Chemostat selection of an Escherichia coli mutant containing permease with enhanced lactose affinity.
Chemostats supplied with limited lactose were used to ask whether it was possible to generate and isolate any mutant of Escherichia coli lactose permease which allowed cells to grow faster. The permease and beta-galactosidase activities of the chemostat culture initially rose together to reach a plateau. After 30 days, the former underwent a second increase alone. From this culture, a faster-growing mutant was isolated. Its permease gene was cloned, sequenced, and found to have a single base pair changed. Thymine at position 199 was changed to guanine, resulting in serine 67 being substituted by alanine. Cells bearing this mutant in the plasmid could grow faster than parents in 10 microM lactose. The Km of the mutant permease toward lactose was 1.4 mM, about half of the wild-type value. Thus, a mutant with higher affinity for substrate could be selected from the chemostat.
Tsen
S D
SD
Graduate Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan, Republic of China.
Lai
S C
SC
Pang
C P
CP
Lee
J I
JI
Wilson
T H
TH
D001431:Bacteriological Techniques; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development*; D029968:Escherichia coli Proteins; D005838:Genotype; D007700:Kinetics; D007763:Lac Operon; D026901:Membrane Transport Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010957:Plasmids; D016133:Polymerase Chain Reaction; D013379:Substrate Specificity; D027981:Symporters; D013997:Time Factors
D017931:DNA Primers; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease