The Mycobacterium abscessus mbtE Analog MAB_2122 Is, in Contrast to the mbtE Analog MAB_2248c, Dispensable for Normal Growth in Low-Iron Conditions

R ecently, we characterized MAB_2248c as an analog of the mbtE gene of Mycobacterium tuberculosis (1).A transposon (Tn) disruption mutant of MAB_2248c grew slowly on media relatively poor in iron, and this slow growth phenotype was salvaged by mycobactin J (MJ), hemin, blood, and also by albumin.The transcription of MAB_2248c (the part before the transposon insertion) was induced in the transposon mutant, as well as in wild-type (WT) bacteria when grown in iron-poor conditions.M. tuberculosis, as well as most other mycobacteria, has one copy of mbtE (2, 3).Interestingly, in Mycobacterium abscessus, there are 2 genes with similarity to the mbtE of M. tuberculosis, and both are annotated as "mbtE" in databases such as Mycobrowser (https://mycobrowser.epfl.ch/).Whereas MAB_2248c has 51.6% similarity to M. tuberculosis mbtE, the other gene, MAB_2122, has 55% similarity-although the similarity between the two is only 42.8%.Genes around MAB_2122 are also designated mbt genes (Fig. 1A).The transcription of MAB_2122 did not increase in the MAB_2248c transposon mutant, nor in WT bacteria when grown in iron-poor conditions (1).All this raised questions about the role of MAB_2122-whether this was a functional mbtE analog, and whether these two genes were redundant or acted in parallel.To test whether MAB_2122 had a role in normal growth and iron acquisition in M. abscessus, we constructed a targeted deletion mutant of this gene.This was done by electroporating a linear deletion construct, with the flanking regions of MAB_2122 and the Zeocin resistance gene (Zeo r ) between them, into M. abscessus (ATCC 19977) bearing plasmid pJV53, used for recombineering (4).Transformants were plated onto 7H9-glycerol-ADS (albumin-dextrose)-MJ agar plates (to ensure that even a strict iron auxotroph would grow) supplemented with Zeocin (66 mg/mL).One of the resulting colonies was examined using PCR, which yielded a 6,530-bp product in WT, but only a 3,700-bp product in the mutant, where most of the MAB_2122 gene was replaced by Zeo r (Fig. 1A shows a graphic representation; Fig. 1B shows the PCR electrophoresis).The colony was thus confirmed to be a MAB_2122 deletion mutant and was named mDB364.The previously created MAB_2248c transposon mutant was named mDB274.To examine whether the DMAB_2122 mutant was an iron auxotroph and was dependent on iron or mature mycobactin J (as is mDB274), we grew WT, mDB274, and mDB364 to an optical density at 600 nm (OD 600 ) of 0.2, washed them twice in phosphatebuffered saline (PBS), diluted them to a concentration of ;2,500 CFU/mL, and spotted 2 mL onto 7H9-glycerol plates supplemented with either ADS, mycobactin J, hemin, or no supplementation (Fig. 1C).All three isolates grew well on plates supplemented with ADS, MJ, or hemin.On the plate with 7H9-glycerol only, the MAB_2248c Tn mutant grew poorly (if at all) (Fig. 1C, bottom right, blue arrow)-as expected.In contrast, the DMAB_2122 mutant did not exhibit any appreciable growth retardation (yellow arrow) compared to the WT.We then analyzed by comparison the sequences of all three genes, MAB_2248c, MAB_2122, and the M. tuberculosis mbtE (Rv2380c).Although all three genes appear to share significant homology, and by length MAB_2122 is actually closer to Rv2380c than MAB_2248c, when

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