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In Mycobacterium tuberculosis, heparin-binding hemagglutinin (HBHAMT) has a relevant role in infection. It is also present in non-virulent mycobacteria and ancient actinobacteria, such as Rhodococcus opacus. To have a better understanding of the underlying mechanisms that shaped the evolutionary divergence of these proteins, we performed a comprehensive phylogenetic analysis of the regulatory sequences that drive the expression of hbha in saprophytic and pathogenic mycobacterial species. The alignment of the hbha loci showed the appeara