Effects of replacement of the 5′-UTR of hfq mRNA with an unrelated 5′-UTR. (A) RNA sequences of 5′-UTR of the wild-type and the mutated hfq gene. The initiation codon is boxed and the sequence corresponding to the SD sequence derived from the plasmid pQE80L is underlined. (B) Overnight cultures of TM589 (Δhfq) cells harboring indicated plasmids were inoculated (1/100-fold) into LB medium containing 1 mM IPTG. Incubation was continued for 60 min and then total proteins were prepared. Protein samples equivalent to 0.0063 or 0.0025 A600 units were subjected to western blotting using anti-His6 monoclonal antibody or anti-GroEL polyclonal antibodies, respectively. Relative levels of Hfq-His6 derivatives are calculated, with the pQE-Hfq-His sample set to one. (C) Effect of expression of the wild-type and mutated hfq-His6 genes on cell growth. Overnight cultures of TM589 (Δhfq) cells harboring indicated plasmids were inoculated (1/1000-fold) into LB medium supplemented with 1 mM IPTG. Incubation was continued for 300 min by measuring A600.
