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ABSTRACT: The survival of a bacterium with a depleted oxygen or nutrient supply is important for its long-term persistence inside the host under stressful conditions. We studied a gene, dps, from Mycobacterium smegmatis, encoding a protein, Dps (for DNA binding protein from starved cells), which is overexpressed under oxidative and nutritional stresses and provides bimodal protection to the bacterial DNA. Characterization of the dps promoter in vivo is therefore important. We cloned a 1-kb putative promoter region of the dps gene of M. smegmatis in an Escherichia coli-Mycobacterium shuttle vector, pSD5B, immediately upstream of the lacZ gene. Promoter activities were assayed in vivo both in solid medium and in liquid cultures by quantitative beta-galactosidase activity measurements. To characterize the minimal promoter region, a 200-bp fragment from the whole 1-kb sequence was further cloned in the same vector, and in a similar way, beta-galactosidase activity was quantitated. Primer extension analysis was performed to determine the +1 transcription start site of the gene. Point mutations were inserted in the putative promoter sequences in the -10 and -20 regions, and the promoter sequence was confirmed. The promoter was not recognized by purified M. smegmatis core RNA polymerase reconstituted with purified Mycobacterium tuberculosis sigmaA or sigmaB during multiple- and single-round in vitro transcription assays. Promoter-specific in vivo pull-down assays with an immobilized 1-kb DNA fragment containing the dps promoter established that extracellular function sigma factors were associated with this starvation-inducible promoter. Single-round transcription at the dps promoter further supported the idea that only core RNA polymerase reconstituted with sigmaF or sigmaH can generate proper transcripts.
Journal of bacteriology 01/2008; 189(24):8973-81. · 3.94 Impact Factor
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ABSTRACT: Mycobacterium smegmatis Dps degrades spontaneously into a species in which 16 C-terminal residues are cleaved away. A second species, in which all 26 residues constituting the tail were deleted, was cloned, expressed and purified. The first did not bind DNA but formed dodecamers like the native protein, while the second did not bind to DNA and failed to assemble into dodecamers, indicating a role in assembly also for the tail. In the crystal structure of the species without the entire C-terminal tail the molecule has an unusual open decameric structure resulting from the removal of two adjacent subunits from the original dodecameric structure of the native form. A Dps dodecamer could assemble with a dimer or one of two trimers (trimer-A and trimer-B) as intermediate. Trimer-A is the intermediate species in the M. smegmatis protein. Estimation of the surface area buried on trimerization indicates that association within trimer-B is weak. It weakens further when the C-terminal tail is removed, leading to the disruption of the dodecameric structure. Thus, the C-terminal tail has a dual role, one in DNA binding and the other in the assembly of the dodecamer. M. smegmatis Dps also has a short N-terminal tail. A species with nine N-terminal residues deleted formed trimers but not dodecamers in solution, unlike wild-type M. smegmatis Dps, under the same conditions. Unlike in solution, the N-terminal mutant forms dodecamers in the crystal. In native Dps, the N-terminal stretch of one subunit and the C-terminal stretch of a neighboring subunit lock each other into ordered positions. The deletion of one stretch results in the disorder of the other. This disorder appears to result in the formation of a trimeric species of the N-terminal deletion mutant contrary to the indication provided by the native structure. The ferroxidation site is intact in the mutants.
Journal of Molecular Biology 08/2007; 370(4):752-67. · 4.00 Impact Factor
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ABSTRACT: Mycobacterium tuberculosis is a successful pathogen that overcomes numerous challenges presented by the immune system of the host. This bacterium usually establishes a chronic infection in the host where it may silently persist inside a granuloma until, a failure in host defenses, leads to manifestation of the disease. None of the conventional anti-tuberculosis drugs are able to target these persisting bacilli. Development of drugs against such persisting bacilli is a constant challenge since the physiology of these dormant bacteria is still not understood at the molecular level. Some evidence suggests that the in vivo environment encountered by the persisting bacteria is anoxic and nutritionally starved. Based on these assumptions, anaerobic and starved cultures are used as models to study the molecular basis of dormancy. This review outlines the problem of persistence of M. tuberculosis and the various in vitro models used to study mycobacterial latency. The basis of selecting the nutritional starvation model has been outlined here. Also, the choice of M. smegmatis as a model suitable for studying mycobacterial latency is discussed. Lastly, general issues related to oxidative stress and bacterial responses to it have been elaborated. We have also discussed general control of OxyR-mediated regulation and emphasized the processes which manifest in the absence of functional OxyR in the bacteria. Lastly, a new class of protein called Dps has been reviewed for its important role in protecting DNA under stress.
International Union of Biochemistry and Molecular Biology Life 04/2005; 57(3):149-59. · 3.51 Impact Factor
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ABSTRACT: The structure of the DNA binding protein from starved cells from Mycobacterium smegmatis has been determined in three crystal forms and has been compared with those of similar proteins from other sources. The dodecameric molecule can be described as a distorted icosahedron. The interfaces among subunits are such that the dodecameric molecule appears to have been made up of stable trimers. The situation is similar in the proteins from Escherichia coli and Agrobacterium tumefaciens, which are closer to the M.smegmatis protein in sequence and structure than those from other sources, which appear to form a dimer first. Trimerisation is aided in the three proteins by the additional N-terminal stretches that they possess. The M.smegmatis protein has an additional C-terminal stretch compared to other related proteins. The stretch, known to be involved in DNA binding, is situated on the surface of the molecule. A comparison of the available structures permits a delineation of the rigid and flexible regions in the molecule. The subunit interfaces around the molecular dyads, where the ferroxidation centres are located, are relatively rigid. Regions in the vicinity of the acidic holes centred around molecular 3-fold axes, are relatively flexible. So are the DNA binding regions. The crystal structures of the protein from M.smegmatis confirm that DNA molecules can occupy spaces within the crystal without disturbing the arrangement of the protein molecules. However, contrary to earlier suggestions, the spaces do not need to be between layers of protein molecules. The cubic form provides an arrangement in which grooves, which could hold DNA molecules, criss-cross the crystal.
Journal of Molecular Biology 07/2004; 339(5):1103-13. · 4.00 Impact Factor
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ABSTRACT: Three crystal forms of a DNA-binding protein from stationary phase cells (Dps) of Mycobacterium smegmatis have been grown. They are hexagonal, tetragonal and cubic. The structure of the cubic form, which has one subunit in the asymmetric unit, has been solved using the molecular-replacement method. The application of the crystallographic symmetry operations on the subunit leads to eight dodecamers with 23 symmetry in the unit cell.
Acta Crystallographica Section D Biological Crystallography 01/2004; 59(Pt 12):2254-6. · 12.62 Impact Factor
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ABSTRACT: Some members of the DNA-binding protein from stationary phase cells (Dps) family of proteins have been shown to play an important role in protecting microorganisms from oxidative or nutritional stress. Dps homologs have been identified in various bacteria such as Escherichia coli, Bacillus subtilis, and Listeria innocua. Recently we have reported the presence of a Dps homolog, Ms-Dps, in Mycobacterium smegmatis. Ms-Dps was found to have a nonspecific DNA binding ability. Here we have detected two stable oligomeric forms of Ms-Dps in vitro, a trimeric and a dodecameric form. Interestingly, the conversion of Dps from a trimeric to a dodecameric form takes place upon incubation at 37 degrees C for 12 h. These two oligomeric forms differ in their DNA binding properties. The dodecameric form is capable of DNA binding and forming large crystalline arrays with DNA, whereas the trimeric form cannot do so. However, even in the absence of DNA binding, the trimeric form has the capacity to protect the DNA against Fenton's-mediated damage. The protection is afforded by the ferroxidase activity of the trimer. However, the trimeric form cannot protect DNA from DNaseI attack, for which a direct physical shielding of DNA by the dodecamer is required. Thus we suggest that Ms-Dps provides a bimodal protection of DNA by its two different oligomeric forms.
Journal of Biological Chemistry 03/2003; 278(7):5235-41. · 4.77 Impact Factor
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ABSTRACT: Mycobacterium tuberculosis is a globally successful pathogen, infecting more than one third of total world's population. These bacteria have the remarkable ability to persist in the host for long periods of time unrecognized by the immune system and then to re-emerge later in life causing the disease. The physiology of such persistent or dormant bacilli is not very well characterized. Some evidence suggests that the dormant bacilli survive in a nutrient-deprived state that is similar to the stationary phase of the bacteria with respect to gene expression and physiology. Under this assumption we have studied the survival of Mycobacterium smegmatis in carbon starvation conditions as a model for mycobacterial persistence. M.smegmatis, being a fast-growing strain, serves as a good model to study starvation responses. Using the two-dimensional electrophoresis-based proteomics approach, we identified a protein which was found to be expressed preferentially under starvation conditions. This protein is homologous to a family of proteins called Dps (DNA binding Protein from Starved cells) that are known to protect DNA under various kinds of environmental stresses and its existence has, so far, not been reported in mycobacteria. Upon expression and purification of this protein, we observed that it has non-specific DNA-binding ability. Formation of a cage-like dodecamer structure is a characteristic feature of Dps. Using comparative modelling we were able to show that Dps from M.smegmatis could form a dodecamer structure similar to the crystal structure of Dps from Escherichia coli.
Protein engineering 07/2002; 15(6):503-12.
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