Type 1 restriction and modification systems

Bacterial Type I restriction and modification (R-M) systems distinguish between their own DNA and that of "foreign" DNA by the methylation state of a target sequence. Previously, Type I systems had been divided into three discrete families (IA, lB and IC) based on DNA hybridisation, antibody reactions and complementation tests. I have added a fourth family, ID, by characterising the system from Salmonella enterica serovar blegdam. I identified the hsd genes; firstly by sequence analysis, then by both functional and mutational analysis. I helped to determine the target recognition sequence (CGA(N6)TACC). I then extended the ID family to include ECOR9I based on sequence similarity and complementation tests. I investigated the diversity and distribution of Type I systems by firstly using the IDspecific probe to screen 37 strains from the ECOR collection; a reference set of Escherichia coli strains that represents the genetic diversity of this species. I found four probe-positive strains. I cloned the hsd genes from one of these strains and found that the specificity of the R-M system was different to that of StySBLI. Secondly, I searched the databases of genomic sequences for amino acid sequences similar to those predicted for the archetypal members of the four Type I families. It was already known that the degree of amino acid identity between families is low (20 30%) whereas within families it is >80%. In three of the screens I identified putative Type I R-M systems across a wide range of bacterial species. Each of the four screens identified HsdM polypeptides with >45% identity in different phyla. The hybridisation of DNA from the ECOR strains and the in silico screen of the databases showed that the distribution of hsd genes among different bacterial populations was not consistent with the relatedness of the strains and therefore suggests horizontal transfer. HsdR contains motifs, characteristic of DEAD-box proteins, that have been identified in ATP-dependent helicases and translocases. I identified two highly conserved sequences (regions X and Y) in HsdR, in addition to the seven motifs (I VII) already identified. Region X was later proven to identify the active site for endonuclease activity and region Y was later identified as DEAD-box motif IV. I showed that two conservative mutations in motif II inactivated the HsdR polypeptide. HsdR is also implicated in restriction alleviation (RA) after DNA damage. I sequenced the hsdR gene from an E. coli strain encoding EcoR124I, but impaired in RA, and found that it contained a mutation. Using site directed mutagenesis I showed that reverting this mutation to wild-type sequence restored the wild-type phenotype. RA is also associated with the acquisition of a new R-M system, whereby the host DNA is protected from restriction until it can be modified with the new specificity. An hsdC derivative of E. co/i C dies after the acquisition of the hsd genes encoding either EcoKI or EcoAl. I helped to show that the hsdC strain had a mutation in c/pX but not clpP. C1pX and C1pP had previously been shown to be necessary for the efficient transmission of EcoKI (Type IA) and EcoAl (Type TB) RM systems by conjugation, transformation and transduction, although they do not affect members of the two families in the same way. I also corrected the published hsdR sequence of EcoRl 241111. The addition of a C near the 3 '-end made a frameshift that added an extra five amino acids to the predicted polypeptide sequence.