Briefly, random insertion of mini-Tn10
into the chromosome containing dnaA46
) (Table ) was accomplished by infection of the mutant with a lambda phage (λ1098) carrying mini-Tn10
), followed by plating of the culture on Luria-Bertani (LB) agar plates containing tetracycline and incubation at 40°C. Twenty-three colonies were isolated. To identify the mini-Tn10
insertion site on the chromosome, Pst
I chromosome fragments containing mini-Tn10
were cloned into the Bluescript plasmid (pSK+) or pUC18 (Table ), and DNA sequences surrounding mini-Tn10
were determined and compared to the E. coli
whole genome sequence (3
) by using the BLAST program (Genetics Computer Group [GCG], University of Wisconsin, Madison, Wis.). To determine these sequences, DNA fragments surrounding mini-Tn10
were amplified by PCR with one primer, P15 (5′ GATCATATGACAAGATGTGTATCCACC), homologous to IS10R and a second primer, P10 (5′ACGCAAACCGCCTCTCCCCG) or P11 (5′ GCGAAAGGGGGATGTGCTGC), homologous to the vectors on either side of the polylinker. These fragments were sequenced with the same primers.
Bacterial strains, phage, and plasmids
The locations were confirmed by Southern hybridization of chromosomal PstI digests with the corresponding probes.
At present, we have identified three sites of mini-Tn10 insertion that suppress the dnaA46 thermosensitivity; they are yccV (in strain M2-24, with the corresponding PstI fragment cloned into pSK+, resulting in plasmid pSK24), rpoN (in strain M2-5, with the corresponding PstI fragment partially deleted after cloning into pUC18, resulting in plasmid pUC5), and mutS (in strain M2-11,with the corresponding PstI fragment also partially deleted after cloning into pSK+, resulting in pSK11), and they are located at 22, 74, and 69 min, respectively, on the genetic map.
The insertion mutants were further analyzed for genetic linkage between the tetracycline-resistant element (mini-Tn10) and the suppressor mutations. P1 phages prepared from M2-24, M2-5, or M2-11 were used for transduction of the tetracycline resistance marker in KA413ΔH at 30°C and then tested for growth at 40°C. The cotransduction frequencies between tetracycline resistance and growth capacity at 40°C were 100% for deletions of yccV and rpoN and 50% for the mutS deletant. Therefore, the mini-Tn10 insertion in either yccV or rpoN is responsible for suppression of dnaA46 thermosensitivity, whereas the insertion in mutS is not sufficient for suppression, indicating that an unidentified mutation closely linked to mutS is necessary.