Figure 1
Analysis of HadDMtb sequence, hadD chromosomic region and Mtb ∆hadD mutant. (A) Sequence alignment of HadDMtb (Rv0504c) with HadDMsm (MSMEG_0948) and HadBMtb (Rv0636) proteins. Black and gray shadings indicate strictly conserved and similar residues, respectively. HadDMtb shares a sequence identity of 63% with HadDMsm (68% using BlastP alignment) and only 19% with HadBMtb (Clustal Omega scores). HadDMtb bears a degenerate hydratase 2 motif ‘F-x(2)-a-x(2)-D-x(2)-P-x-H-x(5)-A’ (uppercase: strictly conserved; lowercase: similar residue) indicated by blue stars; the putative catalytic Asp and His residues are labeled by red stars. The hydratase 2 motif ‘[YF]-x(1,2)-[LIVG]-[STGC]-G-D-x-N-P-[LIV]-H-x(5)-[AS]’ of HadBMtb13 is indicated by black stars. Alignment was performed by using Clustal Omega program, and the figure was shaped with Box shade. Database accession numbers: HadDMtb, P9WFK3 (166 aa); HadDMsm, A0QR13 (177 aa); HadBMtb, I6WYY7 (142 aa). (B) Genomic organization of hadD gene region in M. smegmatis, Mtb H37Rv and Mtb ΔhadD strains. Matching genes are drawn with identical colors. Mtb ΔhadD mutant strain was produced by an in-frame deletion of a 308 bp internal fragment (dashed lines) of hadDMtb gene (501 bp). In Mtb, Rv0502 (1,077 bp), cmaA2 (909 bp), serB1 (1,122 bp) and mmpS2 (444 bp) are annotated as encoding a conserved protein, the mycolic acid cyclopropane synthetase CmaA2, a possible phosphoserine phosphatase SerB1 and a probable conserved membrane protein MmpS2, respectively. The distinct genes in M. smegmatis, MSMEG_0946, MSMEG_0950 and MSMEG_0951, are annotated as encoding a NAD-dependent epimerase/dehydratase family protein, a hypothetical protein and the glutaredoxin 2, respectively. (C) Verification of hadDMtb gene deletion by PCR analysis. The primers (x and y; symbolized by black arrows in panel B) used for the PCR are located outside the hadDMtb gene. The genomic DNA of each strain was used as a template. HadDMtb gene length: 501 bp; ΔhadDMtb gene length: 193 bp. L: DNA ladder.
