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1 Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany
2 Institut für Bakteriologie, Mykologie und Hygiene, Veterinärmedizinische Universität Wien, Veterinärplatz 1, A-1210 Wien, Austria
3 Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 26-32 (IFZ), D-35392 Giessen, Germany
Correspondence
Hans-Jürgen Busse
Hans-Juergen.Busse{at}vu-wien.ac.at
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ABSTRACT |
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7c, summed feature 3 and C16 : 0 as quantitatively the most important constituents and the 16S rRNA gene sequence demonstrated that Pseudomonas sp. B13T indeed belongs to the genus Pseudomonas. The sequence of the Pseudomonas sp. B13T 16S rRNA gene demonstrated a high degree of similarity with that of Pseudomonas citronellolis DSM 50332T (98.9 %), Pseudomonas nitroreducens DSM 14399T (98.7 %), Pseudomonas jinjuensis DSM 16612T (98.1 %) and Pseudomonas multiresinivorans DSM 17553T (98.7 %). Thus it was shown that strain Pseudomonas sp. B13T can be distinguished from related species by the ability/inability to assimilate N-acetylgalactosamine, D-galactose, putrescine, trans-aconitate and mesaconate and some differences in the fatty acid profile. The positioning of Pseudomonas sp. B13T as a separate taxon was finally verified by DNA hybridization, which demonstrated less than 45 % DNA–DNA similarity between strain Pseudomonas sp. B13T and the reference strains. On the basis of these results, Pseudomonas sp. B13T represents a novel species for which the name Pseudomonas knackmussii sp. nov. is proposed. The type strain is B13T (=DSM 6978T=LMG 23759T).
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). The isolate was subsequently used to elaborate the degradative pathway of 3-chlorobenzoate and other halogenated aromatic compounds (e.g. Dorn & Knackmuss, 1978a, b; Reineke & Knackmuss, 1978a, b; Schmidt & Knackmuss, 1980a, b; Kaschabek & Reineke, 1992). Furthermore, Pseudomonas sp. B13T was used for the first experiments that demonstrated the feasibility of in vivo and in vitro approaches for the construction of organisms with new metabolic capabilities (Reineke & Knackmuss, 1979, 1980; Rubio et al., 1986; Ramos et al., 1986; Rojo et al., 1987). Very recently it was demonstrated that the ability to degrade 3- and 4-chlorocatechol is encoded on a self-transferable DNA element in Pseudomonas sp. B13T (Gaillard et al., 2006). Affilation of Pseudomonas sp. B13T to the genus Pseudomonas sensu stricto has already been demonstrated by Busse et al. (1989) based on analysis of the quinone system and polyamine pattern.
Cells of Pseudomonas sp. B13T are Gram-negative, polarly flagellated, motile short rods. The oxidase and catalase reactions were positive. Based on these results and several physiological tests which were performed in accordance with the study of Stanier et al. (1966) on the aerobic pseudomonads (see strain description), it was suggested that the strain belongs to the pseudomonads and was most closely related to Pseudomonas fluorescens, although no fluorescent pigments were observed during growth (Dorn et al., 1974).
The 16S rRNA gene sequence of Pseudomonas sp. B13T containing a continuous stretch of 1528 bp has been deposited at the NCBI database under the accession number AF039489 (Wischnak et al., 1998). Sequence searches at the NCBI database demonstrated that Pseudomonas sp. B13T indeed belongs to the genus Pseudomonas as it is currently defined and the closest relatives of Pseudomonas sp. B13T were found to be Pseudomonas citronellolis DSM 50332T, Pseudomonas nitroreducens DSM 14399T, Pseudomonas jinjuensis DSM 16612T and Pseudomonas multiresinivorans DSM 17553T with 98.1–98.9 % sequence similarity. No other type strain of the genus showed sequence similarity values >97 % to the 16S rRNA gene sequence from Pseudomonas sp. B13T. This clearly demonstrated that Pseudomonas sp. B13T belongs in the respective subcluster of pseudomonads which is part of the Pseudomonas aeruginosa subgroup as defined by 16S rRNA gene sequence comparisons (Anzai et al., 2000). Since results from DNA–DNA hybridizations have demonstrated that P. multiresinivorans is a junior homonym of P. nitroreducens (E. Lang, unpublished), P. multiresinivorans DSM 17553T was not included in all comparative analyses.
Analysis of the carbon substrate utilization for growth of Pseudomonas sp. B13T, P. citronellolis DSM 50332T, P. nitroreducens DSM 14399T, P. jinjuensis DSM 16612T and P. multiresinivorans DSM 17553T demonstrated that all five strains converted a wide range of organic compounds and clearly resembled each other in their metabolic traits. Nevertheless, the five strains could be distinguished by their ability/inability to assimilate N-acetylgalactosamine, D-galactose, putrescine, trans-aconitate and mesaconate (Table 1).
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). Re-examination of the quinone system by HPLC as described previously (Ventosa et al., 1993; Altenburger et al., 1996) but using an HPLC system consisting of a JASCO PU 2080 Plus Pump and JASCO UV-2075 Plus UV/VIS detector, and the polyamine pattern as described by Busse & Auling (1988) and Busse et al. (1997) but employing a JASCO PU 2080 Plus pump, confirmed previous results. The quinone system of Pseudomonas sp. B13T consisted of the major compound Q-9 (94 %) and minor amounts of Q-8. A similar quinone system was detected for P. citronellolis DSM 50332T, P. nitroreducens DSM 14399T and P. jinjuensis DSM 16612T, exhibiting 98, 97 and 98 % Q-9, respectively, and minor amounts of Q-8. The polyamine pattern of Pseudomonas sp. B13T consisted of major amounts of putrescine [94.2 µmol (g dry weight)–1], spermidine [13.5 µmol (g dry weight)–1] and cadaverine [8.8 µmol (g dry weight)–1]. Furthermore, small amounts of 1,3-diaminopropane [1.2 µmol (g dry weight)–1] and spermine [0.3 µmol (g dry weight)–1] were also detected. The reference strains exhibited similar polyamine patterns showing only some remarkable differences in the content of cadaverine. The patterns were as follows [amounts in µmol (g dry weight)–1]: P. citronellolis DSM 50332T: putrescine 95.7, spermidine 12.8, cadaverine 0.8, 1,3-diaminopropane 1.0, spermine 0.9; P. nitroreducens DSM 14399T: putrescine 84.8, spermidine 14.4, cadaverine 10.1, 1,3-diaminopropane 7.9, spermine 1.3; and P. jinjuensis DSM 16612T: putrescine 100.5, spermidine 18.5; cadaverine 1.2, 1,3-diaminopropane 0.6, spermine 3.2. The quinone system and polyamine patterns are in excellent agreement with those reported for other species of the genus Pseudomonas sensu stricto (Busse & Auling, 1988; Auling et al., 1991).
Polar lipids were analysed as described by Tindall (1990). Pseudomonas sp. B13T (Fig. 1) and the reference strains P. citronellolis DSM 50332T, P. nitroreducens DSM 14399T and P. jinjuensis DSM 16612T (results not shown) exhibited almost identical profiles. Differences in the presence of trace amounts of some unknown lipids may occur but could not be unambiguously identified. The polar lipid profile of Pseudomonas sp. B13T consisted of the major compounds phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol, moderate amounts of four unknown phospholipids (PL2–5), an unknown aminolipid (APL1) and two unknown polar lipids (L2, 3) and minor to trace amounts of one phospholipid (PL1) and five unknown polar lipids (L1, 4–7). Two highly hydrophobic lipids were also detected (Fig. 1). These polar lipid profiles contain the same major compounds reported for two other species of the genus, Pseudomonas psychrotolerans DSM 15758T and Pseudomonas oleovorans DSM 1045T, and the misnamed strain Pseudomonas oleovorans ATCC 29347, but they lack phosphatidylcholine, detected in major to moderate amounts in P. psychrotolerans DSM 15758T and P. oleovorans DSM 1045T (Hauser et al., 2004). Also differences in the presence/absence of minor compounds characteristic for representatives of one or the other group were found. These observations indicate that polar lipid profiles may be useful for characterizing subgroups within the genus Pseudomonas sensu stricto and hence their analysis for classification of novel species can be strongly recommended.
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. All four strains had rather similar fatty acid profiles, with C18 : 17c (29.8–41.8 %), summed feature 3 (16.5–24.8 %) and C16 : 0 (19.4–21.2 %) as quantitatively the most important constituents (Table 2).
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. The similarity values (mean values of two individual analyses) determined between the genomic DNA of Pseudomonas sp. B13T and that of the reference strains were as follows: P. citronellolis DSM 50332T, 44.5 %; P. nitroreducens DSM 14399T, 35.2 %; P. jinjuensis DSM 16612T, 38.7 %; and P. multiresinivorans DSM 17553T, 38.0 %.
Organisms sharing a 16S rRNA gene sequence similarity lower than 97 % are usually regarded as belonging to different bacterial species (Stackebrandt & Goebel, 1994). In the genus Pseudomonas, several recently reported Pseudomonas species have shown 16S rRNA gene sequence similarities of more than 99 % to other established species (Achouak et al., 2000; Andersen et al., 2000; Hauser et al., 2004; Kwon et al., 2003; Sikorski et al., 2001). This situation also seems to be relevant for the subgroup of pseudomonads analysed in the present study. Thus from the DNA–DNA hybridization studies and the physiological tests it is evident that Pseudomonas sp. B13T represents a distinct, previously undescribed species within the genus Pseudomonas. In conclusion, Pseudomonas sp. B13T is phylogenetically and physiologically unique and represents a novel species within the genus Pseudomonas, for which we propose the name Pseudomonas knackmussii sp. nov.
Description of Pseudomonas knackmussii sp. nov.
Pseudomonas knackmussii (knack.muss'i.i. N.L. masc. gen. n. knackmussii of Knackmuss, in honour of Hans-Joachim Knackmuss, who initiated the biochemical studies about the degradation of chlorinated aromatics and other xenobiotic compounds by strain B13T and various other micro-organisms).
Cells are Gram-negative, polarly flagellated, motile short rods. Oxidase- and catalase-positive. On solid 3-chlorobenzoate-mineral agar the strain forms round, smooth, colourless, opaque colonies of 1–2 mm diameter when grown at 28 °C for 3 days. Growth is observed at 20–41 °C but not at 4 °C. Good growth is found at pH 6.0 and 8.3; there is no growth at pH 5 or pH 9. No fluorescent or phenazine pigments are formed. Nitrate is not reduced under aerobic or anaerobic conditions. No liquefaction of gelatin, degradation of starch or cellulose, or formation of ethanol from acetate is detected. The strain is positive for: acid formation from D-glucose; utilization of D-gluconate, D-glucose, D-fructose, cis-aconitate, adipate, 4-aminobutyrate, azelate, citrate, fumarate, glutarate, DL-3-hydroxybutyrate, itaconate, DL-lactate, L-malate, pyruvate, 2-oxoglutarate, suberate, L-alanine, 
-alanine, L-aspartate, L-histidine, L-leucine, L-ornithine, L-proline, 4-hydroxybenzoate, phenylacetate and putrescine; hydrolysis of bis-pNP phosphate, pNP phosphorylcholine, L-alanine pNA, L-glutamate-3-carboxy pna and L-proline pNA (pNP, para-nitrophenyl; pNA, para-nitroanilide). Acids are not produced from sucrose, D-mannitol, dulcitol, salicin, adonitol, inositol, D-sorbitol, L-arabinose, raffinose, L-rhamnose, maltose, D-xylose, trehalose, cellobiose, methyl 
-D-glucoside, erythritol, melibiose, D-arabitol, D-mannose, N-acetylgalactosamine, D-galactose, trans-aconitate or mesaconate. The following glycosides are not hydrolysed: pNP -D-galactopyranoside, pNP -D-glucuronide, pNP -D-glucopyranoside, pNP -D-glucopyranoside and pnp -D-xylopyranoside. N-Acetyl-D-glucosamine, L-arabinose, p-arbutin, D-cellobiose, D-trehalose, D-xylose, adonitol, inositol, maltitol, D-mannitol, sorbitol, acetate, propionate and L-tryptophan are not utilized. The quinone system consists predominantly of ubiquinone Q-9. Putrescine and spermidine are the major polyamines. The polar lipid profile consists of the major compounds phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol, moderate amounts of four unknown phospholipids, an unknown aminolipid and two unknown polar lipids, and minor to trace amounts of one phospholipid and five unknown polar lipids. Two highly hydrophobic lipids are also detectable. Fatty acid composition is as follows: C18 : 17c (36.6 %), summed feature 3 (24.8 %) and C16 : 0 (19.4 %), C12 : 0 3-OH (4.0 %), C12 : 0 (3.7 %), C12 : 0 2-OH (3.5 %), C10 : 0 3-OH (3.5 %) and C17 : 0 cyclo (2.7 %).
The type strain is B13T (=DSM 6978T=LMG 23759T), isolated from a sewage treatment plant in Göttingen (Germany) after continuous enrichment with 3-chlorobenzoate.
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ACKNOWLEDGEMENTS |
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P. Kampfer, A. Sessitsch, M. Schloter, B. Huber, H.-J. Busse, and H. C. Scholz Ochrobactrum rhizosphaerae sp. nov. and Ochrobactrum thiophenivorans sp. nov., isolated from the environment Int J Syst Evol Microbiol, June 1, 2008; 58(6): 1426 - 1431. [Abstract] [Full Text] [PDF]
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G. Zhang, F. Niu, H.-J. Busse, X. Ma, W. Liu, M. Dong, H. Feng, L. An, and G. Cheng Hymenobacter psychrotolerans sp. nov., isolated from the Qinghai--Tibet Plateau permafrost region Int J Syst Evol Microbiol, May 1, 2008; 58(5): 1215 - 1220. [Abstract] [Full Text] [PDF]
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N. R. Kumar, S. Nair, S. Langer, H.-J. Busse, and P. Kampfer Altererythrobacter indicus sp. nov., isolated from wild rice (Porteresia coarctata Tateoka) Int J Syst Evol Microbiol, April 1, 2008; 58(4): 839 - 844. [Abstract] [Full Text] [PDF]
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L. D. Sprague, H. C. Scholz, S. Amann, H.-J. Busse, and H. Neubauer Yersinia similis sp. nov. Int J Syst Evol Microbiol, April 1, 2008; 58(4): 952 - 958. [Abstract] [Full Text] [PDF]
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H. C. Scholz, Z. Hubalek, I. Sedlacek, G. Vergnaud, H. Tomaso, S. Al Dahouk, F. Melzer, P. Kampfer, H. Neubauer, A. Cloeckaert, et al. Brucella microti sp. nov., isolated from the common vole Microtus arvalis Int J Syst Evol Microbiol, February 1, 2008; 58(2): 375 - 382. [Abstract] [Full Text] [PDF]
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P. Kampfer, E. Falsen, and H.-J. Busse Reclassification of Pseudomonas mephitica Claydon and Hammer 1939 as a later heterotypic synonym of Janthinobacterium lividum (Eisenberg 1891) De Ley et al. 1978 Int J Syst Evol Microbiol, January 1, 2008; 58(1): 136 - 138. [Abstract] [Full Text] [PDF]
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C. C. Young, M.-J. Ho, A. B. Arun, W.-M. Chen, W.-A. Lai, F.-T. Shen, P. D. Rekha, and A. F. Yassin Sphingobium olei sp. nov., isolated from oil-contaminated soil Int J Syst Evol Microbiol, November 1, 2007; 57(11): 2613 - 2617. [Abstract] [Full Text] [PDF]
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P. Kampfer, U. Meurer, M. Esser, T. Hirsch, and H.-J. Busse Sphingomonas pseudosanguinis sp. nov., isolated from the water reservoir of an air humidifier Int J Syst Evol Microbiol, June 1, 2007; 57(6): 1342 - 1345. [Abstract] [Full Text] [PDF]
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E. Lang, B. Griese, C. Sproer, P. Schumann, M. Steffen, and S. Verbarg Characterization of 'Pseudomonas azelaica' DSM 9128, leading to emended descriptions of Pseudomonas citronellolis Seubert 1960 (Approved Lists 1980) and Pseudomonas nitroreducens Iizuka and Komagata 1964 (Approved Lists 1980), including Pseudomonas multiresinivorans as its later heterotypic synonym Int J Syst Evol Microbiol, April 1, 2007; 57(4): 878 - 882. [Abstract] [Full Text] [PDF]
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