Identification of the DNA region responsible for sulfur-oxidizing ability of Thiosphaera pantotropha

G. Mittenhuber, K. Sonomoto, M. Egert, C. G. Friedrich

研究成果: ジャーナルへの寄稿記事

19 引用 (Scopus)

抄録

For the identification of the DNA region responsible for the sulfur-oxidizing ability (Sox) of Thiosphaera pantotropha, we used previously isolated Tn5-mob insertional Sox- mutants. For seven mutants, the Tn5-mob insertion was localized on the chromosome rather than on the megaplasmids pHG41 or pHG42 by using the Tn5-mob-harboring vehicle pSUP5011 as probe. The specific insertion of Tn5-mob into a sox gene was determined for one Sox- mutant, strain TP19. An 18-kb EcoRI fragment was cloned in Escherichia coli by using the mobilizable plasmid pSUP202 as vector and the kanamycin resistance gene of Tn5 as marker. Conjugal transfer of the resulting hybrid plasmid, pKS3-13, to the wild type resulted in two phenotypically different groups of recombinants. Ninety-five percent of the recombinants were Sox+, kanamycin resistant, and tetracycline resistant; 5% were homogenote recombinants exhibiting the Sox-, kanamycin-resistant, tetracycline-sensitive phenotype, and these indicated the specific insertion. To isolate the respective wild-type sox gene, total DNA from a heterogenote recombinant was partially restricted with EcoRI, religated, and transformed in E. coli. Transformants carrying a pSUP202-derived hybrid plasmid with the intact sox gene were identified by screening for a tetracycline-resistant, kanamycin-sensitive, and chloramphenicol-sensitive phenotype and by complementation of the Sox- mutant TP19. A plasmid of this type, pEG12, contained an insert of 13 kb which gave a positive signal in Southern hybridization with the homologous probe of pKS3-13. pEG12 was used to determine the DNA homology of the sulfur-oxidizing enzyme systems of other thiobacteria. Strong hybridization signals were obtained with total DNA of the neutrophilic sulfur-oxidizing bacteria Paracoccus denitrificans, Thiobacillus versutus, and Rhodobacter capsulatus. No hybridization signal was obtained with DNA of other neutrophilic or acidophilic thiobacteria examined.

元の言語英語
ページ(範囲)7340-7344
ページ数5
ジャーナルJournal of bacteriology
173
発行部数22
DOI
出版物ステータス出版済み - 1 1 1991

Fingerprint

Paracoccus pantotrophus
Sulfur
DNA
Kanamycin
Plasmids
Tetracycline
Genes
Paracoccus denitrificans
Thiobacillus
Rhodobacter capsulatus
Kanamycin Resistance
Escherichia coli
Phenotype
Chloramphenicol
Chromosomes

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology

これを引用

Identification of the DNA region responsible for sulfur-oxidizing ability of Thiosphaera pantotropha. / Mittenhuber, G.; Sonomoto, K.; Egert, M.; Friedrich, C. G.

:: Journal of bacteriology, 巻 173, 番号 22, 01.01.1991, p. 7340-7344.

研究成果: ジャーナルへの寄稿記事

Mittenhuber, G. ; Sonomoto, K. ; Egert, M. ; Friedrich, C. G. / Identification of the DNA region responsible for sulfur-oxidizing ability of Thiosphaera pantotropha. :: Journal of bacteriology. 1991 ; 巻 173, 番号 22. pp. 7340-7344.
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abstract = "For the identification of the DNA region responsible for the sulfur-oxidizing ability (Sox) of Thiosphaera pantotropha, we used previously isolated Tn5-mob insertional Sox- mutants. For seven mutants, the Tn5-mob insertion was localized on the chromosome rather than on the megaplasmids pHG41 or pHG42 by using the Tn5-mob-harboring vehicle pSUP5011 as probe. The specific insertion of Tn5-mob into a sox gene was determined for one Sox- mutant, strain TP19. An 18-kb EcoRI fragment was cloned in Escherichia coli by using the mobilizable plasmid pSUP202 as vector and the kanamycin resistance gene of Tn5 as marker. Conjugal transfer of the resulting hybrid plasmid, pKS3-13, to the wild type resulted in two phenotypically different groups of recombinants. Ninety-five percent of the recombinants were Sox+, kanamycin resistant, and tetracycline resistant; 5{\%} were homogenote recombinants exhibiting the Sox-, kanamycin-resistant, tetracycline-sensitive phenotype, and these indicated the specific insertion. To isolate the respective wild-type sox gene, total DNA from a heterogenote recombinant was partially restricted with EcoRI, religated, and transformed in E. coli. Transformants carrying a pSUP202-derived hybrid plasmid with the intact sox gene were identified by screening for a tetracycline-resistant, kanamycin-sensitive, and chloramphenicol-sensitive phenotype and by complementation of the Sox- mutant TP19. A plasmid of this type, pEG12, contained an insert of 13 kb which gave a positive signal in Southern hybridization with the homologous probe of pKS3-13. pEG12 was used to determine the DNA homology of the sulfur-oxidizing enzyme systems of other thiobacteria. Strong hybridization signals were obtained with total DNA of the neutrophilic sulfur-oxidizing bacteria Paracoccus denitrificans, Thiobacillus versutus, and Rhodobacter capsulatus. No hybridization signal was obtained with DNA of other neutrophilic or acidophilic thiobacteria examined.",
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