| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 IRNASA-CSIC, Apdo 257, 37071 Salamanca, Spain
2 Departamento de Microbiología y Genetica, Universidad de Salamanca, Spain
Correspondence
Encarna Velázquez
evp{at}gugu.usal.es
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
Tables detailing the results of DNA–DNA hybridization studies and fatty acid analyses are available with the online version of this paper.
Present address: Department of Plant Pathology and Microbiology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel. 
Present address: Laboratorium voor Microbiologie, Vakgroep Biochemie, Fysiologie en Microbiologie, Universiteit Gent KL, Ledeganckstraat 35, B-9000 Gent, Belgium.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
) and considered as separate species in the first edition of Bergey's Manual of Systematic Bacteriology by Palleroni, who included the first two species in rRNA group I and P. aurantiaca in group V (Palleroni, 1984). Later, the species P. aureofaciens was proposed as a later heterotypic synonym of P. chlororaphis on the basis of DNA relatedness and phenotypic traits (Johnson & Palleroni, 1989). Recently, in the second edition of the Bergey's Manual of Systematic Bacteriology, Palleroni (2005) proposed that P. aureofaciens and P. chlororaphis may be considered as two subspecies of the same species and that P. aurantiaca remains a separate species. In 1985, a taxonomic study of P. aurantiaca was published outside of the International Journal of Systematic Bacteriology by Kiprianova et al. (1985) who proposed the selection of a neotype strain for this species. Their conclusion was based on phenotypic data from the type strain received from the VKM collection, which showed different characteristics from those recorded in the literature for P. aurantiaca. An analysis of the type strains of P. aurantiaca available from other culture collections was not performed, or at least was not recorded in the cited paper, and therefore the decision to designate a neotype strain was not strongly supported and was never officially approved.
The type strain of P. aurantiaca available at the NCIMB and used in our study fulfils the characteristics described for P. aurantiaca as recorded in the different editions of Bergey's Manual of Systematic Bacteriology. The 16S rRNA gene sequence of P. aurantiaca NCIMB 10068T obtained in this study is 100 % similar to that of strain ATCC 33663T available at GenBank (Anzai et al., 2000). This result is congruent with the fact that the ATCC received the strain from the NCIMB, from where it was also sent to the LMG and the CIP collections. Therefore, the original type strain of P. aurantiaca is currently available in several collections and the selection of a neotype strain is thus not necessary.
The 16S rRNA gene sequence of the type strain of P. aurantiaca showed a similarity greater than 99 % with respect to those of P. chlororaphis DSM 50083T and P. aureofaciens DSM 6698T as obtained in this study. Moreover, a recent study of atpD, recA and carA gene sequences from Pseudomonas species showed that the type strain of P. aurantiaca belongs to the same phylogenetic group as P. chlororaphis and P. aureofaciens, with gene sequence similarities greater than 96 % (Hilario et al., 2004), suggesting that they belong to the same species. Although they show several phenotypic differences that have been pointed out by several authors (Palleroni, 1984, 2005; Kiprianova et al., 1985; Johnson & Palleroni, 1989), the strains belonging to P. aurantiaca, P. chlororaphis and P. aureofaciens are phenotypically close. In this work, we therefore performed a polyphasic study in order to clarify the taxonomic status of P. aurantiaca in comparison with strains of P. chlororaphis and P. aureofaciens. From the results obtained, we conclude that P. aurantiaca is a subspecies of Pseudomonas chlororaphis and propose the name Pseudomonas chlororaphis subsp. aurantiaca subsp. nov., comb. nov. The names P. chlororaphis subsp. chlororaphis subsp. nov. and P. chlororaphis subsp. aureofaciens subsp. nov., comb. nov. are also proposed. The names of these novel subspecies were originally proposed by Palleroni (2005) but have not been validly published.
The 16S rRNA gene sequences of P. aurantiaca ATCC 33663T and P. aureofaciens ATCC 12353T were obtained by Anzai et al. (2000) and deposited in GenBank with accession numbers AB021412 and D84008, respectively. However, both sequences contain several undetermined nucleotides. Other sequences of the same strains held in public databases are not complete and so in this study, the 16S rRNA gene sequences of P. aurantiaca NCIMB 10068T (GenBank accession no. DQ682655) and P. aureofaciens DSM 6698T (AY509898) were determined according to a previously described method (Rivas et al., 2003). The sequences were compared with those deposited in 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.
Fig. 1 shows a phylogenetic tree that includes representative species of the genus Pseudomonas sensu stricto. The 16S rRNA gene sequence of strain DSM 50083T showed 99.5 and 99.4 % similarity to those of strains DSM 6698T and NCIMB 10068T, respectively. Strains DSM 6698T and NCIMB 10068T showed 99.7 % gene sequence similarity to eachother. The three strains formed a separate group from other species of the genus Pseudomonas sensu stricto.
|
, following the recommendations of Willems et al. (2001). Besides the type strains, several strains of P. chlororaphis, P. aurantiaca and P. aureofaciens were also included (see Supplementary Table S1 in IJSEM Online). The DNA–DNA hybridization results showed that, in agreement with the 16S rRNA gene sequence similarities, the highest hybridization values were found between P. aureofaciens DSM 6698T and P. aurantiaca NCIMB 10068T with a mean similarity of 81 %. P. chlororaphis DSM 50083T displayed 75 and 73 % DNA–DNA hybridization with respect to P. aurantiaca NCIMB 10068T and P. aureofaciens DSM 6698T, respectively. Values ranging between 60 and 87 % were obtained for other strains of the three species analysed (see Supplementary Table S1). Although some of these values are slightly lower than the threshold value of 70 % DNA–DNA similarity recommended for the delineation of species (Wayne et al., 1987), the type strains of these species showed values higher than 75 %.
Fatty acid analyses were performed with cultures grown for 24 h in TSA medium (Merck) at 28 °C as already described (Peix et al., 2003). The main non-polar fatty acids detected were C16 : 0 and those summed in summed feature 3 (C16 : 1
7c and C15 : 0 iso 2-OH).The amounts of fatty acid C16 : 0 and summed feature 3 were, respectively, 33.4 % and 34.8 % in strain DSM 50083T, 26.3 % and 34.8 % in strain DSM 6698T and 30.3 % and 25.3 % in strain NCIMB 10068T. The full fatty acid contents of the strains are shown in Supplementary Table S2 in IJSEM Online. Small differences in the amounts of some other fatty acids were found between the three strains. For example, strains DSM 50083T and DSM 6698T differ in the amount of C12 : 0, C16 : 0, C10 : 0 3-OH and C12 : 1 3-OH; strains DSM 50083T and NCIMB 10068T differ in C10 : 0 3-OH, C12 : 1 3-OH, C12 : 0 3-OH, C17 : 0 cyclo and summed feature 3, and strains DSM 6698T and NCIMB 10068T differ in C10 : 0 3-OH, C12 : 0 3-OH, C17 : 0 cyclo and summed feature 3.
The same strains that were included in the DNA–DNA hybridization experiments were also studied phenotypically by using the API 20NE and API 50CH systems as recommended by the manufacturer. The results of the phenotypic characterization concurred with those reported by Johnson & Palleroni (1989), who proposed the reclassification of P. aureofaciens as P. chlororaphis, and with those reported by Doudoroff & Palleroni (1974) and Palleroni (1984, 2005). According to these results, strains of P. aureofaciens use L-arabinose as a carbon source in both API 20NE and API 50CH tests while those strains belonging to P. chlororaphis do not. Nitrate reduction was positive for P. chlororaphis strains, negative in strains of P. aurantiaca and variable for strains of P. aureofaciens. P. aurantiaca strains differed from P. aureofaciens in the use of 5-ketogluconate and from P. chlororaphis strains in the use of both 5-ketogluconate and L-arabinose (Table 1).
|
on the phylogenetic analysis of several housekeeping genes, support the reclassification of P. aurantiaca as a later heterotypic synonym of P. chlororaphis. The results also revealed that strains of P. aurantiaca, P. aureofaciens and P. chlororaphis form three clearly distinguishable groups within P. chlororaphis that merit the status of subspecies. Therefore, we propose the establishment of three novel subspecies within P. chlororaphis; P. chlororaphis subsp. chlororaphis subsp. nov., P. chlororaphis subsp. aureofaciens subsp. nov., comb. nov. and P. chlororaphis subsp. aurantiaca subsp. nov., comb. nov.
Emended description of Pseudomonas chlororaphis (ex Guignard and Sauvageau 1894) Bergey et al. 1930AL
Characteristics in addition to those reported in the original description recorded in Bergey's Manual of Determinative Bacteriology (Doudoroff & Palleroni, 1974) are as follows. The main non-polar fatty acids detected are C16 : 0 and summed feature 3, comprising around 30 % of total fatty acids, followed by C18 : 17c, with amounts of approximately 10 %. A more detailed breakdown of fatty acid content is presented in Supplementary Table S2. Negative result in tests for urease, 
-galactosidase, indole production and aesculin hydrolysis. Nitrate reduction is variable. N-acetylglucosamine, trehalose, raffinose and D-arabitol are used as carbon sources. Assimilation of L-arabinose, phenylacetate and 5-ketogluconate is variable. The use of L-xylose, sorbose, amygdalin, arbutin, salicin, melibiose, melezitose, starch, glycogen, gentiobiose, turanose, lyxose, tagatose, L-fucose, L-arabitol, xylitol, dulcitol, methyl 
-D-glucoside, methyl -D-mannoside and methyl -D-xyloside is negative. The DNA G+C content ranges from 63.5 to 63.6 mol%.
Description of Pseudomonas chlororaphis subsp. chlororaphis subsp. nov.
Pseudomonas chlororaphis subsp. chlororaphis (chlo.ro.ra'phis. Gr. adj. chlorus green; Gr. n. raphis a needle; N.L. fem. n. chlororaphis a green needle).
Displays characteristics typical for the species P. chlororaphis as described above. Chlororaphin, a green insoluble phenazine pigment, is produced. Nitrate reduction is positive. Utilization of L-arabinose is negative. Utilization of 5-ketogluconate is positive. Phylogeny based on 16S rRNA, atpD, recA and carA gene sequences separates this subspecies from the other subspecies of P. chlororaphis.
The type strain is DSM 50083T (=ATCC 9446T=NCIMB 9392T).
Description of Pseudomonas chlororaphis subsp. aureofaciens subsp. nov., comb. nov.
Pseudomonas chlororaphis subsp. aureofaciens (au.re.o.fa'ci.ens. L. adj. aureus golden; L. part. adj. faciens producing; N.L. part. adj. aureofaciens golden-producing, referring to the pigment produced).
Displays characteristics typical for the species P. chlororaphis as described above. Produces a diffusible yellow–orange phenazine pigment. Nitrate reduction is variable. Utilization of L-arabinose is positive. Utilization of 5-ketogluconate is positive or weakly positive. Phylogeny based on 16S rRNA, atpD, recA and carA gene sequences separates this subspecies from the other subspecies of P. chlororaphis.
The type strain is DSM 6698T (=ATCC 13985T=NCIMB 9030T).
Description of Pseudomonas chlororaphis subsp. aurantiaca subsp. nov., comb. nov.
Pseudomonas chlororaphis subsp. aurantiaca (au.ran.ti.a'ca. N.L. fem. adj. aurantiaca orange-coloured).
Displays characteristics typical for the species P. chlororaphis as described above. Green and orange pigments are produced. Nitrate reduction is negative. Utilization of L-arabinose is positive. Utilization of 5-ketogluconate is negative. Phylogeny based on 16S rRNA, atpD, recA and carA gene sequences separates this subspecies from the other subspecies of P. chlororaphis.
The type strain is NCIMB 10068T (=ATCC 33663T=CIP 106718T).
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Anzai, Y., Kim, H., Park, J. Y., Wakabayashi, H. & Oyaizu, H. (2000). Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence. Int J Syst Evol Microbiol 50, 1563–1589.[Abstract]
Doudoroff, M. & Palleroni, N. (1974). Gram negative aerobic rods and cocci. Genus I. Pseudomonas Migula 1894, 23. In Bergey's Manual of Determinative Bacteriology, 8th edition, edited by R. E. Buchanan & N. E. Gibbons, pp. 217–249. Baltimore: Williams & Wilkins.
Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.
Hilario, E., Buckley, T. R. & Young, J. M. (2004). Improved resolution on the phylogenetic relationships among Pseudomonas by the combined analysis of atpD, carA, recA and 16S rDNA. Antonie van Leeuwenhoek 86, 51–64.[CrossRef][Medline]
Johnson, J. L. & Palleroni, N. (1989). Deoxyribonucleic acid similarities among Pseudomonas species. Int J Syst Bacteriol 39, 230–235.
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]
Kiprianova, E. A., Levanova, G. F., Novova, E. V., Smirnov, V. V., Garagulya, A. D. & Boiko, O. I. (1985). Taxonomic study of Pseudomonas aurantiaca Nakhimovskaya, 1948 and the proposal of a neotype strain of this species. Mikrobiologiia 54, 434–440 (in Russian).[Medline]
Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2001). Molecular Evolutionary Genetics Analysis software. Arizona State University. Tempe, AZ, USA.
Palleroni, N. J. (1984). Family I. Pseudomonadaceae Winslow, Broadhurst, Buchanan, Krumwiede, Rogers and Smith 1917, 555. In Bergey's Manual of Systematic Bacteriology, 1st edn, pp. 141–218. Edited by P. H. A. Sneath, N. S. Mair, M. E. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins.
Palleroni, N. J. (2005). Genus I. Pseudomonas. In Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 2, pp. 323–379. Edited by D. J. Brenner, N. R. Krieg, J. T. Staley & G. M. Garrity. East Lansing: Springer.
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., Willems, A., Subba-Rao, N. S., Mateos, P. F., Dazzo, F. B., Martínez-Molina, E., Gillis, M. & Velázquez, E. (2003). Description of Devosia neptuniae sp. nov. that nodulates and fixes nitrogen in symbiosis with Neptunia natans, an aquatic legume from India. Syst Appl Microbiol 26, 47–53.[CrossRef][Medline]
Saitou, N. & Nei, M. (1987). A neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 44, 406–425.
Skerman, V. B. D., McGowan, V. & Sneath, P. H. A. (editors) (1980). Approved lists of bacterial names. Int J Syst Bacteriol 30, 225–420.
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.
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. & other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
Willems, A., Doignon-Bourcier, F., Goris, J., Coopman, R., de Lajudie, P., De Vos, P. & Gillis, M. (2001). DNA-DNA hybridization study of Bradyrhizobium strains. Int J Syst Evol Microbiol 51, 1315–1322.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  E. P. Ivanova, R. Christen, C. Bizet, D. Clermont, L. Motreff, C. Bouchier, N. V. Zhukova, R. J. Crawford, and E. A. Kiprianova Pseudomonas brassicacearum subsp. neoaurantiaca subsp. nov., orange-pigmented bacteria isolated from soil and the rhizosphere of agricultural plants Int J Syst Evol Microbiol, October 1, 2009; 59(10): 2476 - 2481. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Behrendt, P. Schumann, J.-M. Meyer, and A. Ulrich Pseudomonas cedrina subsp. fulgida subsp. nov., a fluorescent bacterium isolated from the phyllosphere of grasses; emended description of Pseudomonas cedrina and description of Pseudomonas cedrina subsp. cedrina subsp. nov. Int J Syst Evol Microbiol, June 1, 2009; 59(6): 1331 - 1335. [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 | |
