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1 Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstraße 3, 48149 Münster, Germany
2 Laboratory of Microbiology, Department of Biochemistry, Physiology and Microbiology, University of Ghent, K. L. Ledeganckstraat 35, 9000 Ghent, Belgium
Correspondence
Alexander Steinbüchel
steinbu{at}uni-muenster.de
| ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain DPN7T is AY880023.
A phase-contrast micrograph of cells of strain DPN7T is available as supplementary material in IJSEM Online.
Present addresss: Massachusetts Institute of Technology, Department of Chemical Engineering, 77 Massachusetts Avenue 56-422, Cambridge, MA 02139, USA. ![]()
| MAIN TEXT |
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R. eutropha and E. coli are unable to utilize these sulfur-containing substrates as sole carbon sources for growth. In addition, polythioesters currently cannot be synthesized de novo from simple carbon sources and sulphate: their biosynthesis depends on the use of the organic sulfur compounds mentioned above. Little is known (or published) about the catabolic pathways of organic sulfur compounds; exceptions are the catabolism of cysteine and methionine as well that as of dimethylsulfoxide, dimethylsulfoniopropionate and dimethylsulfide as intermediates of the sulfur cycle (Kertesz, 2000
; Kiene et al., 2000
; Lomans et al., 2002
; Yoch, 2002
) and the biodesulfurization of benzothiophenes (McFarland, 1999
) and of the fluorinated organic sulfur compound bis-(3-pentafluorophenylpropyl)-sulfide (Van Hamme et al., 2004
).
To investigate the unknown catabolism of DTDP (Fig. 1
), which is one of the precursor substrates currently used for the production of 3-mercaptopropionic acid-containing polythioesters (Lütke-Eversloh & Steinbüchel, 2003
), bacterial strains capable of utilizing this organic sulfur compound as a carbon source for growth are desirable for the understanding, and potential application, of DTDP-converting enzymes. In this study, we describe the taxonomic and biochemical characterization of a newly isolated bacterium, strain DPN7T, which is able to grow with DTDP as the sole carbon source.
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Colonies of strain DPN7T were circular, white to lime-yellowish in colour and were more flat than convex. Older colonies on MSM-DTDP agar developed a more yellowish colour. On complex medium, colonies of DPN7T grew in 12 days and appeared more white than yellow in colour. The cells were cultivated at 30 °C in liquid MSM containing 0.5 % (w/v) DTDP and samples were withdrawn from different growth stages of the culture. Cells of DPN7T were Gram-negative, coccoid rods approximately 1.52 µm in length and 1.5 µm in width and often occurred in pairs, as revealed under a Zeiss light microscope (see Supplementary Fig. S1 available in IJSEM Online). Cell motility was observed: after flagella staining performed according to Blenden & Goldberg (1964)
, one or two flagella could be detected.
Gram-staining performed according to Gerhardt et al. (1994)
gave a negative reaction. Strain DPN7T was oxidase-positive, as revealed by using Bactident oxidase test strips (Merck), and was catalase-positive, as revealed by adding 3 % (v/v) H2O2 to freshly grown colonies. API 20NE (bioMérieux) was used to determine the assimilation of some carbon sources and the presence of important enzymes. The utilization of different carbon sources and the growth at different temperatures were investigated in liquid MSM and on MSM agar plates containing 0.2 % (w/v) of the respective carbon source (Table 1
), unless indicated otherwise. Besides having the physiological characteristics shown in Table 1
, both strains were positive for utilization of D-glucose, sodium gluconate, acetic acid, propionic acid, butyric acid and succinic acid by conventional cultivation and according to the API 20NE system both strains were positive for utilization of L-arabinose, adipic acid, citric acid and malic acid and negative for D-mannose, D-mannitol, D-maltose, N-acetylglucosamine, capric acid and phenylacetic acid. Additional biochemical characteristics (shown in Table 1
) according to the API 20NE system were as follows: both strains were positive for urease activity and negative with regard to the fermentative degradation of glucose, indole production and activity of arginine dihydrolase,
-glucosidase, protease and
-galactosidase. This strain had no specific requirement for vitamins and also showed no enhanced growth in the presence of vitamin solution (Mohn, 1995
) in defined medium. Growth was typically observed after 24 days on MSM agar plates containing 0.5 % (w/v) DTDP when incubated at 2037 °C. In liquid MSM containing 0.20.5 % (w/v) DTDP as the sole carbon source, strain DPN7T exhibited a characteristic lag phase of approximately 48 h. After that period of time, the exponential phase followed, with a reproducible duration of more than 50 h. To the best of our knowledge, this is the first bacterial isolate found to utilize DTDP as the sole carbon and energy source for growth. Whereas good growth occurred with DTDP, no growth was observed with the C4 analogue 4,4'-dithiodibutyric acid or with 3,3'-thiodipropionic acid. The putative cleavage product of DTDP, 3-mercaptopropionic acid, could be utilized only at concentrations of less than 0.2 %, because of its toxicity (Table 1
).
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Antibiotic-susceptibility testing on nutrient broth agar (Sambrook et al., 1989
) containing different concentrations of antibiotics showed good growth of strain DPN7T on plates containing 5 µg chloramphenicol ml1 and poor growth with 50 µg chloramphenicol ml1, but no growth with 500 µg chloramphenicol ml1. Growth also occurred in the presence of 50 µg thiostrepton ml1. The strain showed no growth on plates containing 5 µg kanamycin ml1, 5 µg gentamicin ml1, 6.25 µg tetracycline ml1 or 50 µg streptomycin ml1 (the lowest concentrations tested). Strain DPN7T was able to grow slowly on agar plates containing ampicillin at concentrations up to the standard concentration for E. coli (75 µg ml1).
To determine the sequence of the 16S rRNA gene, genomic DNA was extracted according to Marmur (1961)
. The gene was amplified by a PCR using Pfx DNA polymerase (MBI Fermentas) and total genomic DNA as template for the oligonucleotide primers 27f and 1525r, which were complementary to conserved regions of the 16S rRNA gene sequence of E. coli. The 1502 bp PCR product was purified using the NucleoTrap kit (Macherey-Nagel) and then sequenced. The PCR product was also ligated to EcoRV-linearized pBluescript II SK() (Stratagene) DNA and transformed into competent cells of E. coli TOP10. Plasmid DNA was isolated as described by Birnboim & Doly (1979)
.
DNA sequencing was performed by applying the SequiTherm long-read cycle sequencing kit (Epicenter Technologies), with the following oligonucleotides as sequencing primers: 27f (5'-GAGTTTGATCCTGGCTCAG-3'), 343r (5'-CTGCTGCCTCCCGTA-3'), 357f (5'-TACGGGAGGCAGCAG-3'), 519r [5'-G(T/A)-ATTACCGCGGC(T/G)GCTG-3'], 536f [5'-CAGC(C/A)GCCGCGGTAAT(T/A)C-3'], 803f (5'-ATTAGATACCCTGGTAG-3'), 907r (5'-CCGTCAATTCATTTGAGTTT-3'), 1114f (5'-GCAACGAGCGCAACCC-3'), 1385r [5'-CGGTGTGT(A/G)CAAGGCCC-3'] and 1525r (5'-AGAAAGGAGGTGATCCAGCC-3'), as well as the universal primer 5'-GTAAAACGACGGCCAGT-3' and the reverse primer 5'-CAGGAAACAGCTATGAC-3' that hybridize to pBluescript II SK(). Sequence reactions were accomplished by using the GeneReadIR 4200 DNA analyser (LI-COR). 16S rRNA gene sequences were analysed using the program BLAST (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov) by running the BLASTN program. The nucleotide sequence of the 16S rRNA gene of strain DPN7T and of related type strains belonging to the same phylogenetic group and also to well-known representatives of the Betaproteobacteria were aligned using CLUSTAL X (Thompson et al., 1997
). 16S rRNA gene sequences were retrieved from the EMBL database and from the Ribosomal Database Project (Maidak et al., 1997
). The resulting trees, displayed with TreeView (Page, 1996
), were calculated by using the neighbour-joining method (Saitou & Nei, 1987
).
The nucleotide sequence of the 16S rRNA gene of strain DPN7T shows high levels of sequence similarity (98.698.0 %) to several Alcaligenes sp. strains (Hiraishi et al., 2003
; Boivin-Jahns et al., 1996
), but these strains could not be analysed further since they are no longer available (R. Christen, personal communication). A sequence similarity of 98.5 % with respect to the type strain of the recently isolated and classified species Tetrathiobacter kashmirensis (Ghosh et al., 2005
) was found, whereas lower similarity values were found with respect to the type strains of Castellaniella defragrans (strain 54PinT) (95.1 %), Alcaligenes faecalis (the type species of the genus Alcaligenes) (94.0 %), Alcaligenes latus (88.9 %) and Taylorella equigenitalis (94.7 %). The phylogenetic position of strain DPN7T is illustrated in a neighbour-joining dendrogram with Burkholderia kururiensis as the outgroup (Fig. 2
).
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phage DNA (Sigma) was used as the calibration reference. The DNA G+C content of strain DPN7T was 55.1 mol%, which is the same as that reported for Tetrathiobacter kashmirensis LMG 22695T. The separated phylogenetic position of strain DPN7T was confirmed by DNADNA hybridization. Experiments were performed at 42 °C with photobiotin-labelled probes in microplate wells as described previously (Goris et al., 1998
Agarose gel electrophoresis of crude extracts of cells of strain DPN7T obtained by the method of Birnboim & Doly (1979)
revealed the presence of a 22 kbp plasmid. In preliminary experiments, this plasmid could not be connected with the capacity to utilize DTDP or other organic sulfur compounds. No plasmids were detected in Tetrathiobacter kashmirensis LMG 22695T or Tetrathiobacter kashmirensis LMG 22696.
Fatty acid analysis was carried out with a loopful of well-grown cells after an incubation period of 48 h. Fatty acid methyl esters were prepared, separated and identified using the Microbial Identification System (Microbial ID) as described previously (Vandamme et al., 1992
). Strain DPN7T contained the following fatty acid components: 12 : 0 (4.5 %), 16 : 0 (18.2 %), 17 : 0 cyclo (4.8 %), 16 : 0 3-OH (3.3 %), 18 : 1
7c (26.5 %), 18 : 0 (2.1 %), 19 : 0
8c cyclo (2.6 %), summed feature 2 (13.3 %, comprising 12 : 0 ALDE, 16 : 1 iso I and/or 14 : 0 3-OH) and summed feature 3 (23.3 %, comprising 16 : 1
7c and/or iso-15 : 0 2-OH) (summed features are groups of two or three fatty acids that cannot be distinguished using the Microbial Identification System). Members of this phylogenetic lineage typically contain 14 : 0 3-OH and 16 : 1
7c; therefore, the peaks designated as summed features 2 and 3 probably corresponded to these fatty acids. A comparison with the fatty acid profile of Tetrathiobacter kashmirensis LMG 22695T (data available in Ghosh et al., 2005
) revealed only small variations in composition.
The whole-cell protein profile of strain DPN7T was determined by using SDS-PAGE (Pot et al., 1994
) and was compared with those of reference taxa belonging to this bacterial lineage. Data for reference strains were taken from previous studies (Coenye et al., 2003
; Jang et al., 2001
). The protein profile of strain DPN7T was clearly different from those of Alcaligenes, Achromobacter, Bordetella, Castellaniella and Taylorella reference strains, as well as from the type species of the genus Tetrathiobacter (Fig. 3
).
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Description of Tetrathiobacter mimigardefordensis sp. nov.
Tetrathiobacter mimigardefordensis (mi.mi.gar.de.for.den'sis. M.L. masc. adj. mimigardefordensis of Mimegardefordum, a medieval name of Münster, where the type strain was isolated).
Cells are motile, Gram-negative, non-spore-forming coccoid rods (approx. 1.52 µm in length). They are oxidase- and catalase-positive, showing an oxidative metabolism. The temperature range for growth is 1540 °C and the best growth occurs at 3037 °C. Colonies on DTDP-containing agar plates are more flat than convex, up to 3 mm in diameter and white to lime-yellowish in colour, showing an increase in yellow colour with culture age. On complex medium, colonies grow in 12 days and appear more white than yellow and are up to 5 mm in diameter. Susceptible to kanamycin, gentamicin, tetracycline and streptomycin, with no significant antibiotic resistance. Fatty acid profile largely comprises 18 : 1
7c (26.5 %), 16 : 0 (18.2 %) and of the two fatty acids 16 : 1
7c and/or iso-15 : 0 2-OH (together constituting 23.3 %), which cannot be distinguished using the Microbial Identification System. The DNA G+C content of the type strain is 55.1 mol%. Carbon sources utilized are indicated in Table 1
.
The type strain, DPN7T (=DSM 17166T=LMG 22922T), was isolated from a sample of matured compost from the compost plant in Münster (Germany).
| ACKNOWLEDGEMENTS |
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