GCVA, A LYSR-TYPE TRANSCRIPTIONAL REGULATOR PROTEIN, ACTIVATES EXPRESSION OF THE CLONED CITROBACTER-FREUNDII AMPC BETA-LACTAMASE GENE IN ESCHERICHIA-COLI - CROSS-TALK BETWEEN DNA-BINDING PROTEINS

Escherichia coli JRG582 is an ampD ampE deletion derivative of strain HfrH and accordingly it is derepressed for expression of the cloned in ducible beta-lactamase gene of Citrobacter freundii, carried on plasmi d pNU305. Following chemical mutagenesis of JRG582(pNU305) a cefotaxim e sensitive mutant was isolated, CS51(pNU305), which produced low leve ls of beta-lactamase due to a mutation in the host chromosome. Two rec ombinant plasmids containing genomic DNA from E. coli HfrH, namely pUB 5608 and pUB5611, were isolated as a consequence of their ability to r estore the beta-lactam resistant phenotype to CS51(pNU305). This abili ty was due to direct transcriptional activation of the beta-lactamase gene, ampC, rather than complementation of the CS51 mutation. Transpos on mutagenesis and subcloning showed that restoration of ampicillin re sistance to CS51(pNU305) was the function of a single gene, which maps at 60.3 min on the E. coli chromosome. The gene encodes a 33 kDa prot ein with significant homology to members of the LysR family of bacteri al activator proteins, in particular the AmpR protein from C. freundii . Homology is especially strong over the N-terminal region which inclu des the helix-turn-helix DNA-binding motif. This gene was shown to com plement the gcvA1 mutation at 60.3 min on the E. coli chromosome, and the DNA sequence agrees exactly with the published sequence of gcvA wh ich encodes the transcriptional activator of the inducible glycine cle avage enzyme system. It is suggested that GcvA can activate transcript ion of ampC by binding to the AmpR binding region upstream of ampC so as to mimic the activated state of AmpR and hence provides an example of cross-talk between DNA-binding proteins of different inducible enzy me systems.