Site-directed mutagenesis of putative initiation codons (see M1 and M2 in Fig.  1 ) was performed using plasmid pX5 and the ‘QuickChange’ site-directed mutagenesis kit (Stratagene).

Two potential start codons, M1 and M2, were found in the chiB gene (Fig.  1 ).

Three cDNA clones resulted from a splicing that excised a 171-bp intron extending from 43 to 213 bp upstream of M2 (Fig.  1 , S1–S3), and the other eight clones resulted from an alternative splicing that excised a 138-bp intron extending from 43 to 180 bp upstream of M2 (Fig.  1 , S2–S3).

Indeed, we found high levels of the enzymatically active protein in the culture medium (Fig.  3 ), but the determination of the initiation codon revealed that ChiB lacks the hydrophobic N-terminal region upstream of M2 (Figs.  1 , 2 ), a sequence that could function as a respective secretion signal.