Characterization of a restriction modification system from the commensal Escherichia coli strain A0 34/86 (O83:K24:H31)

Abstract

Background: Type I restriction-modification (R-M) systems are the most complex restriction enzymes discovered to date. Recent years have witnessed a renaissance of interest in R-M enzymes Type I. The massive ongoing sequencing programmes leading to discovery of, so far, more than 1 000 putative enzymes in a broad range of microorganisms including pathogenic bacteria, revealed that these enzymes are widely represented in nature. The aim of this study was characterisation of a putative R-M system EcoA0ORF42P identified in the commensal Escherichia coli A0 34/86 (O83: K24: H31) strain, which is efficiently used at Czech paediatric clinics for prophylaxis and treatment of nosocomial infections and diarrhoea of preterm and newborn infants.

Results: We have characterised a restriction-modification system EcoA0ORF42P of the commensal Escherichia coli strain A0 34/86 (O83: K24: H31). This system, designated as EcoAO83I, is a new functional member of the Type IB family, whose specificity differs from those of known Type IB enzymes, as was demonstrated by an immunological cross-reactivity and a complementation assay. Using the plasmid transformation method and the RM search computer program, we identified the DNA recognition sequence of the EcoAO83I as GGA(8N)ATGC. In consistence with the amino acids alignment data, the 3' TRD component of the recognition sequence is identical to the sequence recognized by the EcoEI enzyme. The A-T (modified adenine) distance is identical to that in the EcoAI and EcoEI recognition sites, which also indicates that this system is a Type IB member. Interestingly, the recognition sequence we determined here is identical to the previously reported prototype sequence for Eco377I and its isoschizomers.

Conclusion: Putative restriction-modification system EcoA0ORF42P in the commensal Escherichia coli strain A0 34/86 (O83: K24: H31) was found to be a member of the Type IB family and was designated as EcoAO83I. Combination of the classical biochemical and bacterial genetics approaches with comparative genomics might contribute effectively to further classification of many other putative Type-I enzymes, especially in clinical samples.

Figure 1

Detection of Hsd polypeptides in total cell extracts using the anti-EcoAI antibody. lane 1, purified EcoAI enzyme – standard; lane 2, extract from E. coli DH10B [pFFP30] expressing the EcoAI system; lanes 3, 4, extracts from E. coli DH10B [BAC-C4/1] expressing the EcoAO83I; lane 5,E. coli A0 34/86 (O83: K24: H31), EcoAO83I expressed from the chromosome; lane 6,E. coli DH10B [BAC] – control with no R-M system; The position of the Hsd subunits is indicated, molecular weights in kD are in bracket.

Figure 2

Specific recognition site of the EcoAO83I R-M enzyme. EcoAO83I site, GGA(8N)ATGC and surrounding DNA sequences on the pL series lambda clones and two pE series clones. pEco377I [22] contains 20 mer oligoduplex shown above, which is cloned at the unique EcoRV site of pMECA [24]