Construction and verification of a null allele of the Shewanella oneidensis MR-1 hfq gene. (A) Knockout strategy for the MR-1 hfq gene. Most of the hfq gene coding sequence (all but the first 9 codons and last 6 codons) was replaced with a cassette containing a promoterless lacZ gene and a gentamicin resistance marker. (B) Agarose gel of analytical PCR reactions using wild type MR-1 (lanes 2–4) or hfq∆ mutant (lanes 5–7) templates and primers A, B, C, and D (see Materials and Methods for primer sequences) indicated with arrows on the diagram in panel (A) The size standard (M) in lane 1 is 1kb plus DNA ladder (Invitrogen). (C) Western blot of SDS-PAGE-fractionated total protein from various Shewanella strains probed with a polyclonal antibody raised against E. coli Hfq protein. Lanes 1 and 2: MR-1 containing pBBR1-MCS-2 (vector) or hfq rescue construct (phfq), respectively. Lanes 3 and 4: hfq∆ containing vector or phfq, respectively. The antibody detects both putative Hfq monomers (~10kDa) as well as putative Hfq homohexamers (~60kDa).
Brennan et al. BMC Microbiology 2013 13:33 doi:10.1186/1471-2180-13-33