Escherichia coli K-12 tolZ mutants tolerant to colicins E2, E3, D, Ia, and Ib: defect in generation of the electrochemical proton gradient

Abstract

Spontaneous Escherichia coli K-12 mutants tolerant to colicin E3 were isolated, and on the basis of their tolerance patterns to 19 kinds of colicins, a new phenotypic class of toLZ mutants was found. The tolZ gene was located between min 77 and 78 on the E. coli K-12 genetic map. The tolZ mutants were tolerant to colicins E2, E3, D, Ta, and lb, and showed an increased sensitivity to ampicillin, neomycin, and EDTA, but not to deoxycholate; they were able to grow on glucose miniinal medium, but not on nonfermentable carbon sources (succinate, acetate, pyruvate, lactate, malate, etc.). The pleiotropic phenotype of the tolZ mutant was due to a single mutation. Both respiration and membrane ATPase activity of the tolZ mutant were normal. The tolZ mutant had a defect in the uptake of proline, glutamine, thiomethyl-IB-D-galactoside, and triphenylmethylphosphonium ion; these uptake systems are driven by an electrochemical proton gradient (AljH+) or a membrane potential (A*). In contrast, the uptake of methionine and a-methyl-D-glucoside, which is not dependent on A-tH+ and A*, was kormal in the tolZ nmutant. Glucose 6-phosphate uptake at pH 5.5, which is driven by a transmembrane pH gradient, in the tolZ mutant was similar to the parent level. These results indicate that the tolZ mutant has a defect in the generation of AlR"+ and A*.

Balanced biosynthesis of major membrane components through regulated degradation of the committed enzyme of lipid A biosynthesis by the AAA protease FtsH (HflB) in Escherichia coli

“…The Std phenotype was examined according to the method described previously (Akiyama et al ., 1994b), using pKY251 instead of pKY221. The Tol and Nfc phenotypes were defined as a strain's sensitivity to colicins E2 and E3 (Matsuzawa et al ., 1984; Qu et al ., 1996) and its ability to grow on an M9 plate containing 30 mM succinate at 37°C (Qu et al ., 1996). To test the sensitivity to CIII, p tacc III was introduced into the strains for test, and the strains carrying p tacc III was grown on an L plate containing 1 mM IPTG to induce expression of c III under the tac promoter.…”

“…Several lines of evidence have also suggested that FtsH has a chaperone‐like activity. It has been reported that ftsH mutants show defects in membrane functions, such as anchoring of integral membrane proteins (Akiyama et al ., 1994b), export of secretory proteins (Akiyama et al ., 1994a, 1994b), colicin tolerance (Matsuzawa et al ., 1984; Qu et al ., 1996) and the membrane potential (ΔΨ) (Matsuzawa et al ., 1984). Some of these defects are partially suppressed by overproduction of the molecular chaperones GroEL/ES (Hsp60/10) or HtpG (Hsp90) (Shirai et al ., 1996).…”

“…This substitution of His‐418 by Tyr must greatly reduce the protease activity of FtsH. The tolZ21 mutant is viable, although it is temperature sensitive (Matsuzawa et al ., 1984; Qu et al ., 1996). These findings suggest that the proteolytic function of FtsH is not essential for viability and, therefore, another function such as the proposed chaperone activity must be essential in wild‐type cells.…”

FtsH – a single-chain charonin?

“…If the second step cannot occur due to a mutation within the gene encoding the periplasmic protein, a colicin-tolerant phenotype will result. One of these colicin-tolerant mutants received the designation tolZ [10].…”

The proton‐motive force is required for translocation of CDI toxins across the inner membrane of target bacteria

“…FtsH is a membrane-bound, ATP-dependent protease that functions primarily in the turnover of integral membrane proteins. Mutant alleles of ftsH (tolZ) confer resistance to some colicins (Matsuzawa et al, 1984;Qu et al, 1996), and de Zamaroczy and colleagues have proposed that FtsH releases the C-terminal toxin domains of colicins through its protease activity (Chauleau et al, 2011). Kleanthous and colleagues have speculated that colicin molten globules may be recognized as misfolded membrane proteins and pulled into the cytoplasm by FtsH (Walker et al, 2007).…”