Genome dynamics of Campylobacter jejuni in response to bacteriophage predation.

Campylobacter jejuni is a leading cause of food-borne illness. Although a natural reservoir of the pathogen is domestic poultry, the degree of genomic diversity exhibited by the species limits the application of epidemiological methods to trace specific infection sources. Bacteriophage predation is a common burden placed upon C. jejuni populations in the avian gut, and we show that amongst C. jejuni that survive bacteriophage predation in broiler chickens are bacteriophage-resistant types that display clear evidence of genomic rearrangements. These rearrangements were identified as intra-genomic inversions between Mu-like prophage DNA sequences to invert genomic segments up to 590 kb in size, the equivalent of one-third of the genome. The resulting strains exhibit three clear phenotypes: resistance to infection by virulent bacteriophage, inefficient colonisation of the broiler chicken intestine, and the production of infectious bacteriophage CampMu. These genotypes were recovered from chickens in the presence of virulent bacteriophage but not in vitro. Reintroduction of these strains into chickens in the absence of bacteriophage results in further genomic rearrangements at the same locations, leading to reversion to bacteriophage sensitivity and colonisation proficiency. These findings indicate a previously unsuspected method by which C. jejuni can generate genomic diversity associated with selective phenotypes. Genomic instability of C. jejuni in the avian gut has been adopted as a mechanism to temporarily survive bacteriophage predation and subsequent competition for resources, and would suggest that C. jejuni exists in vivo as families of related meta-genomes generated to survive local environmental pressures.

dnaC mutations suppress defects in DNA replication- and recombination-associated functions in priB and priC double mutants in Escherichia coli K-12.

PriA, PriB and PriC were originally discovered as proteins essential for the PhiX174 in vitro DNA replication system. Recent studies have shown that PriA mutants are poorly viable, have high basal levels of SOS expression (SOSH), are recombination deficient (Rec-), sensitive to UV irradiation (UVS) and sensitive to rich media. These data suggest that priA's role may be more complex than previously thought and may involve both DNA replication and homologous recombination. Based on the PhiX174 system, mutations in priB and priC should cause phenotypes like those seen in priA2:kan mutants. To test this, mutations in priB and priC were constructed. We found that, contrary to the PhiX174 model, del(priB)302 and priC303:kan mutants have almost wild-type phenotypes. Most unexpectedly, we then found that the priBC double mutant had very poor viability and/or a slow growth rate (even less than a priA2:kan mutant). This suggests that priB and priC have a redundant and important role in Escherichia coli. The priA2:kan suppressor, dnaC809, partially suppressed the poor viability/slow growth phenotype of the priBC double mutant. The resulting triple mutant (priBC dnaC809 ) had small colony size, recombination deficiency and levels of SOS expression similar to a priA2:kan mutant. The priBC dnaC809 mutant, however, was moderately UVR and had good viability, unlike a priA2:kan mutant. Additional mutations in the triple mutant were selected to suppress the slow growth phenotype. One suppressor restored all phenotypes tested to nearly wild-type levels. This mutation was identified as dnaC820 (K178N) [mapping just downstream of dnaC809 (E176G)]. Experiments suggest that dnaC820 makes dnaC809 suppression of priA and or priBC mutants priB and or priC independent. A model is proposed for the roles of these proteins in terms of restarting collapsed replication forks from recombinational intermediates.

A new component of bacteriophage Mu replicative transposition machinery: the Escherichia coli ClpX protein.

We have shown previously that some particular mutations in bacteriophage Mu repressor, the frameshift vir mutations, made the protein very sensitive to the Escherichia coli ATP-dependent Clp protease. This enzyme is formed by the association between a protease subunit (ClpP) and an ATPase subunit. ClpA, the best characterized of these ATPases, is not required for the degradation of the mutant Mu repressors. Recently, a new potential ClpP associated ATPase, ClpX, has been described. We show here that this new subunit is required for Mu vir repressor degradation. Moreover, ClpX (but not ClpP) was found to be required for normal Mu replication. Thus ClpX has activities that do not require its association with ClpP. In the pathway of Mu replicative transposition, the block resides beyond the strand transfer reaction, i.e. after the transposition reaction per se is completed, suggesting that ClpX is required for the transition to the formation of the active replication complex at one Mu end. This is a new clear-cut case of the versatile activity of polypeptides that form multi-component ATP-dependent proteases.

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.