A colicin-tolerant Escherichia coli mutant that confers hfl phenotype carries two mutations in the region coding for the C-terminal domain of FtsH (HflB)

An Escherichia coli mutant, ER437, which was originally isolated for colicin tolerance, was found to carry two amino acid changes in the C-terminal portion of FtsH (HflB). These mutations were demonstrated to reduce the ability of FtsH to degrade the phage lambda CII protein in vivo and in vitro, providing a rationalization for the mutant Hfl phenotype.     

The Escherichia coli Fis protein stimulates bacteriophage lambda integrative recombination in vitro

The Escherichia coli nucleoid-associated protein Fis was previously shown to be involved in bacteriophage lambda site-specific recombination in vivo, enhancing the levels of both integrative recombination and excisive recombination. While purified Fis protein was shown to stimulate in vitro excision, Fis appeared to have no effect on in vitro integration reactions even though a 15-fold drop in lysogenization frequency had previously been observed in fis mutants. We demonstrate here that E. coli Fis protein does stimulate integrative lambda recombination in vitro but only under specific conditions which likely mimic natural in vivo recombination more closely than the standard conditions used in vitro. In the presence of suboptimal concentrations of Int protein, Fis stimulates the rate of integrative recombination significantly. In addition, Fis enhances the recombination of substrates with nonstandard topologies which may be more relevant to the process of in vivo phage lambda recombination. These data support the hypothesis that Fis may play an essential role in lambda recombination in the host cell.     

hflB, a new Escherichia coli locus regulating lysogeny and the level of bacteriophage lambda cII protein

The level of the viral cII protein has been proposed to be the crucial determinant in the lysis-lysogeny decision of bacteriophage lambda. A new Escherichia coli locus (hflB) has been identified in which a mutation (hflB29) leads to high frequency of lysogeny by lambda. A double mutant defective in both hflB and the previously identified hflA gene displays a more severe Hfl- phenotype than either single mutant. The hflB locus is at 69 minutes on the E. coli map, 85% co-transducible with argG. The hflB29 mutation results in increased stability of the phage cII protein (increasing its half-life twofold) and is recessive to hflB+. We conclude that the hflB+ locus is a negative regulator of cII, perhaps coding for or regulating a protease that acts on cII. In addition, we observe that the can1 mutation, an alteration of the cII gene that results in enhanced lysogenization, leads to increased stability of cII protein. These observations reinforce the view that the level of cII is a key factor in the lysis-lysogeny decision of lambda.     

Characterization of a conserved alpha-helical, coiled-coil motif at the C-terminal domain of the ATP-dependent FtsH (HflB) protease of Escherichia coli

FtsH (HflB) is an ATP-dependent protease found in prokaryotic cells, mitochondria and chloroplasts. Here, we have identified, in the carboxy-terminal region of FtsH (HfIB), a short alpha helix predicted of forming a coiled-coil, leucine zipper, structure. This region appears to be structurally conserved. The presence of the coiled-coil motif in the Escherichia coli FtsH (HflB) was demonstrated by circular dichroism and cross-linking experiments. Mutational analysis showed that three highly conserved leucine residues are essential for FtsH (HfIB) activity in vivo and in vitro. Purified proteins mutated in the conserved leucine residues, were found to be defective in the degradation of E. coli sigma(32) and the bacteriophage lambda CII proteins. In addition, the mutant proteins were defective in the binding of CII The mutations did not interfere with the ATPase activity of FtsH (HflB). Finally, the mutant proteins were found to be more sensitive to trypsin degradation than the wild-type enzyme suggesting that the alpha helical region is an important structural element of FtsH (HflB).