Pathogenic mycobacteria possess two homologous chaperones encoded by cpn60.1 and cpn60.2. Cpn60.2 is essential for survival, providing the basic chaperone function, while Cpn60.1 is not. In the present study we show that inactivation of the Mycobacterium bovis BCG cpn60.1 (Mb3451c) gene does not significantly affect bacterial growth in 7H9 broth, but that this knockout mutant (cpn60.1) forms smaller colonies on solid 7H11 medium than the parental and complemented strains. When growing on Sauton medium, the cpn60.1 mutant exhibits a thinner surface pellicle and is associated with higher culture filtrate protein content and, coincidently, with less protein in its outermost cell-envelope in comparison with the parental and complemented strains. Interestingly, in this culture condition the cpn60.1 mutant is devoid of phthiocerol dimycocerosates and its mycolates are two carbon-atoms longer than those of the wild type, a phenotype that is fully reversed by complementation. In addition, cpn60.1 bacteria are more sensitive to stress induced by H2O2 but not by SDS, high temperature or acidic pH. Taken together, all these data indicate that the cell wall of the cpn60.1 mutant is impaired. Analysis by 2-dimensional gel electrophoresis-mass spectrometry reveals the upregulation of a few proteins such as FadA2 and ICL in the cell extract of the mutant, whereas more profound differences are found in the composition of the mycobacterial culture filtrate e.g. the well-known hsp65 chaperonin Cpn60.2 is particularly abundant and increases about 200-fold in the filtrate of the cpn60.1 mutant. In mice, the cpn60.1 mutant is less persistent in lungs and, to a lesser extent, in spleen, but it induces a comparable mycobacteria-specific IFN-gamma production and protection against Mycobacterium tuberculosis H37Rv challenge as the parental and complemented BCG strains. Thus, by inactivating the cpn60.1 gene in M. bovis BCG we show that Cpn60.1 is necessary for the integrity of the bacterial cell wall, is involved in resistance to H2O2 induced stress but not essential for its vaccine potential
