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. 2010 Oct;192(19):4868-75.
doi: 10.1128/JB.00550-10. Epub 2010 Jul 30.

Different roles of DosS and DosT in the hypoxic adaptation of Mycobacteria

Min-Ju Kim  1 , Kwang-Jin Park, In-Jeong Ko, Young Min Kim, Jeong-Il Oh
Affiliations

Different roles of DosS and DosT in the hypoxic adaptation of Mycobacteria

Min-Ju Kim et al. J Bacteriol. 2010 Oct.

Abstract

The DosS (DevS) and DosT histidine kinases form a two-component system together with the DosR (DevR) response regulator in Mycobacterium tuberculosis. DosS and DosT, which have high sequence similarity to each other over the length of their amino acid sequences, contain two GAF domains (GAF-A and GAF-B) in their N-terminal sensory domains. Complementation tests in conjunction with phylogenetic analysis showed that DevS of Mycobacterium smegmatis is more closely related to DosT than DosS. We also demonstrated in vivo that DosS and DosT of M. tuberculosis play a differential role in hypoxic adaptation. DosT responds to a decrease in oxygen tension more sensitively and strongly than DosS, which might be attributable to their different autooxidation rates. The different responsiveness of DosS and DosT to hypoxia is due to the difference in their GAF-A domains accommodating the hemes. Multiple alignment analysis of the GAF-A domains of mycobacterial DosS (DosT) homologs and subsequent site-directed mutagenesis revealed that just one substitution of E87, D90, H97, L118, or T169 of DosS with the corresponding residue of DosT is sufficient to convert DosS to DosT with regard to the responsiveness to changes in oxygen tension.

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Figures

FIG. 1.
FIG. 1.
Phylogenetic analysis of the GAF-A domains of mycobacterial DevS (DosS, DosT) homologs. Phylogenetic analysis was performed using the neighbor-joining method. Bootstrap values, expressed as percentages of 1,000 replications, are given at the nodes. The scale bar indicates 0.05 nucleotide substitution per nucleotide position. The strains relevant to this study are underlined. The GenBank accession numbers of the amino acid sequences are given in parentheses.
FIG. 2.
FIG. 2.
Expression of hspX in M. smegmatis strains grown under aerobic or hypoxic conditions. M. smegmatis strains with the expression plasmid pNBV1SHis (pNBV1::devS), pNBV1DosS (pNBV1::dosS), or pNBV1DosT (pNBV1::dosT) were used for complementation analysis. The complementation test was performed by determining the expression levels of hspX in M. smegmatis strains grown under either aerobic (O2+) or hypoxic (O2−) conditions for 20 h by means of RT-PCR. As controls, the wild-type (WT) and ΔdevS mutant strains of M. smegmatis containing the empty vector pNBV1 were included in the test. RT-PCR for the 16S rRNA gene was performed to ensure that the same amounts of total RNA were employed for RT-PCR.
FIG. 3.
FIG. 3.
Different responsiveness of DosS and DosT to hypoxia. The expression levels of hspX in the ΔdevS mutant strains of M. smegmatis containing the expression plasmid pNBV1, pNBV1SHis (pNBV1::devS), pNBV1DosS (pNBV1::dosS), or pNBV1DosT (pNBV1::dosT) were determined by means of RT-PCR (A) and qRT-PCR (B). M. smegmatis strains were grown under either aerobic (+O2) or hypoxic (−O2) conditions for 20 and 50 h. As controls, the ΔdevS mutant strains of M. smegmatis containing either the empty vector pNBV1 or pNBV1SHis were included in the experiment. The levels of mRNA specific for hspX were determined by qRT-PCR and normalized to those of 16S rRNA. Fold induction of hspX expression indicates the level of hspX mRNA in hypoxic culture relative to that in aerobic culture.
FIG. 4.
FIG. 4.
Schematic diagram depicting the chimeric DosST and DosTS HKs (A) and complementation analysis using the ΔdevS mutant strain of M. smegmatis with DosST and DosTS (B). The chimeric DosST HK consists of the GAF-A domain of DosS (M1 to A218) and the GAF-B and kinase domains of DosT (T217 to R573). The chimeric DosTS HK consists of the GAF-A domain of DosT (M1 to A216) and the GAF-B and kinase domains of DosS (T219 to Q578). The plasmids pNBV1DosST and pNBV1DosTS, containing the genes encoding DosST and DosTS, respectively, were introduced into the ΔdevS mutant strain of M. smegmatis, and the complementation test was performed by determining the expression levels of hspX in M. smegmatis strains grown under either aerobic (+O2) or hypoxic (−O2) conditions for 20 and 50 h by means of RT-PCR. RT-PCR for the 16S rRNA gene was performed to ensure that the same amounts of total RNA were employed in RT-PCR.
FIG. 5.
FIG. 5.
Multiple alignment of the GAF-A domains of mycobacterial DosS and DosT homologs. Multiple alignment was generated by using ClustalW. Identical and conservatively substituted residues are indicated by asterisks and colons, respectively. The arrows and coils indicate the positions of α-helices and β-strands, respectively. The amino acid residues conserved differentially between the DosS and DosT subclades are highlighted in black. The DosT homologs are shaded by the gray boxes. Abbreviations: Mbo, M. bovis; Mgi, M. gilvum; Mka, M. kasasii; Mma, M. marinum; Msm, M. smegmatis; Mtb, M. tuberculosis; Mul, M. ulcerans; Mva, M. vanbaalenii.
FIG. 6.
FIG. 6.
Effect of E87G, H89R, D90G, H97E, V108R, L118R, and T169N mutations on the sensory function of DosS in vivo. Complementation tests were performed by determining the expression levels of hspX in M. smegmatis strains grown under either aerobic (O2+) or hypoxic (O2−) conditions for 20 h by means of RT-PCR (A) and qRT-PCR (B). The ΔdevS mutant strains were complemented with the wild-type dosS and dosT as well as the E87G, H89R, D90G, H97E, V108R, L118R, and T169N mutant forms of dosS. As a control, the ΔdevS mutant strain of M. smegmatis containing the empty vector pNBV1 was included in the experiment. The strains were grown under either aerobic (O2+) or hypoxic (O2−) conditions for 20 h. The levels of mRNA specific for hspX were determined by qRT-PCR and normalized to those of 16S rRNA. Fold induction of hspX expression indicates the level of hspX mRNA of hypoxic culture relative to that of aerobic culture. All values provided are the averages of results of two independent determinations. Error bars indicate standard deviations.
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