Transcriptional profiling of Mycobacterium tuberculosis Rv3852 (hns) knockout mutant. Mycobacterium tuberculosis H37Rv

A handful of nucleoid-associated proteins (NAPs) regulate the vast majority of genes in a bacterial cell. H-NS, the Histone-like Nucleoid-Structuring protein, is one of these NAPs and protects Escherichia coli from foreign gene expression. Though lacking any sequence similarity with E. coli H-NS, Rv3852 was annotated as the H-NS ortholog in Mycobacterium tuberculosis, as it resembles human histone H1. The role of H-NS was thoroughly investigated by immunoblotting, subcellular localization, construction of an unmarked hns deletion in the M. tuberculosis genome and subsequent analysis of the resulting Δhns strain. We found that H-NS was predominantly present in the logarithmic growth phase with a decrease in protein abundance in stationary phase. Furthermore, it was strongly associated with the cell membrane and not detected in the cytosolic fraction, nor was it secreted. The Δhns strain displayed no growth defect or morphological abnormalities. Quantitative measurement of nucleoid localization in Δhns compared to the parental H37Rv strain showed no difference in nucleoid position or spread. Infection of macrophages as well as severe combined immunodeficient (SCID) mice demonstrated that loss of H-NS had no detectable influence on the virulence of M. tuberculosis. Only few genes wre differentially expressed in the Δhns strain. We thus conclude that M. tuberculosis H-NS is not involved in pathogenesis and is not a typical NAP. Overall design: The hns knockout mutant (named Δhns) was constructed using a two-step allelic exchange method based on the suicide vector pJG1100. Phenotypic properties and transcriptional profile of Δhns was compared to the parental wild-type strain (H37Rv). Since hns was reported to be part of an operon with the downstream gene rraA, we first mapped the 5’-end of the bicistronic mRNA to ensure that the promoter region was not deleted and rraA expression was not affected in the mutant.