We present a new method for diparental mating with the outstanding advantage that counterselection of the Escherichia coli donor strain is not required. This improved method uses a new donor strain, E. coli ST18, a hemA deletion mutant defective in tetrapyrrole biosynthesis. The hemA mutation can be complemented by addition of 5-aminolevulinic acid. Therefore, counterselection is carried out only using standard media and growth conditions optimal for the recipient strain. Consequently, recipient strains are isolated in a significantly shorter period.