Bacteriophage lambda’s high mutation rate is not solely due to MMR inefficiency

A single study estimated that the mutation rate of lambda is 400-fold higher than that of its host, Escherichia coli (7.8x10-8 per base replicated for lambda vs. 2x10-10 for E. coli). Mutation rate is determined by replication fidelity and correction of errors (mainly by mismatch repair, MMR). Because E. coli DNA polymerase III is assumed to replicate both genomes, the high mutation rate of lambda was supposed to be due to MMR inefficiency during infection. This inefficiency was suspected to results from uncomplete adenine methylation of GATC sites in lambda’s DNA by Dam, as methylation of GATC  is important for strand recognition and repair of mismatches by the MMR system.

In this work, we re-evaluated lambda mutation rate, and investigated the mechanisms of mutagenesis in lambda, notably the effect of Dam methylation. First, we estimated lambda mutation rate by a whole genome sequencing approach (Duplex Sequencing or DS), that gave a slightly lower lambda mutation rate than previously estimated (1.5x10-8) and confirmed the poor efficiency of MMR on lambda DNA (2-fold increase for lambda vs. 150-fold increase for the host). In addition, we performed Fluctuation Assays (FA), that consist in determining the number of mutations in a target locus (here cII lambda gene) in a large number of parallel cultures, in different E. coli backgrounds. We found that the mutation rate of lambda with FA is close to that obtained with the DS experiment in the WT cells (1.8x10-8), but not in MutS E. coli cells (1.8x10-7). We also found that dam deletion increased lambda’s mutation rate to an intermediate level compared to the MutS deletion, as observed in E. coli. In addition, dam overexpression did not diminished lambda mutation rate, suggesting that a default of methylation does not explain lambda mutation rate. Finally, we determined the mutation spectrum of lambda by sequencing of a large number of cII mutants. This revealed that lambda mutation rate is different from that of either WT of MMR deficient E. coli, and is not indicative of any known pathway of mutagenesis in E. coli.

Altogether these results indicate that lambda’s high mutation rate is not explained by MMR inactivity, as previously thought,but most likely by molecular mechanisms that modify the rate and the nature of Pol III replication errors on phage DNA. We are currently performing a Mutation Accumulation assay, to confirm the mutation spectrum on the whole genome of lambda.