Mycobacterial phage D29 has a broad host range which includes pathogenic members of the genus Mycobacterium.  Despite being well studied, the nature of the host cell receptor is not known and resistance to this phage is not well described. Here a phage-resistant mutant of M. smegmatis MC² 155 was isolated by continuous exposure to low numbers of D29. Analysis of the genome sequences of the wildtype and resistant mutant identified one SNP (G insertion) in an unassigned ORF. Protein prediction tools indicated that the protein encoded by this ORF has two hydrophobic a-helices close to the N-terminus which may form a membrane spanning domain. Analysis of sequences upstream of the candidate gene identified a second ORF which encodes a homologue of the IclR (Isocitrate Lyase Regulator)-family transcriptional regulators, suggesting these two genes may form a two-component signal transduction system. Sequence analysis of this potential two gene operon identified a candidate transcriptional terminator after the second gene.  Analysis of mRNA using qPCR probes targeting each of the ORFs and the intergenic region indicated these genes are expressed and do form a bicistronic operon. Interestingly, despite the known broad host range of phage D29, homologues of these two genes are not found in genomes of pathogenic mycobacteria that the phage is also known to infect. This suggests this gene system may regulate expression of the phage receptor protein in M. smegmatis rather than being the target for D29 infection.