Expression of a cloned beta-glucanase gene from Bacillus amyloliquefaciens in an Escherichia coli relA strain after plasmid amplification.
Amino acid starvation of cells of the Escherichia coli relA strain, CP79, which cannot accumulate guanosine tetraphosphate (ppGpp) in response to amino acid limitation, increased the pEG1 plasmid content about 5- to 7-fold in comparison with exponentially growing cells (pEG1:pBR322 with an insertion of Bacillus amyloliquefaciens DNA coding for beta-glucanase). In contrast, no pEG1 amplification occurred in E. coli CP78, the stringently controlled counterpart, after amino acid starvation. In order to verify these results, the plasmid DNA content was monitored by measuring the expression of pEG1-encoded beta-glucanase from B. amyloliquefaciens both before and after plasmid amplification. When amino acid starved CP79 cells were given an additional dose of amino acids, a more than 10-fold increase in pEG1-encoded beta-glucanase activity (per cell mass) was measured. This increase in enzyme activity correlates with pEG1 amplification during amino acid limitation. Under comparable conditions the activity of beta-glucanase was not increased in strain CP78, which did not amplify the plasmid. We suggest that the replication of pEG1 in amino acid starved E. coli cells is somehow under negative control by ppGpp. Moreover, we found the Bacillus beta-glucanase in E. coli relA cells to be excreted into the growth medium after starvation and overexpression.
Hecker
M
M
Department of General Microbiology, Ernst-Moritz-Arndt-University, DDR, Greifswald.
Riethdorf
S
S
Bauer
C
C
Schroeter
A
A
Borriss
R
R
D000596:Amino Acids / Q000494:pharmacology; D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D005784:Gene Amplification; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000235:genetics*; D006159:Guanosine Tetraphosphate / Q000235:genetics; D010957:Plasmids
D000596:Amino Acids; D006159:Guanosine Tetraphosphate; D006026:Glycoside Hydrolases; C028020:licheninase