In some cases, the products of the first PCR were further amplified with repeated alternation of one high annealing temperature (58°C) cycle and one moderate annealing temperature (44°C) cycle in which the randomized primer was replaced with primer Fix5-29-2 (5′ CTA CAC
GAG TCA CTG CAG 3′), a primer sequence that was identical to selleck compound 18 of the 21 5′ terminal nucleotides of the randomized primer. DNA sequences obtained were used as query probes to search the E. coli K-12 genome sequence database for identifying transposon insertion sites. Lethality of environmental stresses The susceptibility of bacterial cells to UV irradiation was tested by applying serial dilutions of mid-log phase (OD600 = 0.3 ~0.5) cultures to agar plates that were irradiated
with an Ultraviolet Crosslinker CL-1000 (UVP) at a dose of 2000 μJ/cm2 in a dark room. The plates were then covered with aluminium foil and incubated overnight at 37°C. A1155463 For other stressors, mid-log phase cells were treated with 2 mM H2O2 (cells were resuspended in 0.9% saline before treatment), 10% sodium dodecyl sulfate (SDS), or high temperature (52°C) for 15 min. Serial dilutions were then prepared, and 10-μl of aliquots from the dilutions were spotted in triplicate on plates and incubated at 37°C overnight. The sensitivity of cells to the lethal effects of these stressors was expressed as percent survival of treated cells relative to that of Sepantronium molecular weight untreated cells determined at the time of treatment (LD90 could not be used because many of the mutant-stressor combinations did not reduce survival sufficiently). Complementation of hyperlethality by cloned genes All DNA manipulations were carried out according to procedures described previously Farnesyltransferase . The emrK and ycjU genes
with their promoter regions were amplified by PCR using chromosomal DNA isolated from DM4100 as templates and cloned into pBR322. The primers used were 5′-TAG GAA TTC ATC TCC CTT CTC CCT GTA GT-3′ and 5′-TAA GTC GAC ATT CTT TGT GCC AAC CTG-3′ for emrK, and 5′-TGC GAA TTC CTG CTG ACC CAA AGT TAT-3′ and 5′-TAG CTG CAG TCA CCT CTT TGG CGA TT-3′ for ycjU. Plasmids containing wild-type ycjW, yrbB, and ybcM were from the ASKA library . The plasmids were placed in the corresponding mutant strains, as well as in the wild-type strain DM4100, by electroporation. The strains harboring the plasmids were then tested for nalidixic acid lethality. For ycjW, yrbB, and ybcM, the expression was induced by adding 1 mM of IPTG 2 hr before nalidixic acid treatment. Results and Discussion Screening for mutants exhibiting hyperlethality to nalidixic acid During the course of evolution, bacteria have acquired a variety of genetic networks that provide protection from stress. For example, in E. coli more than 30 two-component systems detect the environment and cause changes in the expression of large numbers of genes .