EXPRESSION IN ESCHERICHIA-COLI OF Y5-MUTANT AND N-TERMINAL DOMAIN-DELETED DNA GYRASE-B PROTEINS AFFECTS STRONGLY PLASMID MAINTENANCE

Escherichia coli DNA gyrase B subunit (GyrB) is composed of a 43-kDa. N-terminal domain containing an ATP-binding site and a 47-kDa C-termin al domain involved in the interaction with the gyrase A subunit (GyrA) . Site-directed mutagenesis was used to substitute, in both the entire GyrB subunit and its 43-kDa N-terminal fragment, the amino acid Y5 by either a serine (Y5S) or a phenylalanine residue (Y5F). Under standar d conditions, cells bearing Y5S or Y5F mutant GyrB expression plasmids produced significantly less recombinant proteins than cells transform ed with the wild-type plasmid. This dramatic decrease in expression of mutant GyrB proteins was not observed when the corresponding N-termin al 43-kDa mutant plasmids were used. Examination of the plasmid conten t of the transformed cells after induction showed that the Y5F and Y5S GyrB protein level was correlated with the plasmid copy number. By re pressing tightly the promoter activity encoded by these expression vec tors during cell growth, it was possible to restore the normal level o f the mutant GyrB encoding plasmids in the transformed bacteria. Treat ment with chloramphenicol before protein induction enabled large overe xpression of the GyrB mutant Y5F and Y5S proteins. Ln addition, the de crease in plasmid copy number was also observed when the 47-kDa C-term inal fragment of the GyrB subunit was expressed in bacteria grown unde r standard culture conditions. Analysis of DNA supercoiling and relaxa tion activities in the presence of GyrA demonstrated that purified Y5- mutant GyrB proteins were deficient for ATP-dependent gyrase activitie s. Taken together, these results show that Y5F and Y5S mutant GyrB pro teins, but not the corresponding 43-kDa N-terminal fragments, compete in vivo with the bacterial endogenous GyrB subunit of DNA gyrase, ther eby reducing the plasmid copy number in the transformed bacteria by pr obably acting on the level of negative DNA supercoiling in vivo. This competition could be mediated by the presence of the intact 47-kDa C-t erminal domain in the Y5F and Y5S mutant GyrB subunits. This study dem onstrates also that the amino acid Y5 is a crucial residue for the exp ression of the gyrase B activity in vivo. Thus, our in vivo approach m ay also be useful for detecting other important amino acids for DNA gy rase activity, as mutations affecting the ATPase activity or the GyrB/ GyrB or GyrB/GyrA protein interactions. (C) 1997 Academic Press.