Abstract
Drug-resistance element Tn5 coding for kanamycin resistance was used for mutagenesis of Alcaligenes eutrophus strain H16. The vehicle for introducing Tn5 into A. eutrophus was plasmid pJB4JI harboured by Escherichia coli. Kanamycin-resistant transconjugants occurred at a frequency of approximately 5×10-8. One third of the transconjugants exhibited other plasmid-coded resistances such as gentamycin and spectinomycin. However, the latter markers were not stably maintained in the new host. Among the kanamycin-resistant transconjugants three classes of mutants were found: (i) Auxotrophic mutants occurred at a frequency of 0.8% and showed requirements for histidine, methionine, aspartate orisoleucine. Out of eleven auxotrophic mutants examined eight reverted to prototrophy. However, none of the revertants was kanamycin-sensitive. (ii) Mutants unable to grow with fructose as the carbon source occurred at a frequency of almost 10%. (iii) Mutants which had lost the ability to grow autotrophically with hydrogen and carbon dioxide were found at a frequency of 1%. Further analyses revealed that this class of mutants was either defective in carbon dioxide fixation or impaired in hydrogen metabolism.
Similar content being viewed by others
References
Berg DE (1977) Insertion and excision of the transposable kanamycin resistance determinant Tn5. In: AT Bukhari, JA Shapiro, S Adhya (eds) DNA Insertion elements, plasmids, and episomes. Cold Spring Harbor Laboratory, New York, pp 205–212
Berg DE, Weiss A, Crossland L (1980) Polarity of Tn5 insertion mutations in Escherichia coli. J Bacteriol 142:439–446
Beringer JE, Beynon JL, Buchanon-Wallaston AV, Johnston AWB (1978) Transfer of the drug-resistance transposon Tn5 to Rhizobium. Nature 276:633–634
Biek D, Roth JR (1980) Regulation of Tn5 transposition in Salmonella typhinurium. Proc Natl Acad Sci USA 77:6047–6051
Boucher C, Bergeron B, Barate De Bertalmio M, Dénarié J (1977) Introduction of bacteriophage Mu into Pseudomonas solanacearum and Rhizobium meliloti using the R factor RP4. J Gen Microbiol 98:253–263
Duncan MJ (1981) Properties of Tn5-induced carbohydrate mutants in Rhizobium meliloti. J Gen Microbiol 122:61–67
Friedrich B, Schlegel HG (1975) Aromatic amino acid biosynthesis in Alcaligenes eutrophus H16. II. The isolation and characterization of mutants auxotrophic for phenylalanine and tyrosine. Arch Microbiol 103:141–149
Friedrich CG, Bowien B, Friedrich B (1979) Formate and oxalate metabolism in Alcaligenes eutrophus. J Gen Microbiol 115:185–192
Friedrich B, Heine E, Finck A, Friedrich CG (1981a) Nickel requirement for active hydrogenase formation in Alcaligenes eutrophus. J Bacteriol 145:1144–1149
Friedrich B, Hogrefe C, Schlegel HG (1981b) Naturally occurring genetic transfer of hydrogen-oxidizing ability between strains of Alcaligenes eutrophus. J Bacteriol 147:198–205
Howe MM, Bade EG (1975) Molecular biology of bacteriophage Mu. Science 190:624–632
Kleckner N, Roth J, Botstein D (1977) Genetic engineering in vivo using translocatable drug-resistance elements. J Mol Biol 116:125–159
Merrick M, Filser M, Kennedy C, Dixon R (1978) Polarity of mutations induced by insertion of transposons Tn5, Tn7 and Tn10 into the nif gene cluster of Klebsiella pneumoniae. Molec Gen Genetics 165:103–111
Merrick M, Filser M, Dixon R, Elmerich C, Sibold L, Houmard J (1980) The use of translocatable genetic elements to construct a fine-structure map of Klebsiella pneumoniae nitrogen fixation (nif) gene cluster. J Gen Microbiol 117:509–520
Schink B, Schlegel HG (1978) Mutants of Alcaligenes eutrophus defective in autotrophic metabolism. Arch Microbiol 117:123–129
Schink B, Schlegel HG (1979) The membrane-bound hydrogenase of Alcaligenes eutrophus. I. Solubilization and purification and biochemical properties. Biochim Biophys Acta 567:315–324
Schlegel HG, Eberhardt U (1972) Regulatory phenomena in the metabolism of Knallgasbacteria. Adv Microb Physiol 7:205–242
Schlegel HG, Kaltwasser H, Gottschalk G (1961) Ein Submersverfahren zur Kultur wasserstoffoxidierender Bakterien: Wachstumsphysiologische Untersuchungen. Arch Mikrobiol 38:209–222
Schneider K, Cammack R, Schlegel HG, Hall DO (1979) The iron-sulfur centres of soluble hydrogenase from Alcaligenes eutrophus. Biochim Biophys Acta 578:445–461
Shaw KJ, Berg CM (1979) Escherichia coli auxotrophs induced by insertion of the transposable element Tn5. Genetics 92:741–747
Van Vliet F, Silva B, Van Montagu M, Schell J (1978) Transfer of RP4::Mu Plasmids to Agrobacterium tumefaciens. Plasmid 1:446–455
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Srivastava, S., Urban, M. & Friedrich, B. Mutagenesis of Alcaligenes eutrophus by insertion of the drug-resistance transposon Tn5. Arch. Microbiol. 131, 203–207 (1982). https://doi.org/10.1007/BF00405879
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00405879