RifR mutations in the beginning of the Escherichia coli rpoB gene

In Escherichia coli, mutations conferring rifampicin (Rif) resistance map to the rpoB gene, which encodes the 1342-amino acid subunit of RNA polymerase. Almost all sequenced RifR mutations occur within the Rif region, encompassing rpoB codons 500–575. A strong Rif^R mutation lying outside the Rif region, which changed Val¹⁴⁶ to Phe was previously reported, but was not recovered in subsequent studies. Here, we used site-directed mutagenesis followed by selection on Rif to search for Rif^R mutations in the evolutionarily conserved segment of rpoB around codon 146. Strong Rif^R mutations were obtained when Val¹⁴⁶ was mutated, and several weak Rif^R mutations were also isolated near position 146. The results define a new, N-terminal cluster of Rif^R mutations, in addition to the classical central Rif region.