Isolation of Escherichia coli mutants defective in uptake of molybdate.
For the study of molybdenum uptake by Escherichia coli, we generated Tn5lac transposition mutants, which were screened for the pleiotropic loss of molybdoenzyme activities. Three mutants A1, A4, and M22 were finally selected for further analysis. Even in the presence of 100 microM molybdate in the growth medium, no active nitrate reductase, formate dehydrogenase, and trimethylamine-N-oxide reductase were detected in these mutants, indicating that the intracellular supply of molybdenum was not sufficient. This was also supported by the observation that introduction of plasmid pWK225 carrying the complete nif regulon of Klebsiella pneumoniae did not lead to a functional expression of nitrogenase. Finally, molybdenum determination by induced coupled plasma mass spectroscopy confirmed a significant reduction of cell-bound molybdenum in the mutants compared with that in wild-type E. coli, even at high molybdate concentrations in the medium. A genomic library established with the plasmid mini-F-derived cop(ts) vector pJE258 allowed the isolation of cosmid pBK229 complementing the molybdate uptake deficiency of the chlD mutant and the Tn5lac-induced mutants. Certain subfragments of pBK229 which do not contain the chlD gene are still able to complement the Tn5lac mutants. Mapping experiments showed that the Tn5lac insertions did not occur within the chromosomal region present in pBK229 but did occur very close to that region. We assume that the Tn5lac insertions have a polar effect, thus preventing the expression of transport genes, or that a positively acting regulatory element was inactivated.
Hemschemeier
S
S
Gentechnologie/Mikrobiologie, Fakultät für Biologie, Universität Bielefeld, Germany.
Grund
M
M
Keuntje
B
B
Eichenlaub
R
R
D015139:Blotting, Southern; D004251:DNA Transposable Elements / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005816:Genetic Complementation Test; D015698:Genomic Library; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000235:genetics; D013058:Mass Spectrometry; D008982:Molybdenum / Q000032:analysis / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism; D009591:Nitrogenase / Q000235:genetics; D009876:Operon / Q000235:genetics; D010957:Plasmids / Q000235:genetics
D004251:DNA Transposable Elements; C044659:molybdate; D008982:Molybdenum; D009591:Nitrogenase; D009565:Nitrate Reductases; D050901:Nitrate Reductase