Antisense downregulation of sigma(32) as a transient metabolic controller in Escherichia coli: effects on yield of active organophosphorus hydrolase.
Plasmids containing an antisense fragment of the sigma(32) gene were constructed and introduced into Escherichia coli cells. Downregulation of the sigma(32)-mediated stress response was evaluated under heat shock and ethanol stress and during the production of organophosphorus hydrolase (OPH). Northern blot analyses revealed that sigma(32) sense mRNA was virtually undetected in antisense-producing cultures from 5 to 20 min after antisense induction. However, lower-molecular-weight bands were found, presumably due to partial degradation of sigma(32) mRNA. While a >10-fold increase in sigma(32) protein level was found under ethanol stress in the control cultures, antisense producing cultures resulted in a <3-fold increase, indicating downregulation of sigma(32). Correspondingly, antisense synthesis resulted in a decreased level of a sigma(32) regulated chaperone (GroEL) for the first 2 h after induction relative to control cultures without sigma(32) antisense mRNA. The total yield of OPH in the presence of sigma(32) antisense was, on average, 62% of the yield without antisense. However, during sigma(32) antisense production, a sixfold-higher specific OPH activity was observed compared to non-antisense-producing cultures.
Srivastava
R
R
Department of Chemical Engineering, University of Maryland, College Park, 20742, USA.
Cha
H J
HJ
Peterson
M S
MS
Bentley
W E
WE
D043303:Aryldialkylphosphatase; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D004950:Esterases / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology*; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D012808:Sigma Factor / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
D006360:Heat-Shock Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; D012808:Sigma Factor; D014157:Transcription Factors; C069774:heat-shock sigma factor 32; D004950:Esterases; D043303:Aryldialkylphosphatase