Novel Role of Phosphorylation-Dependent Interaction between FtsZ and FipA in Mycobacterial Cell Division

The bacterial divisome is a multiprotein complex. Specific protein-protein interactions specify whether cell division occurs optimally, or whether division is arrested. Little is known about these protein-protein interactions and their regulation in mycobacteria. We have investigated the interrelationship between the products of the Mycobacterium tuberculosis gene cluster Rv0014c-Rv0019c, namely PknA (encoded by Rv0014c) and FtsZ-interacting protein A, FipA (encoded by Rv0019c) and the products of the division cell wall (dcw) cluster, namely FtsZ and FtsQ. M. smegmatis strains depleted in components of the two gene clusters have been complemented with orthologs of the respective genes of M. tuberculosis. Here we identify FipA as an interacting partner of FtsZ and FtsQ and establish that PknA-dependent phosphorylation of FipA on T77 and FtsZ on T343 is required for cell division under oxidative stress. A fipA knockout strain of M. smegmatis is less capable of withstanding oxidative stress than the wild type and showed elongation of cells due to a defect in septum formation. Localization of FtsQ, FtsZ and FipA at mid-cell was also compromised. Growth and survival defects under oxidative stress could be functionally complemented by fipA of M. tuberculosis but not its T77A mutant. Merodiploid strains of M. smegmatis expressing the FtsZ(T343A) showed inhibition of FtsZ-FipA interaction and Z ring formation under oxidative stress. Knockdown of FipA led to elongation of M. tuberculosis cells grown in macrophages and reduced intramacrophage growth. These data reveal a novel role of phosphorylation-dependent protein-protein interactions involving FipA, in the sustenance of mycobacterial cell division under oxidative stress

Solvation dynamics in DMPC vesicle in the presence of a protein

Solvation dynamics of DCM is studied in DMPC vesicle in the presence of a protein, human serum albumin (HSA). In bulk water, solvation dynamics of DCM bound to HSA displays a 10 ns component. This component is absent when HSA is entrapped in the vesicle. This suggests that the protein does not undergo tumbling inside the vesicle. In the vesicle, solvation dynamics in the presence of HSA is slower compared to that in its absence. Solvation dynamics below the gel transition temperature (Tc≈23 °C) is about two times slower than that above it

Solvation dynamics in a worm-like CTAB micelle

The solvation dynamics of 4-(dicyanomethylene)-2-methyl-(p-dimethyl-aminostyryl) 4Hpyran (DCM) is studied in a worm-like micelle consisting of cetyl trimethylammonium bromide (CTAB) and sodium salicylate (NaSal). Solvation dynamics in the worm-like micelle is found to be about 4-times slower than that in ordinary CTAB micelle. This indicates that the fluorescent probe molecule (DCM) experience a different microenvironment inside the worm-like micellar aggregates compared to that in ordinary spherical CTAB micelle. The spectral width (Γ) of the time resolved emission spectra of DCM in the micellar aggregates is found to be time dependent. This is ascribed to the diffusion of the probe (DCM)

Solvation dynamics in the water pool of an aerosol-OT microemulsion. Effect of sodium salicylate and sodium cholate

The effect of sodium salicylate (Na-sal) and sodium cholate (Na-cholate) on the solvation dynamics in the water pool of aerosol-OT (AOT, sodium dioctylsulfosuccinate) microemulsion in n-heptane is reported. In the absence of any additive, the solvation dynamics of 4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran (DCM) in AOT microemulsions is found to be biexponential with an average solvation time (&#964;<SUB>s</SUB>) of 710 ps. In the water pool, &#964;<SUB>s</SUB> decreases to 330 ps on addition of 1 mM Na-sal and increases to 3050 ps in the presence of 100 mM Na-cholate. The spectral width (&#915;) of the time-resolved emission spectra of DCM in the water pool is found to be time dependent. This is ascribed to diffusion of the probe (DCM). The Na-sal-induced acceleration of the solvation dynamics is ascribed to the increase in the size of the water pool. In bulk water, in the presence of 100 mM Na-cholate (a bile salt), &#964;<SUB>s</SUB> is ~830 ps. This is 4 times shorter than &#964;<SUB>s</SUB> in the presence of Na-cholate in the water pool. This is ascribed to extreme crowding in the water pool because of the presence of Na-cholate aggregates. In bulk water, the emission spectral width displays a very small time dependence in the presence of Na-cholate aggregates. This suggests that in this case self-diffusion is unimportant and the slow solvation arises entirely from the dynamic exchange

Solvation dynamics in a protein-surfactant complex

Solvation dynamics in the denatured state of a protein, lysozyme (denatured by sodium dodecyl sulfate, SDS) is markedly slower than that in the native state. For coumarin 153 bound to lysozyme, the average solvation time, &lt;τs&gt; is 330 ps. In the lysozyme-SDS complex, the solvation dynamics is markedly slower with &lt;τs&gt;=7250 ps. On addition of dithiothreitol (DTT) to the lysozyme-SDS complex, when the di-sulfide bonds are destroyed, &lt;τs&gt; is found to be 1140 ps. The slow dynamics in the denatured protein is attributed to the polymer chain dynamics and the exchange of bound and free water molecules

Excited state proton transfer of 1-naphthol in a hydroxypropylcellulose/sodium dodecyl sulfate system

Excited state proton transfer (ESPT) of 1-naphthol has been studied in a polymer (hydroxypropylcellulose, HPC)-surfactant (sodium dodecyl sulfate, SDS) aggregate. The ESPT process of 1-naphthol is drastically retarded in the HPC-SDS aggregate above the critical association concentration (cac). The cac of SDS for HPC is found to be 2 mM, which is much smaller than its cmc (8 mM). The time-resolved emission data indicate that at a low SDS concentration (&lt;8 mM) the decay of the neutral emission and the rise and decay of the anion emission are very different from those in SDS micelles. Above the cmc of SDS, 1-naphthol molecules are distributed between the HPC-SDS aggregate and free SDS micelles

Interaction between FtsW and penicillin-binding protein 3 (PBP3) directs PBP3 to mid-cell, controls cell septation and mediates the formation of a trimeric complex involving FtsZ, FtsW and PBP3 in mycobacteria

In bacteria, biogenesis of cell wall at the division site requires penicillin-binding protein 3 (PBP3) (or FtsI). Using pull-down, bacterial two-hybrid, and peptide-based interaction assays, we provide evidence that FtsW of Mycobacterium tuberculosis (Fts<SUB>WMTB</SUB>) interacts with PBP3 through two extracytoplasmic loops. Pro<SUP>306</SUP> in the larger loop and Pro<SUP>386</SUP> in the smaller loop of FtsW are crucial for these interactions. Fluorescence microscopy shows that conditional silencing of ftsW in Mycobacterium smegmatis prevents cell septation and positioning of PBP3 at mid-cell. Pull-down assays and conditional depletion of FtsW in M. smegmatis provide evidence that FtsZ, FtsW and PBP3 of mycobacteria are capable of forming a ternary complex, with FtsW acting as a bridging molecule. Bacterial three-hybrid analysis suggests that in M. tuberculosis, the interaction (unique to mycobacteria) of FtsZ with the cytosolic C-tail of FtsW strengthens the interaction of FtsW with PBP3. ftsW of M. smegmatis could be replaced by ftsW of M. tuberculosis. FtsW<SUB>MTB</SUB> could support formation of the FtsZ-FtsW-PBP3 ternary complex in M. smegmatis. Our findings raise the possibility that in the genus Mycobacterium binding of FtsZ to the C-tail of FtsW may modulate its interactions with PBP3, thereby potentially regulating septal peptidoglycan biogenesis