to membrane filter hybridization inwhich radioisotopes are used to determine genetic relatedness amongbacterial strains. Int J Syst Bacteriol 39, 224–229.Goris, J., Suzuki, K., De Vos, P., Nakase, T. & Kersters, K. (1998).Evaluation of a microplate DNA-DNA hybridization methodcompared with the initial renaturation method. Can J Microbiol 44,1148–1153.Fig. 1. Phylogenetic tree obtained by neighbour-joining analysis of 16S rRNA gene sequences, showing the position of theAntarctic isolates M8T and M6 among neighbouring species of the genus Pseudomonas. Bootstrap values .70 % (based on1000 replications) are shown at branch points.Pseudomonas guineae sp. nov.http://ijs.sgmjournals.org 2611Kersters, K., Ludwig, W., Vancanneyt, M., De Vos, P., Gillis, M. &Schleifer, K. H. (1996). Recent changes in the classification of thepseudomonads: an overview. Syst Appl Microbiol 19, 465–477.King, E. O., Ward, M. K. & Rainey, D. E. (1954). Two simple media forthe demonstration of pyocyanin and fluorescein. J Lab Clin Med 44,301–307.Kriss, A. E., Mitskevich, I. N., Rozanova, E. P. & Osnitskaia, L. K. (1976).Microbiological studies of the Wanda Lake (Antarctica). Microbiology(English translation of Mikrobiologiya) 45, 1075–1081 (in Russian).Ma, Y., Wang, L. & Shao, Z. (2006). Pseudomonas, the dominantpolycyclic aromatic hydrocarbon-degrading bacteria isolated fromAntarctic soils and the role of large plasmids in horizontal genetransfer. Environ Microbiol 8, 455–465.Maugeri, T. L., Gugliandolo, C. & Bruni, V. (1996). Heterotrophicbacteria in the Ross Sea (Terra Nova Bay, Antarctica). New Microbiol19, 67–76.Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precisemeasurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.Niemann, S., Puehler, A., Tichi, H. V., Simon, R. & Selbitschka, W.(1997). Evaluation of the resolving power of the three differentfingerprinting methods to discriminate among isolates of a naturalRhizobium meliloti population. J Appl Microbiol 82, 477–484.Reddy, G. S. N., Matsumoto, G. I., Schumann, P., Stackebrandt, E. &Shivaji, S. (2004). Psychrophilic pseudomonads from Antarctica:Pseudomonas antarctica sp. nov. and Pseudomonas proteolytica sp.nov. Int J Syst Evol Microbiol 54, 713–719.Shivaji, S., Rao, N. S., Saisree, L., Sheth, V., Reddy, G. S. N. &Bhargava, P. M. (1989). Isolation and identification of Pseudomonasspp. from Schirmacher Oasis, Antarctica. Appl Environ Microbiol 55,767–770.Vanparys, B., Heylen, K., Lebbe, L. & De Vos, P. (2006). Pseudomonaspeli sp. nov. and Pseudomonas borbori sp.nov., isolated from anitrifying inoculum. Int J Syst Evol Microbiol 56, 1875–1881.Wakabayashi, H. & Egusa, S. (1972). Characteristics of aPseudomonas sp. from an epizootic of pound-cultured eels (Anguillajaponica). Bull Jpn Soc Sci Fish 38, 577–587.Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O.,Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & otherauthors (1987). International Committee on Systematic Bacteriology.Report of the ad hoc committee on reconciliation of approaches tobacterial systematics. Int J Syst Bacteriol 37, 463–464.Wilson, K. (1987). Preparation of genomic DNA from bacteria. InCurrent Protocols in Molecular Biology, pp. 2.4.1–2.4.5. Edited by F. M.Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A.Smith & K. Struhl. New York: Green Publishing & Wiley-Interscience.N. Bozal, M. J. Montes and E. Mercadé2612 International Journal of Systematic and Evolutionary Microbiology 57

Pseudomonas guineae sp.nov, a novel psychrotolerant bacterium from an Antarctic environment

https://diposit.ub.edu/dspace/bitstream/2445/214717/1/152177.pdf

Pseudomonas guineae sp.nov, a novel psychrotolerant bacterium from an Antarctic environment

Abstract

Similar works

Full text

Available Versions

Diposit Digital de la Universitat de Barcelona