sed of three subunitsbut both are dispensable for growth. In contrast, the number of RNases H encoded in bacterial genomes is more variable, and more complicated in terms of being essential for viability. For example, Escherichia coli possesses one RNase HI and one RNase HII, while the genome of Bacillus subtilis contains two RNase H type 2- RNase HII and RNase HIII. In both of these species, there are contradictory reports regarding essentiality of RNase H enzymes, however, most recent studies show that they are dispensable under certain conditions. RNase HI has also been shown dispensable in Haemophilus influenzae. The genome of Mycobacterium tuberculosis contains one gene encoding an RNase H type II, rnhB, and one gene encoding a bifunctional protein, Rv2228c. Its N-terminal domain is homologous with eukaryotic and prokaryotic RNases H type I, while C-terminal domain is homologous with alpha ribazole phosphatase involved in cobalamin biosynthesis. Recombinant protein expression confirmed the activity of both SB203580 web domains in vitro . In turn, the genome of Mycobacterium smegmatis seems to PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19786614 encode four RNases H. Two of them belong to RNases H type I. The first one is encoded by rnhA and the RNase HI activity of the derived protein has been confirmed in vitro. The second is a homolog of Rv2228c of M. tuberculosis, MSMEG4305. Further, the genome of M. smegmatis encodes an RNase H type II, through gene rnhB. Additionally, the gene MSMEG5849 of M. smegmatis encodes a protein which presents RNase HII activity through domain Duf429 . A protein with limited homology to Duf429 can be found in M. tuberculosis. The summary of presumable RNase H encoding genes in E. coli, M. tuberculosis and M. smegmatis is presented in 2 / 20 RNase HI Is Essential in M. smegmatis Fig 1. Schematic representation of a mode of action of RNases H. RNases H cleave RNA from the RNA/DNA duplex. RNases H type I recognize two strands of the heteroduplex and cleave RNA when at least four ribonucleotides are present. In contrast, RNases H type II cleave even single ribonucleotides and recognize transition from DNA to RNA on a single strand. Materials and Methods In silico analysis Genes presumably encoding RNases H in M. smegmatis mc2 155, E. coli K12_MG1655 and M. tuberculosis H37Rv were identified and obtained from National Center for Biotechnology Information database. The span of 
the domains was defined using Simple Modular Architecture Research Tool . The homology between the domains was estimated using Basic PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19785045 Local Alignment Search Tool of NCBI. The alignments were visualized using MultiAlin and ESPript 3.0. Bacteria and culture conditions Cultures of E. coli T10 were carried out at 37C for 1820 h in liquid Luria-Bertani broth, and when necessary supplemented with antibiotics or other supplements or both at the following concentrations: kanamycin 50 g/ml; ampicillin 100 g/ml; X-gal 40 g/ml, IPTG 0.4 mM. Cultures of M. smegmatis during gene replacement were carried out in nutrient broth or 7H9 broth with or without oleic albumin dextrose catalase growth supplement and 0.05% Tween 80 at 37C or 28C. When necessary, media were supplemented with antibiotics or other supplements or both at the following concentrations: kanamycin 25 g/ml; X-gal 40 g/ml, 0.4% succinate, sucrose 2%, vitamin B12 10 g/ml. A list of M. smegmatis strains used in this study is presented in doi:10.1371/journal.pone.0126260.t001 3 / 20 RNase HI Is Essential in M. smegmatis 2 Characteristics ~~ Beta-amyloid