Supplemental Experimental Procedures Construction of the ΔfadD22 Mutant

The fadD22 deletion (ΔfadD22) mutant was engineered using the p2NIL/pGOAL method as reported (Parish and Stoker, 2000). A ΔfadD22 deletion cassette was constructed containing a 5´ arm (1.2-kb region upstream of fadD22 and fadD22’s start codon) and a 3 ´ arm (last ten codons of fadD22 and the downstream 810-bp segment). The C-terminal codons were preserved due to sequence overlap with pks15/1. Each arm was generated by PCR using M. bovis genomic DNA as template. Primers fadD22opL (5´-aaattcggtgctgtcaagcaaatccgcatcggcaccccaaataa-3´) and fadD22ipL (5´-gtcatccaataaatgcagctaatcgattcgattccgaacttgggaagtga-3’) were used to generate the 3 ’ arm. Primers fadD22opR (5´-agatttccagcgcaccgaatttcggtttcgaattggcggtacgca-3´) and fadD22ipR (5´-ggaatcgattagctgcattattggatgaccgccctagcgcgcca-3´) were used to generate the 5´ arm. Each PCR-generated arm was cloned into pCR2.1TOPO (Invitrogen) and sequenced to verify sequence fidelity. The 5 ´ arm fragment was recovered from the pCR2.1TOPO construct as a ClaI-XhoI fragment and ligated to the pCR2.1TOPO-3 ´ arm construct digested with ClaI and XhoI to fuse the 5 ´ and 3 ´ arms. The deletion cassette was then excised from this pCR2.1TOPO construct using NotI and SalI and ligated to p2NIL linearized by NotI and SalI digestion. The resulting p2NIL-∆fadD22 deletion cassette construct and the pGOAL19 plasmi