FIG. 4.

Northern blot analysis of sigC operon RNA from the wild type and Δlmo0421, Δlmo0422, and ΔsigC mutants. Overnight cultures of the wild-type 10403S and derived Δlmo0421, Δlmo0422, and ΔsigC mutants were diluted 1:200 into fresh BHI, grown to mid-log phase (OD₆₀₀, ∼0.4), and shifted to 48°C. RNA was extracted from samples harvested before (37) and 30 min after (48) the shift, and 50 μg of RNA was electrophoresed in formaldehyde-agarose gels and transferred to nylon membranes. The membranes were probed with labeled PCR products derived from internal segments of the sigC (+121 to +414 of the lmo0423 coding region) or lmo0421 (+735 to +1191 of the lmo0421 coding region) genes. The observed hybridizing transcripts are shorter in length in the mutant strains due to the length of the individual deletions (sigC deletion = 224 bp; lmo0422 deletion = 219 bp; lmo0421 deletion = 963 bp). To confirm equal loading of RNA, the blots were stripped and reprobed with a probe from a temperature-independent housekeeping gene, lmo0265 (dapE) gene (extending from +190 to +319), which encodes succinyl-diaminopimelate desuccinylase (39).