Co-overproduction and localization of the Escherichia coli motility proteins motA and motB.
The motility genes motA and motB of Escherichia coli were placed under control of the Serratia marcescens trp promoter. After induction with beta-indoleacrylic acid, the levels of MotA and MotB rose over about a 3-h period, reaching plateau levels approximately 50-fold higher than wild-type levels. Both overproduced proteins inserted into the cytoplasmic membrane. Growth and motility were essentially normal, suggesting that although the motor is a proton-conducting device, MotA and MotB together do not constitute a major proton leak. Derivative plasmids which maintained an intact version of motB but had the motA coding region deleted in various ways were constructed. With these, the levels of MotB were much lower, reaching a peak within 30 min after induction and declining thereafter; pulse-chase measurements indicated that a contributing factor was MotB degradation. The low levels of MotB occurred even with an in-frame internal deletion of motA, whose translational initiation and termination sites were intact, suggesting that it is the MotA protein, rather than the process of MotA synthesis, that is important for MotB stability. Termination at the usual site of overlap with the start of motB (ATGA) was not an absolute requirement for MotB synthesis but did result in higher rates of synthesis than when translation of motA information terminated prematurely. Even in the total absence of MotA, the MotB that was synthesized was found exclusively in the cytoplasmic membrane fraction. In wild-type cells, MotA was estimated by immunoprecipitation to be in about fourfold excess over MotB; a previous estimate of 600 +/- 250 copies of MotA per cell then yielded an estimate of 150 +/- 70 copies of MotB per cell.
Wilson
M L
ML
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
Macnab
R M
RM
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D002872:Chromosome Deletion; D004274:DNA, Recombinant / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000502:physiology; D005407:Flagella / Q000502:physiology; D015870:Gene Expression; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005838:Genotype; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012706:Serratia marcescens / Q000235:genetics
D001426:Bacterial Proteins; D004274:DNA, Recombinant; C063010:MotA protein, Bacteria; C064779:MotB protein, Bacteria