CUG as a mutant start codon for cat-86 and xylE in Bacillus subtilis

The cat-86 gene specifies chloramphenicol acetyltransferase (CAT). The cat-86 start codon is UUG, although related genes have AUG as the start codon. Changing the start codon to AUG increased expression of cat-86 by 36% in Bacillus subtilis. Changing the start codon to GUG and CUG decreased expression to 65% and 30%, respectively, of the level obtained when AUG was the start codon. CUG has not been previously shown to function as a start codon in B. subtilis. N-terminal sequencing of purified CAT protein specified by the CUG mutant, revealed that CUG was indeed the start codon and specified methionine. The gene xylE, which specifies catechol 2,3-dioxygenase, has AUG as its start codon. Changing the start codon for xylE to CUG decreased expression by 98%. However, when the ribosome-binding site sequence for xylE was optimized and the spacing between it and the start codon was increased to 8 nucleotides, xylE activity increased to 13% of the activity observed for AUG. CUG did not function efficiently as a start codon for cat-86 in Escherichia coli. These data suggest conditions under which CUG can function, with modest efficiency, as a start codon in B. subtilis.