Lucie Desmet

Virulence in bacteriophage Mu: a case of trans-dominant proteolysis by the Escherichia coli Clp serine protease.

The importance of proteases in gene regulation is well documented in both prokaryotic and eukaryotic systems. Here we describe the first example of genetic regulation controlled by the Escherichia coli Clp ATP‐dependent serine protease. Virulent mutants of bacteriophage Mu, which carry a particular mutation in their repressor gene (vir mutation), successfully infect Mu lysogens and induce the resident Mu prophage. We show that the mutated repressors have an abnormally short half‐life due to an increased susceptibility to Clp‐dependent degradation. This susceptibility is communicated to the wild type repressor present in the same cell, which provides the Muvir phages with their trans‐dominant phenotype. To our knowledge this is the first case where the instability of a mutant protein is shown to trigger the degradation of its wild type parent.

Bacteriophage Mu repressor as a target for the Escherichia coli ATP-dependent Clp Protease.

Bacteriophage Mu repressor, which is stable in its wildtype form, can mutate to become sensitive to its Escherichia coli host ATP‐dependent ClpXP protease. We further investigated the determinants of the mutant repressors sensitivity to Clp. We show the crucial importance of a C‐terminal, seven amino acid long sequence in which a single change is sufficient to decrease the rate of degradation of the protein. The sequence was fused at the C‐terminal end of the CcdB and CcdA proteins encoded by plasmid F. CcdB, which is naturally stable, was unaffected, while CcdA, which is normally degraded by the Lon protease, became a substrate for ClpXP while remaining a substrate for Lon. In agreement with the current hypothesis on the mechanism of recognition of their substrates by energy‐ dependent proteases, these results support the existence, on the substrate polypeptides, of separate motifs responsible for recognition and cleavage by the protease.

Virulent mutants of temperate phage Mu-1

SummaryVirulent mutants of phage Mu have been isolated after mutagenesis. The virulent phenotype results from most probably 2 mutations located in the c-A region of the Mu genome.Vir mutants are trans-dominant; they induce the resident prophage upon infection in broth of any Mu lysogen. They however form plaques only on certain lysogens, that are monolysogenic for a mutant prophage. We further isolated secondary mutations in Mu Vir which suppress the virulent phenotype.

The products of gene A of the related phages Mu and D108 differ in their specificities.

SummaryBy recombination between different mutants of mutator phages Mu and D108, we isolated a set of viable hybrids. The structure of the hybrids was analyzed by digestion with different restriction enzymes. Genetic studies show that hybrids which carry the left end of the Mu genome complement a mini-Mu deleted from within the A gene as well as Mu while hybrids with the left end of the D108 genome or D108 do not. Vice versa, hybrids with the left end of the D108 genome or D108, but not hybrids with the left end of the Mu genome or Mu complement a mini-D108 deleted from within the A gene. The nucleotide sequence of the A gene of Mu and its equivalent on D108 are mainly similar except on their left end. These observations demonstrate that the two pA products, although only partially different, have different specificities.

Mapping of the Modification Function of Temperate Phage Mu-1

SummaryUsing internal deletions in the Mu genome, we have mapped the gene coding for Mu modification in the β segment of Mu DNA.

Characterization of amber mutations in bacteriophage Mu transposase: A functional analysis of the protein

We have characterized a series of amber mutations in the A gene of bacteriophage Mu encoding the phage transposase. We tested different activities of these mutant proteins either in a sup0 strain or in different sup bacteria. In conjunction with the results described in the accompanying paper by Bétermier et al. (1989) we find that the C‐terminus of the protein is not absolutely essential for global transposase function, but is essential for phage growth. Specific binding to Mu ends is defined by a more central domain. Our results also reinforce the previous findings (Bétermier et al., 1987) that more than one protein may be specified by the A gene.

Genetic analysis of Mu or mini-Mu containing F′ pro lac episomes after prophage induction

SummaryWe have investigated the fate of different F pro lac episomes carrying a Mu or mini-Mu, after induction of the Mu or mini-Mu prophage, by looking at the frequencies of transfer of the episome and of one chromosomal marker. During the first 10 min after induction the frequency of chromosome mobilization increases while the frequency of episome transfer decreases. This suggests that the F interacts with the chromosome through some kind of Mu mediated process. Later the transfer of both the episome and chromosomal markers is inhibited. Possible reasons for this inhibition are discussed.

The Escherichia coli DnaK chaperone machine and bacteriophage Mu late transcription

Bacteriophage Mu does not grow on temperature‐sensitive E. coli dnaK mutants at elevated temperatures because of a defect in late transcription. As the Mu‐encoded C protein is required for activation of transcription from the phage late promoters, we attempted to determine if DnaK and its accessory proteins DnaJ and GrpE are required for synthesis of C protein or at a later step. We found that the chaperones act in Mu late transcription beyond C‐protein synthesis, and that C‐protein stability is decreased in the mutant hosts. This suggests that the DnaK chaperone machine may be required for the proper folding and/or multi‐merization of C protein.

Simultaneous expression of a bacteriophage-mu transposase and repressor - a way of preventing killing due to mini-mu replication

In vitro studies of bacteriophage Mu transposition have shown that the phage‐encoded transposase and repressor bind the same sequences on the phage genome. We attempted to test that prediction in vivo and found that Mu repressor directly inhibits transposition. We also found that, in the absence of repressor, constitutive expression of Mu transposition functions pA and pB is lethal in Escherichia coli strains lysogenic for a mini‐Mu and that this is the result of intensive replication of the mini‐Mu. These findings have important consequences where such mini‐Mus are used as genetic tools. We also tested whether in Erwinia chrysanthemi the effect of transposition functions on a resident mini‐Mu was the same as in E. coli. We observed that expression of pA alone was lethal in E. chrysanthemi and that a large fraction of the survivors underwent precise excision of the mini‐Mu.

Amber suppressors of Erwinia chrysanthemi

Mutations trp1 and thyA1, both of a polyauxotrophic derivative of the Erwinia chrysanthemi strain B374, were characterized as amber mutations with an Escherichia coli suppressor, supA1P2, which inserts a glutamine in response to UAG. Simultaneous reversion of both mutations allowed us to isolate amber suppressor mutants of E. chrysanthemi. These suppressors were tested with a set of amber mutants of bacteriophage Mu which had been previously characterized on E. coli. The two independently isolated suppressors behaved as supD and supE mutants, respectively, of E. coli.