Overproduction and localization of Mycobacterium tuberculosis ParA and ParB proteins

Abstract

The ParA and ParB family proteins are required for accurate partitioning of replicated chromosomes. The Mycobacterium tuberculosis genome contains parB, parA and two parA homologs, Rv1708 and Rv3213c. It is unknown if parA and its homologs are functionally related. To understand the roles of ParA and ParB proteins in M. tuberculosis cell cycle, we have evaluated the consequences of their overproduction and visualized their localization patterns in M. smegmatis. We show that cells overproducing ParA, Rv1708 and Rv3213c and ParB are filamentous and multinucleoidal indicating defects in cell-cycle progression. Visualization of green-fluorescent protein fusions of ParA and its homologues showed similar localization patterns with foci at poles, quarter-cell, midcell positions and spiral-like structures indicating that they are functionally related. On the other hand, the ParB- GFP fusion protein localized only to the cell poles. The cyan- and yellow-fluorescent fusion proteins of ParA and ParB, respectively, colocalized at the cell poles indicating that these proteins interact and possibly associate with the chromosomal origin of replication. Collectively our results suggest that the M. tuberculosis Par proteins play important roles in cell-cycle progression.

Fig. 1

Sequence alignment of Mycobacterium tuberculosis ParA and the ParA homologs Rv1708 and Rv3213c. Analysis revealed that the Walker A and Walker B motifs, associated with ATPase activity, are found in both ParA and the ParA homologs. * -amino acid residues that are identical in all sequences, : - conserved amino acid residues, and . -residues that are semi-conserved.

Fig. 2

Septal and nucleoid staining of Par overproduction strains. M. smegmatis strains overproducing M. tuberculosis ParA, the ParA homologs Rv1708 and Rv3213, and ParB were stained during log phase growth with FM4-64 (FM) and DAPI to show cell septa and nucleoids, respectively. The grayscale images are brightfield (BF); black bars - 4 μm. Magnification is 100x. Overlay panels were created by combining the fluorescent images. (1a-d) Wild-type M. smegmatis with mid-cell septum and well separated nucleoids. (5ab) Wild-type M. smegmatis stained with DAPI to show well separated nucleoids. (2a-d) ParA overproducing strain with asymmetric septum and nucleoids in both cell compartments. (3a-d) ParA overproducing strain without apparent septa formation; nucleoids are distributed throughout the cell. (4a-d) ParB overproducing strain with asymmetric septum; nucleoids are only seen in one cell compartment. (6a, b) Anucleate cell stained with DAPI (ParB overproducing strain).

Fig. 3

Viability of strains overproducing Par proteins. The Par overproduction strains were grown in the presence of the 0.2% acetamide for 6 hours, then serially diluted and plated on Middlebrook 7H10 agar plates without acetamide; counts of viable colonies were used to calculate viability. Viabiity of the ParB overproduction strain was reduced by about 0.5 log compared with wild-type M. smegmatis. Overproducing ParA and the ParA homologs Rv1708 and Rv3213 did not lead to a significant loss of viability.

Fig. 4

Localization of M. tuberculosis Par protein fusions with GFP in M. tuberculosis and M. smegmatis. M. tuberculosis strains overproducing the Par:GFP fusions were grown for 24 hours with 0.2% acetamide; M. smegmatis Par:GFP overproduction strains were grown with 0.2% acetamide for 3 hrs. Cells were imaged during log phase. Grayscale images are brightfield (BF) and green pseudocolor - Par:GFP fusions; black bars - 4 μm. Magnification is 100x. (IA,A’) ParB:GFP localization in M. tuberculosis at cell poles. (IB,B’) ParA:GFP localization at cell poles and midcell in M. tuberculosis. (IIAA’-EE’) Localization patterns of ParA:GFP in M. smegmatis. (IIFF’) Membrane-like distribution of Rv3213c:GFP in M. smegmatis. (IIGG’-KK’) Localization patterns of ParB:GFP in M. smegmatis.

Fig. 5

Colocalization of M.tb Par:GFP fusions with nucleoids and ParA:CFP colocalization with ParB:YFP. For colocalization of Par:GFP fusions and nucleoids, M. smegmatis Par:GFP strains were grown with 0.2% acetamide for 3 hrs in log phase. Cells were stained with ethidium bromide and imaged. Grayscale images are brightfield (BF); green pseudocolor - Par:GFP fusions (GFP) and red pseudocolor - nucleoids stained with ethidium bromide (EtBr); black bars – 4 μm. Magnification is 100x. An overlay panel was created from the fluorescence images. For colocalization of ParA:CFP and ParB:YFP, the M. smegmatis double overproduction strain was grown with 0.2% acetamide for 3hrs during logarithmic growth, then cells were imaged. Green pseudocolor - ParA:CFP and red pseudocolor - ParB:YFP; an overlay panel was created from the fluorescence images. (Ia-d) ParA:GFP shown with stained nucleoids in M. smegmatis. The arrow indicates colocalization. (Ie-h) ParB:GFP shown with stained nucleoids in M. smegmatis. (IIa-d and e-h) ParA:CFP and ParB:YFP foci in M. smegmatis. The arrow indicates colocalization.