ABSTRACT RNase H enzymes participate in various processes that require processing of RNA:DNA hybrids, including DNA replication, transcription, and ribonucleotide excision from DNA. Mycobacteria encode multiple RNase H enzymes and prior data indicates that RNase HI activity is essential for mycobacterial viability. However, the additional roles of mycobacterial RNase Hs are unknown, including whether RNase HII (RnhB and RnhD) excises chromosomal ribonucleotides misincorporated during DNA replication and whether individual RNase HI enzymes (RnhA and RnhC) mediate additional phenotypes. We find that loss of RNase HII activity in M. smegmatis (through combined deletion of rnhB/rnhD ) or individual RNase HI enzymes, does not affect growth, hydroxyurea sensitivity, or mutagenesis, whereas overexpression of either RNase HII severely compromises bacterial viability. We also show that deletion of rnhC , which encodes a protein with an N terminal RNase HI domain and a C terminal acid phosphatase domain, confers sensitivity to rifampicin and oxidative stress as well as loss of light induced carotenoid pigmentation. These phenotypes are due to loss of the activity of the C terminal acid phosphatase domain rather than the RNase HI activity, suggesting that the acid phosphatase activity may confer rifampicin resistance through the antioxidant properties of carotenoid pigment production.

The C-terminal acid phosphatase module of the RNase HI enzyme RnhC controls rifampicin sensitivity and light-dependent colony pigmentation of<i>Mycobacterium smegmatis</i>

The C-terminal acid phosphatase module of the RNase HI enzyme RnhC controls rifampicin sensitivity and light-dependent colony pigmentation ofMycobacterium smegmatis