Molecular Microbiology (1992) 6(11). 1547-1553 nov: a new genetic locus that affects the response of Escherichia co/i K-12 to novobiocin Jasna Rakonjac,^^ Mrrjana Milic,^ Oragana Ajdic- Predic/^ Diogenes Santos,^ Radmila Ivanisevic' and Dragutin J. Savic^* ' Institute of Molecular Genetics and Genetic Engineering. Vojvode Stepe 282. PO Box 794, 11000 Belgrade, Yugostavia. ^Imperiat Cancer Research Fund, institute of Molecular Medicine. University of Oxford. Oxford OX2 9DU, UK. Summary We have identified a new gene locus {nov) affecting the resistance of Escherichia co//K-12 to novobiocin. The gene aiso affects, although to a iesser extent, toi- erance to another gyrase inhibitor coumermycin. Transductionai and complementation analysis show that nov is located between att08O and the osmZ {has) genes at minute 27 of the E. coli K-12 genetic map. In standard iaboratory strains of E. co//K-12 nov exists at least in two aiielic forms. Introduction The antibiotics novobiocin (Nov) and coumermycin (Cou) inhibit DNA replication by blocking the p subunit of DNA gyrase (Driica and Franco, 1988). Most Escherichia coli mutants conferring resistance to Nov and Cou map within the gyrB gene (Gellert et al., 1976). However, mutations in dnaA and rpoB can also change the bacterial response to these antibiotics. A decrease in hypersensitivity to Nov and Cou in dnaAiTs) mutants by a class of rpoB mutants may be explained by a bypass of the requirement of supercoiling in the initiation of DNA replication, although other explanations unrelated to DNA supercoiling are fea- sible (Filutowicz and Jonczyck, 1981; 1983). E. coli K-12 has high natural resistance to Nov due to the low permeability of the outer membrane lipopolysacharide (LPS) layer (Nikaido and Vaara, 1987). Mutations that impair synthesis or stability of LPS (e.g. acrA and rfaD) Increase sensitivity to Nov about 100-fold (Coleman and Leive, 1979). Unexpectedly. E. coli is much less resistant to Cou. which is essentially a dimer of Nov (Gellert ef a/., 1976). The molecular basis of this dif- ference remains unknown. In this work we report identification, characterization and mapping of a new genetic locus (nov) whose prod- uct(s) influences the tolerance of E coli to gyrase inhibitors novobiccin and coumermycin. The gene is located between attOSO and the osmZ genes at minute 27 of the E. coli K-12 genetic map immediately clockwise from the opp operon. Results Novobiocin-resistant reeombinants in the trp region of E. coli An analysis of Trp* transductants in a Pi-mediated cross between the widely used E. co//K-12 laboratory strains, C600 (donor) and SY209, trp derivative of AB1157, (recipient) (Table 1), yielded a proportion of transductants (36%) significantly more resistant to Nov than either of the parental strains (Table 2). In a controi experiment. His* and Pro* transductants obtained from the same transduc- tionai mixtures showed the same level of resistance to Nov as the native resistance of the recipient strain (100 ng mr^). This indicated that a locus linked to trp has an effect on Nov resistance. Very similar results were obtained with other donor strains and one additional recipient (Table 2). Each of the parental strains, and approximately 60% of the transductants, failed to grow on plates supplemented with 300 fig mp^ of Nov. C600 and SY209 did not grow on concentrations higher than 75 and 100 ^ g m ^ ^ respectively (Table 2). In contrast, approxi- mately 40% of the Trp* reeombinants grew well on such plates. Furthermore, the Nov resistance (Ncv'^) character couid be crossed out with Pi phages grown on the origi- nal, Nov sensitive (Nov^) recipient. In these backcross transductions (SY333 x SY353 and AB1157 x SY339) the percentage of Nov® reeombinants among Tet" and Trp* transductants was similar to the percentage of Nov" transductants in the original crosses (34% and 38% respectively). Received 20 July. 1991; revised 1 February, 1992; accepted 20 February, 1992. Present addresses: •\JhB Rockefeller University. New York. New York 10021, USA; §Departmenlot Microbiology and Immunology. Univer- sity of Oklahoma, Oklahoma City, Oklahoma 73190, USA. *For corre- spondence. Tel. (11) 491 395; Fax (492) 492 397. Novobiocin resistance is not due to suppressor mutations Several sup genes are linked to the trp operon (Bach- mann, 1990). Although neither the donor or recipient