rhlB gene disruption and verification of degradosome component protein depletion in mutant strains. (
A) Western blot-based verification of degradosome mutant protein expression. Shown are the amido-black-stained poly(vinylidene difluoride) (PVDF) membrane (
Left) and corresponding Western blots (
Right) conducted to verify mutant protein expression. PAGE and Western blotting were carried out as described in
Materials and Methods. The target of antibodies used to probe each lane is indicated above the lanes corresponding to each parental/mutant strain pair. Deletion mutants YHC012 and SU02 failed to express PNPase and RhlB helicase (lanes 2 and 4, respectively). Strain DF261 contains a nonsense point mutation in the
eno gene that also resulted in the absence of detectable protein expression (lane 6). Strain BZ453, carrying a chromosomal deletion of the C terminus of RNase E expressed an appropriately truncated RNase E protein (1–602 aa; lane 8). (
B) A schematic diagram of the
rhlB locus and flanking ORFs before and after deletion by homologous recombination. H1 and H2 indicate sequences complementary to the RhlBKO 5′ and 3′ primers. FRT is the Flp recombination target site. Arrowheads indicate the direction of ORFs. (
C and
D) PCR verification of
rhlB disruption in strains SU01 and SU02. Agarose gels (2%) assessing PCR products are shown. Primer pairs
rhlB and
rhlBKO are as described in
Materials and Methods; they amplify the
rhlB locus, 1.2 kbp, and the
cassette, 1.6 kbp, respectively. M indicates lanes containing a 1-kbp DNA ladder. In lanes marked BW, N, and P, template DNA for PCR was from strains BW25113, N3433, and plasmid pKD4, respectively. In lanes marked 1 and 2, template DNA for PCR was from candidate recombinant colonies; in
C these are derivatives of BW25113 after introduction of the rhlBKO PCR construct, and in
D these are derivatives of N3433 after P1 transduction. For each candidate kanamycin-resistant colony, in contrast to the parental strains BW25113 and N3433, the
rhlB gene-specific primer pair failed to produce a PCR product, whereas the rhlBKO primers resulted in a DNA fragment with an expected DNA size of a PCR fragment containing the kanamycin gene encoded in the plasmid pKD4.