Distinct properties of Mycobacterium tuberculosis single-stranded DNA binding protein and its functional characterization in Escherichia coli

Abstract

Single-stranded DNA binding proteins (SSBs) play an essential role in various DNA functions. Characterization of SSB from Mycobacterium tuberculosis, which infects nearly one-third of the world's population and kills about 2-3 million people every year, showed that its oligomeric state and various in vitro DNA binding properties were similar to those of the SSB from Escherichia coli. In this study, use of the yeast two-hybrid assay suggests that the ECO:SSB and the MTU:SSB are even capable of heterooligomerization. However, the MTU:SSB failed to complement a Deltassb strain of E. coli. The sequence comparison suggested that MTU:SSB contained a distinct C-terminal domain. The C-terminal domain of ECO:SSB interacts with various cellular proteins. The chimeric constructs between the N- and C-terminal domains of the MTU:SSB and ECO:SSB exist as homotetramers and demonstrate DNA binding properties similar to the wild-type counterparts. Despite similar biochemical properties, the chimeric SSBs also failed to complement the Deltassb strain of E.coli. These data allude to the occurrence of a 'cross talk' between the N- and the C-terminal domains of the SSBs for their in vivo function. Further, compared with those of the ECO:SSB, the secondary/tertiary interactions within MTU:SSB were found to be less susceptible to disruption by guanidinium hydrochloride. Such structural differences could be exploited for utilizing such essential proteins as crucial molecular targets for controlling the growth of the pathogen.

Figure 1

Interaction between MtuSSB and EcoSSB using the yeast two-hybrid system. The host strain alone and the transformants were streaked on medium containing histidine, tryptophan and leucine (A) or on medium lacking histidine, tryptophan and leucine but containing 10 mM 3-aminotriazole (B). Sector 1, HF7c; sector 2, pGBT9EcoSSB and pGAD424EcoSSB; sector 3, pGBT9EcoSSB and pGAD424MtuSSB; sector 4, pGBT9EcoSSB; sector 5, pGBT9MtuSSB and pGAD424MtuSSB; sector 6, pGBT9MtuSSB and pGAD424EcoSSB.

Figure 2

(A) Comparison of EcoSSB and MtuSSB sequences. Shading indicates residues that are identical/conserved between the two SSBs. (B) Schematic representation of the chimeric constructs as shown with respect to EcoSSB and MtuSSB (shaded and hatched regions, respectively). Dashed box, linker region missing in MtuSSB and EcoMtuSSB; unfilled box, the corresponding region in EcoSSB, EcoSSBG129S and MtuEcoSSB; downward arrow, BamHI site, found in MtuSSB, EcoSSBG129S and the fusion constructs. Sizes of the SSBs (amino acids) are shown on the right.

Figure 2

(A) Comparison of EcoSSB and MtuSSB sequences. Shading indicates residues that are identical/conserved between the two SSBs. (B) Schematic representation of the chimeric constructs as shown with respect to EcoSSB and MtuSSB (shaded and hatched regions, respectively). Dashed box, linker region missing in MtuSSB and EcoMtuSSB; unfilled box, the corresponding region in EcoSSB, EcoSSBG129S and MtuEcoSSB; downward arrow, BamHI site, found in MtuSSB, EcoSSBG129S and the fusion constructs. Sizes of the SSBs (amino acids) are shown on the right.

Figure 3

Analysis of purified SSBs on 15% SDS–PAGE (∼1.5 µg protein was analyzed). Lane 2, MtuSSB; lane 3, EcoSSB; lane 4, MtuEcoSSB; lane 5, EcoMtuSSB. Prestained molecular weight markers (lane 1, BioRad) correspond to bovine serum albumin (∼79.5 kDa), ovalbumin (∼49.5 kDa), carbonic anhydrase (∼34.8 kDa), soybean trypsin inhibitor (∼28.3 kDa), lysozyme (∼20.4 kDa) and aprotinin (7.2 kDa).

Figure 4

Analysis of the SSBs on 15% native PAGE (∼7.5 µg protein was analyzed). Lane 1, MtuSSB; lane 2, EcoSSB; lane 3, MtuEcoSSB; lane 4, EcoMtuSSB.

Figure 5

Electrophoretic mobility shift assay (see Materials and Methods). Lane 1, free 27mer DNA; lanes 2–5, complexes of the 27mer DNA with MtuSSB (lane 2), EcoSSB (lane 3), MtuEcoSSB (lane 4) and EcoMtuSSB (lane 5).

Figure 6

Relative fluorescence intensity changes in EcoSSB (open circles) and MtuSSB (filled circles) upon isothermal denaturation with varying concentrations of guanidinium hydrochloride (see Materials and Methods).