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1 GBF – Gesellschaft für Biotechnologische Forschung mbH, Mascheroder Weg 1, D-38124 Braunschweig, Germany
2 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg 1b, D-38124 Braunschweig, Germany
Correspondence
Hanno Biebl
hbi{at}gbf.de
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rDNA sequence of Propionispora hippei KST is AJ508927.
Details of the time-course of polymer degradation by P. hippei are available as supplementary material in IJSEM Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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described a novel genus and species of anaerobes, Propionispora vibrioides, which belongs to the Sporomusa–Pectinatus–Selenomonas group, as defined by Strömpl et al. (1999). The type species of Propionispora resembles Sporomusa in cell shape and spore formation (Möller et al., 1984), but its fermentation type is that of propionic-acid bacteria and is not acetogenic. In the course of a screening programme for the anaerobic degradation of natural and synthetic polyesters (Abou-Zeid et al., 2001; Abou-Zeid, 2001), a strain was isolated that proved to be morphologically and physiologically very similar to P. vibrioides. However, on the basis of low DNA–DNA hybridization values with P. vibrioides, and differences in 16S rRNA gene sequence, substrate utilization and spore morphology, we propose that the isolated strain be classified as a novel species within the genus Propionispora.
The isolate was obtained from an enrichment culture set up to examine the biological degradability of the aliphatic copolyester SP 3/6, a condensate of 1,3-propanediol and adipic acid, under anaerobic conditions. A mineral salts medium was used, and was prepared according to the anaerobic cultivation techniques recommended by Hungate (Abou-Zeid et al., 2001; Holdeman et al., 1977). The heat-labile polymer was prepared as a thin-film disc of 2·5 cm diameter (Witt et al., 1994), sterilized by UV radiation and introduced aseptically into cold, autoclaved medium under sterile nitrogen. Sewage sludge from the municipal treatment plant in Gifhorn (Lower Saxony, Germany) was used as inoculum.
For isolation, 0·1 ml of the culture was plated on three complex agar media (anaerobic TVLS, Brewer's anaerobic and thioglycolate medium; all from Merck). The plates were incubated anaerobically at 35 °C in an anaerobic chamber. A first attempt after 3 months of incubation failed, but, after another 15 months, colonies developed on all three media. Fifteen colonies were checked on mineral salts agar with emulsified SP 3/6 polymer (for preparation see Witt et al., 1994); nine of them formed clear zones indicating degradation of the polymer. They were purified on complex agar medium and rechecked on polymer agar. One strain, KS, attracted attention by its curved cells and conspicuous spores and was characterized further; the others were straight rods and were not investigated further.
Cells of strain KST were vibrio-shaped in exponentially growing cultures and formed slightly curved rods during the fermentation phase. Spores were readily formed in peptone-yeast extract-glucose, thioglycolate medium and mineral salts medium containing fructose. The sporangia were distinctly swollen, either dark or refractive, and appeared in terminal position. Vegetative cells could not be discriminated from P. vibrioides (Biebl et al., 2000), while spore-forming cells were somewhat thicker (Fig. 1).
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. Growth was monitored by measuring the optical density of the entire tube at 600 nm. Strain KST behaved as a mesophilic organism, with good growth between 20 and 50 °C, and an optimum at 37 °C. A maximum growth rate of 0·55 h–1 was measured. The pH range for growth was between 5·0 and 8·5, with an optimum at 6·8. Yeast extract was required at a minimum of 0·5 g l–1.
Only a limited number of carbon and energy sources was utilized by strain KST, mainly sugars and sugar alcohols, including glycerol (see species description). 1,3-Propanediol and adipate, the depolymerization products of the polyester SP 3/6, did not support growth, nor were the products of other polymers decomposed by the strain (see below): 1,4-butanediol, caproate and terephthalate. The fermentation products were determined by GC (Biebl et al., 2000). It was shown that all utilized substrates were fermented to propionic and acetic acid, CO2 and H2. The fermentation balances, as determined in tightly sealed Hungate tubes, are shown in Table 1.
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-caprolactone). The copolymer of 1,4-butanediol, terephthalic acid and adipic acid (BTA), which exhibits better material properties than the aliphatic polyesters, was weakly degraded, as long as the aromatic component did not exceed 20 % of the acidic components. Natural polyesters, such as polyhydroxybutyrate (PHB) and poly(hydroyxybutyrate-hydroxyvalerate) (PHBS), were not attacked. The course of depolymerization of these compounds, shown as the increase in the clear zone diameter in agar, can be viewed as Supplementary Fig. A in IJSEM Online. Although the ability to depolymerize synthetic polyesters may be significant for their degradation in nature, it appears to be without significance for the metabolism of the organism, as the products are not further decomposed. The depolymerizing enzyme seems to be a non-specific enzyme, probably a lipase, which affects the ester bonds of the synthetic polymer.
Genomic-DNA extraction, PCR-mediated amplification of the 16S rRNA gene and sequencing of PCR products was carried out as described by Rainey et al. (1996). Purified PCR products were sequenced directly using the Taq DyeDeoxy Terminator Cycle Sequencing kit (Applied Biosystems), according to the manufacturer's instructions. An Applied Biosystems 373A DNA genetic analyser was used for electrophoresis of the sequence reaction products. Although nearly complete sequence determination was achieved by this method, part of the 5' region of the gene, corresponding to helix 6 (De Rijk et al., 1992), could not be resolved. In accordance with the characterization of P. vibrioides (Biebl et al., 2000), the 16S rRNA gene PCR product had to be cloned to resolve the sequence in this region. Insertions of about 119 nt were found in several individual clones (data not shown). For alignment and subsequent analysis, sequences from clones carrying no insertion were used.
Using the ae2 editor (Maidak et al., 1999), the almost complete 16S rRNA gene sequence of one clone of strain KST was aligned manually with those of all currently available nucleotide sequences of the Sporomusa–Selenomonas–Pectinatus group retrieved from GenBank and EMBL. The method of Jukes & Cantor (1969) was used to calculate evolutionary distances. Phylogenetic dendrograms were reconstructed according to the method of De Soete (1983) and the neighbour-joining and maximum-likelihood methods contained in the PHYLIP package (Felsenstein, 1993). The highest binary similarity value was found to the sequence of P. vibrioides (97·9 %). Lower values, ranging between 92·2 and 89·8 %, were found for the type strains of Sporomusa species. Different treeing algorithms (De Soete, 1983; Felsenstein, 1993) placed the novel strain on a separate line of descent together with P. vibrioides (Fig. 2).
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With the isolation of strain KST, a second representative of the genus Propionispora (Biebl et al., 2000) was found. This genus possesses properties that also occur in other related genera belonging to the Selenomonas–Sporomusa–Pectinatus group (Strömpl et al., 1999), but in an unusual combination. It shares the typical cellular organization, i.e. vibrio-shaped cells with flagella inserted at the concave side, with Selenomonas and Sporomusa, and the ability to form spores with Sporomusa and Dendrosporobacter. The strain ferments propionic acid, in common with many other members of the group, including Selenomonas, but not Sporomusa, which is acetogenic (Möller et al., 1984).
Strain KST matches the type strain (DSM 13304T) of P. vibrioides in essential criteria. Cells are vibrio-shaped and form terminal, round spores. They produce propionic and acetic acid from a limited number of sugars and sugar alcohols. However, there are differences in the utilization of glucose and glycerol, which are not attacked by the P. vibriodes strain. Also, spore-forming KST cells are distinctly thicker than those of P. vibrioides. The 16S rRNA gene sequence similarity between KS and P. vibrioides of 97·9 % corresponds to values found among Sporomusa species (mean, 97 %). The DNA–DNA hybridization was far below 70 %, indicating an evolutionary distance at the species level (Johnson, 1984). We therefore propose to classify strain KST as a separate species within the genus Propionispora as Propionispora hippei sp. nov.
Description of Propionispora hippei sp. nov.
Propionispora hippei (hip'.pe.i. N.L. gen. n. hippei named after Dr Hans-H. Hippe, DSMZ, for his numerous contributions to the cultivation and taxonomy of anaerobes).
Cells appear as vibrios to slightly curved rods in phase-contrast microscopy. Spores are round and in terminal position; spore-forming cells are distinctly swollen. Growth and fermentation substrates are glucose, fructose, mannitol, xylitol, erythritol and glycerol. Propionic and acetic acids are the fermentation products. Lactose, lactate, pyruvate, 3-hydroxybutyrate, 2,3-butanediol, ethanol, methanol and H2/CO2 are not used. Yeast extract is required. The species differs from P. vibrioides in its ability to ferment glucose and glycerol. It depolymerizes the aliphatic polyesters of 1,3-propanediol or 1,4-butanediol and adipic acid, as well as poly(-caprolactone), without degrading the monomers.
The type strain is strain KST (=DSM 15287T=ATCC BAA-665T), isolated from an enrichment culture for decomposition of the aliphatic copolyester of 1,3-propanediol and adipic acid.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Abou-Zeid, D. M., Müller, R.-J. & Deckwer, W.-D. (2001). Degradation of natural and synthetic polyesters under anaerobic conditions. J Biotechnol 86, 113–126.[CrossRef][Medline]
Biebl, H., Schwab-Hanisch, H., Spröer, C. & Lünsdorf, H. (2000). Propionispora vibrioides, nov. gen., nov. sp., a new gram-negative, spore-forming anaerobe that ferments sugar alcohols. Arch Microbiol 174, 239–247.[CrossRef][Medline]
De Rijk, P., Neefs, J.-M., Van de Peer, Y. & De Wachter, R. (1992). Compilation of small ribosomal subunit RNA sequences. Nucleic Acids Res 20, 2075–2089.
De Soete, G. (1983). A least squares algorithm for fitting additive trees to proximity data. Psychometrica 48, 621–626.[CrossRef]
Felsenstein, J. (1993). PHYLIP (phylogenetic inference package), version 3.5c. Department of Genetics, University of Washington, Seattle, USA
Holdeman, L. V., Cato, E. P. & Moore, W. E. C. (1977). Anaerobe Laboratory Manual, 4th edn. Blacksburg, VA: Virginia Polytechnic Institute and State University.
Johnson, J. L. (1984). Nucleic acids in bacteria classification. In Bergey's Manual of Systematic Bacteriology, vol. 1, pp. 8–11. Edited by N. R. Krieg & J. G. Holt. Baltimore: Williams & Wilkins.
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In Mammalian Protein Metabolism, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
Maidak, B. L., Cole, J. R., Parker, C. T. & 11 other authors (1999). A new version of the RDP (Ribosomal Database Project). Nucleic Acids Res 27, 171–173.
Möller, B., Oßmer, R., Howard, B. H., Gottschalk, G. & Hippe, H. (1984). Sporomusa, a genus of gram-negative anaerobic bacteria including Sporomusa sphaeroides sp. nov., and Sporomusa ovata, sp. nov. Arch Microbiol 139, 388–396.[CrossRef]
Rainey, F. A., Ward-Rainey, N., Kroppenstedt, R. M. & Stackebrandt, E. (1996). The genus Nocardiopsis represents a phylogenetically coherent taxon and a distinct actinomycete lineage: proposal of Nocardiopsaceae fam. nov. Int J Syst Bacteriol 46, 1088–1092.
Strömpl, C., Tindall, B. J., Jarvis, G. N., Lünsdorf, H., Moore, E. R. B. & Hippe, H. (1999). A re-evaluation of the taxonomy of the genus Anaerovibrio, with the reclassification of Anaerovibrio glycerini as Anaerosinus glycerini gen. nov., comb. nov., and Anaerovibrio burkinabensis as Anaeroarcus burkinensis [corrig.], gen. nov., comb. nov. Int J Syst Bacteriol 49, 1861–1872.
Witt, U., Müller, R.-J. & Deckwer, W.-D. (1994). Biodegradation of polyester copolymers containing aromatic compounds. J Macromol Sci Pure Appl Chem A 32, 851–856.
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