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-termina l 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 standard conditions, cells bearing Y5S or Y5F mutant GyrB expression plasmids produced significantly less recombinant proteins than cells transforme d with the wild-type plasmid. This dramatic decrease in expression of mutant GyrB proteins was not observed when the corresponding N-termina l 43 kDa mutant plasmids were used. Examination of the plasmid content of the transformed cells after induction showed that the Y5F and Y5S GyrB protein level was correlated with the plasmid copy number. By rep ressing tightly the promoter activity encoded by these expression vect ors during cell growth, it was possible to restore the normal level of the mutant GyrB encoding plasmids in the transformed bacteria. Treatm ent with chloramphenicol before protein induction enabled large overex pression of the GyrB mutant Y5F and Y5S proteins. In addition, the dec rease in plasmid copy number was also observed when the 47-kDa C-termi nal fragment of the GyrB subunit was expressed in bacteria grown under standard culture conditions. Analysis of DNA supercoiling and relaxat ion activities in the presence of GyrA demonstrated that purified YS-m utant GyrB proteins were deficient for ATP-dependent gyrase activities . Taken together, these results show that Y5F and Y5S mutant GyrB prot eins, but not the corresponding 43-kDa N-terminal fragments, compete i n vivo with the bacterial endogenous GyrB subunit of DNA gyrase, there by reducing the plasmid copy number in the transformed bacteria by pro bably acting on the level of negative DNA supercoiling in vivo. This c ompetition could be mediated by the presence of the intact 47-kDa C-te rminal domain in the Y5F and Y5S mutant GyrB subunits. This study demo nstrates also that the amino acid Y5 is a crucial residue for the expr ession of the gyrase B activity in vivo. Thus, our in vivo approach ma y also be useful for detecting other important amino acids for DNA gyr ase activity, as mutations affecting the ATPase activity or GyrB/GyrB, or GyrB/GyrA protein interactions. (C) 1998 Academic Press.