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1 Fachbereich Biologie der Universität, D-78457 Konstanz, Germany
2 DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, D-38124 Braunschweig, Germany
3 CSICUIBInstitut Mediterrani d'Estudis Avançats, E-07190 Esporles, Mallorca, Spain
Correspondence
Alasdair M. Cook
alasdair.cook{at}uni-konstanz.de
| ABSTRACT |
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-oxygenate the commercial surfactant linear alkylbenzenesulfonate (LAS) and shorten the side chain by
-oxidation to yield sulfophenylcarboxylates. The morphotype is widespread in cultures able to utilize LAS, and a second organism with similar characteristics, strain AN-8, is now available. Utilization of LAS is concomitant with formation of a biofilm, and cells were non-motile. Many surfactants were utilized. The organisms also grew with acetate or octane, but required no biofilm and were motile. Analysis of the gene encoding 16S rRNA placed the organisms in the
-subclass of the Proteobacteria with a sequence divergence of >8 % from any species whose name has been validly published. 16S rRNA gene sequence comparison with entries in the GenBank database showed 98 % similarity to an
-protobacterial marine isolate, JP57: strain JP57 displayed the same morphotype as strain DS-1T, but it was unable to utilize surfactants or any single source of carbon tested. The lipid components of strains DS-1T and JP57 were virtually identical. The fatty acids contained ester- and putative amide-linked hydroxy fatty acids, in a combination that is currently unique in the
-Proteobacteria. The major respiratory quinone present in both strains was Q11. The polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and two unidentified aminolipids. Data on the 16S rRNA gene sequence and the lipid composition indicated that strains DS-1T and JP57 should be placed in a new genus, for which the name Parvibaculum is proposed. The differences between these strains, supported by DNA hybridizations, lead to the conclusion that strain DS-1T (=DSM 13023T=NCIMB 13966T) is the type strain of a species in the genus Parvibaculum, for which the name Parvibaculum lavamentivorans gen. nov., sp. nov. is proposed.
Published online ahead of print on 20 February 2004 as DOI 10.1099/ijs.0.03020-0.
The GenBank accession number for the 16S rRNA gene sequence of strain DS-1T is AY387398.
| INTRODUCTION |
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-oxygenation of the LAS side chain and about three rounds of
-oxidation (Fig. 1
,
-unsaturated sulfophenylcarboxylates, is formed from commercial LAS, which nominally comprises 20 compounds (Dong et al., 2004
,
-unsaturated sulfophenylcarboxylates (Dong et al., 2004
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-subclass of the Proteobacteria and was suspected to represent a novel genus (Schleheck et al., 2000
-proteobacterium strain JP57 (Eilers et al., 2001
-Proteobacteria, from freshwater and marine enrichment cultures that degrade hydrocarbons, also have similar rRNA genes (9597 % identity to strain JP57) (Chang et al., 2000| METHODS |
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Morphology, physiology and biofilm staining.
Culture purity, cell morphology, motility and spore formation were examined microscopically. The Gram reaction was assayed using the KOH test (Gregersen, 1978
). Oxidase and catalase tests were carried out using standard methods (Gerhardt et al., 1994
). Strain DS-1T grew in a biofilm on the polyester fleece when utilizing LAS. The biofilm on a section of fleece from 0·5 mM LAS-salts medium was visualized in situ by staining with ruthenium red (2·5 µg ml1), whereas the cells were stained with 4',6-diamidino-2-phenylindole (1 µg ml1).
Analytical methods.
Respiratory lipoquinones and polar lipids were extracted from freeze-dried cell material (100 mg) using a two-stage method (Tindall, 1990a
, b
). The lipoquinones were separated by TLC, and UV-absorbing bands corresponding to menaquinones or ubiquinones were removed from the plate and further analysed by reversed-phase HPLC. Polar lipids were separated by two-dimensional, silica-gel TLC; total lipid material and specific functional groups were detected using dodecamolybdophosphoric acid (total lipids), Zinzadze reagent (phosphate), ninhydrin (free amino groups), periodateSchiff (
-glycols), Dragendorff (quaternary nitrogen) and anisaldehyde-sulfuric acid (glycolipids) as described previously (Tindall, 1990a
, b
). Fatty acids were analysed as the methyl ester derivatives prepared from 10 mg dry cell material. Cells were subjected to differential hydrolysis to detect ester-linked and non-ester-linked (amide-bound) fatty acids (Labrenz et al., 1998
). Fatty acid methyl esters were analysed by GC using a 0·2 µmx25 m non-polar capillary column and flame-ionization detection. The run conditions were as follows: injection and detector port temperature, 300 °C; inlet pressure, 60 kPa; split ratio, 50 : 1; injection volume, 1 µl; temperature program, 130310 °C at a rate of 4 °C min1. The identity of each fatty acid was confirmed by GC-MS, using conditions described in Strömpl et al. (1999)
. Hydroxy fatty acids were confirmed by either the presence of a major peak at m/z 103 (3-OH fatty acids) or the loss of 59 mass units from the mole peak (i.e. m/z M+59, for 2-OH fatty acids). The position of double bonds was confirmed using dimethysulfide adducts (Nichols et al., 1986
).
LAS and sulfophenylcarboxylates were determined by HPLC (Schleheck et al., 2000
). Sulfate was quantified turbidimetrically (Sörbo, 1987
). Protein solubilized from whole cells was quantified in a Lowry-type reaction (Kennedy & Fewson, 1968
).
Extraction of genomic DNA, PCR-mediated amplification of the 16S rRNA gene, purification of the PCR products and sequence analysis were done as described elsewhere (Rainey et al., 1996
). Sequence reactions were analysed using the Applied Biosystems 373A DNA sequencer. The sequence was aligned manually to 16S rRNA gene sequences of representative micro-organisms belonging to the domain Bacteria by using the alignment editor ae2 (Maidak et al., 1996
). A phylogenetic tree was generated using the algorithm of De Soete (1983)
(carried out by the DSMZ). DNADNA hybridizations and DNA G+C content were determined as described previously (Ziemke et al., 1998
).
| RESULTS AND DISCUSSION |
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In contrast to the generality of needing a solid support for growth with surfactants, strain DS-1T utilized some alkanes, alkanoates, alcohols and short-chain acids (e.g. acetate), without the requirement for a solid support (Table 1
). Under these latter conditions, the organism grew in suspension (Fig. 3a
) and was motile. Strain DS-1T grew slowly in complex medium without a solid support, e.g. in peptone-salts medium (5 days): the organism was motile and was found as single cells or in short chains (two to five organisms). The sugars tested were not utilized (Table 1
).
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-oxidation was hindered by a (polar) substituent, as indicated in Fig. 1
-oxidation for growth. In contrast, the growth yield with dodecyl sulfate indicated complete utilization and, correspondingly, sulfate was recovered at high yields. Growth of strain AN-8 with LAS also showed an absolute requirement for a solid support, concomitant with formation of a biofilm and the presence of non-motile cells. Growth with e.g. acetate or octane involved motile cells; no solid support was necessary and no biofilm was formed. Growth in peptone-salts medium involved single cells and short chains. Sugars did not support growth. The cell morphology was indistinguishable from that of strain DS-1T.
Marine isolate strain JP57 grew on synthetic-sea-water agar plates without an additional carbon source in pinpoint colonies, as described in the original publication (Eilers et al., 2001
). The organism grew in peptone medium after 5 days incubation and was morphologically indistinguishable from strains DS-1T and AN-8. Strain JP57 failed to grow with LAS, sodium dodecyl sulfate, methylestersulfonate, octane, dodecane, hexadecane, hexadecanol, hexadecanoate, acetate, succinate, pyruvate, ethanol or sugars as carbon source, either when supplied in minimal-salts medium or when supplied in supplemented artificial sea-salts medium. We found no single source of carbon for the organism. Neither the addition of solid support nor a vitamin supplement had any effect.
Formation of biofilm
The first visible indication of growth of strain DS-1T in LAS-salts medium was the formation of biofilm on the solid support; later during growth, bacteria were also found in suspension. Two forms of biofilm could be observed on polyester fibres (Fig. 3b, c
). The first form was a densely packed sheath of cells around a fibre (Fig. 3b
). Fig. 3(b)
was generated with ruthenium red as the stain, but 4',6-diamidino-2-phenylindole, methylene blue or Congo red gave similar results (not shown). The second one was a gossamer form (Fig. 3c
), usually found linking two or more fibres. Each form could be removed from the support by, for example, vortexing and was of a soft consistency. The gossamer form only was observed on glass fibres (not shown) and negligible amounts of biofilm (gossamer) were observed on glass particles, e.g. in the sediment from the culture fluid: the bacteria grew predominantly in suspension and were non-motile. We assume that the biofilm observed on glass particles was continuously disrupted during incubation on a roller.
Growth in acetate-salts medium involved motile cells in suspension (see above). When LAS was added to an acetate-salts culture, a solid support was needed for growth; a biofilm was formed and non-motile cells were observed in the presence of LAS.
Some growth substrates were precipitated in the medium. In the case of methylestersulfonate surfactant (Table 1
), a biofilm could be detected on the particles by using staining (ruthenium red, not shown) and only non-motile cells were observed in the medium. In contrast, growth with the insoluble substrates hexadecanoate or hexadecanol did not involve a biofilm (ruthenium red) and cells in the culture were motile. We presume that the formation of biofilm with strains DS-1T and AN-8, and the switch from motile to non-motile, are protective responses to the risks inherent in growing with membrane-solubilizing agents.
16S rDNA sequence analysis, chemotaxonomy and DNADNA hybridization
Published data on the sequences of 16S rRNA genes place strains DS-1T (1451 nt) and JP57 (1356 nt), which share 98 % similarity, in the
-Proteobacteria, where the nearest, well-described organism is Rhodobium (formerly Rhodobacter) marinum, which shares only 92 % similarity (Eilers et al., 2001
; Schleheck et al., 2000
). The morphological similarities of these two organisms support the implication from the sequence that they are closely related, but differences (e.g. substrate range) are also apparent. A 400 bp sequence of the 16S rRNA gene from strain AN-8 was identical to the corresponding sequence of strain DS-1T; this, together with the essentially identical morphology and physiology, supports the hypothesis that they are members of one species.
Examination of the respiratory lipoquinone composition of both strain JP57 and strain DS-1T showed that ubiquinones are the sole respiratory quinones present. Furthermore, the major lipoquinone had 11 isoprenologues in the side chain, i.e. Q11.
The fatty acids comprised both saturated and unsaturated straight-chain fatty acids, as well as hydroxylated fatty acids, strains DS-1T and JP57 showing only quantitative differences (Table 2
). Differential hydrolysis of the cells indicated that some of the hydroxy fatty acids were probably amide-linked (Table 2
). The polar lipids comprised the phospholipids phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine (Fig. 4
). In addition, two aminolipids were present which were detected between phosphatidyl glycerol and phosphatidyl choline (Fig. 4
). There were quantitative differences in the ratio of these two lipids, with one predominating in strain JP57. This causes the lower lipid to be partially obscured by the upper lipid, but this was simply a crowding effect on the TLC plate.
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The use of a combination of the 16S rRNA sequence data and the lipid composition provides a convenient way of quickly determining the differences from, and/or similarities to, other organisms. The presence of ubiquinones as the sole respiratory quinones is indicative of the fact that strains DS-1T and JP57 are members of the
-,
- or
-subclass of the Proteobacteria. The presence of Q11 is distinctive in that the presence of this compound as the sole major quinone has previously been found only in members of the genus Hyphomonas (Sittig & Hirsch, 1992
; Urakami & Komagata, 1987
), in the
-subclass of the Proteobacteria. Q11 is also known to occur in members of the genus Legionella (a member of the
-subclass of the Proteobacteria), but it is usually accompanied by additional isoprenologues (see Collins & Gilbart, 1983
). The fatty acid patterns of the two organisms, showing large amounts of 18 : 1
7c (Table 2
), are not atypical of members of the
-subclass of the Proteobacteria. However, the distribution of the hydroxy fatty acids does appear to be a unique feature of these two organisms. In particular, the fact that some of the hydroxy fatty acids are ester-linked, while others are presumably amide-linked, allows further differentiation of these two strains from other members of the
-subclass of the Proteobacteria. The presence of the phospholipids phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine is also characteristic, together with the respiratory lipoquinone and fatty acid data, of certain subgroups within the
-subclass of the Proteobacteria. However, the presence of the two additional aminolipids appears to be distinctive for these two organisms.
DNADNA hybridization with strains DS-1T and JP57 showed 42 % binding, which is similar to the value obtained against a strain of Pseudomonas putida (35 %), whereas the positive control was 100 %. We thus conclude that strains DS-1T and JP57 represent different strains of the same higher taxon within the
-subclass of the Proteobacteria. In the present work, we have concentrated on the taxonomic position of strain DS-1T; a taxonomic treatment of strain JP57 may appear at a later date.
Description of Parvibaculum lavamentivorans gen. nov., sp. nov.
Parvibaculum lavamentivorans [Par.vi.ba'cu.lum. L. adj. parvus small; L. neut. n. baculum stick; N.L. neut. n. Parvibaculum small stick. la.va.men.ti.vo'rans. L. v. lavo to wash, L. neut. n. suffix -mentum agent of (specified) action; L. v. voro to consume; N.L. neut. part. adj. lavamentivorans consuming (chemicals) used for washing].
Cells are aerobic, uniform, Gram-negative, short rods (0·8 µm in length and 0·2 µm in diameter). Oxidase- and catalase-positive. Mesophilic. Motile when growing with acetate, octane or in complex medium. Q11 is the major respiratory quinone. Straight-chain saturated and unsaturated, as well as ester- and (presumptively) amide-linked hydroxy-fatty acids are present in membrane fractions (see Table 2
). The major polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and two unidentified aminolipids. 16S rRNA gene sequence analysis indicates that this taxon is a member of the
-subclass of the Proteobacteria, with >8 % sequence divergence with respect to any other species, within this subclass, whose name has been validly published. The G+C content is 64 mol%. Strain DS-1T grows with LAS and other surfactants via
-oxygenation and
-oxidation of the alkyl chain to produce short-chain sulfophenylcarboxylates. Growth in surfactant medium involves biofilm formation on solid support (polyester fleece, glass particles) and non-motile cells.
The type species of the genus is Parvibaculum lavamentivorans. Strain DS-1T (=DSM 13023T=NCIMB 13966T) is the type strain of Parvibaculum lavamentivorans.
| ACKNOWLEDGEMENTS |
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