Phosphorylation of <i>Mycobacterium tuberculosis</i> ParB Participates in Regulating the ParABS Chromosome Segregation System

<div><p>Here, we present for the first time that <i>Mycobacterium tuberculosis</i> ParB is phosphorylated by several mycobacterial Ser/Thr protein kinases <i>in vitro</i>. ParB and ParA are the key components of bacterial chromosome segregation apparatus. ParB is a cytosolic conserved protein that binds specifically to centromere-like DNA <i>parS</i> sequences and interacts with ParA, a weak ATPase required for its proper localization. Mass spectrometry identified the presence of ten phosphate groups, thus indicating that ParB is phosphorylated on eight threonines, Thr32, Thr41, Thr53, Thr110, Thr195, and Thr254, Thr300, Thr303 as well as on two serines, Ser5 and Ser239. The phosphorylation sites were further substituted either by alanine to prevent phosphorylation or aspartate to mimic constitutive phosphorylation. Electrophoretic mobility shift assays revealed a drastic inhibition of DNA-binding by ParB phosphomimetic mutant compared to wild type. In addition, bacterial two-hybrid experiments showed a loss of ParA-ParB interaction with the phosphomimetic mutant, indicating that phosphorylation is regulating the recruitment of the partitioning complex. Moreover, fluorescence microscopy experiments performed in the surrogate <i>Mycobacterium smegmatis ΔparB</i> strain revealed that in contrast to wild type Mtb ParB, which formed subpolar foci similar to <i>M</i>. <i>smegmatis</i> ParB, phoshomimetic Mtb ParB was delocalized. Thus, our findings highlight a novel regulatory role of the different isoforms of ParB representing a molecular switch in localization and functioning of partitioning protein in <i>Mycobacterium tuberculosis</i>.</p></div

Localization of ParB and derivatives.

<p><b>(A)</b> Subcellular localization of ParB-GFP in <i>M</i>. <i>smegmatis mc</i>^<i>2</i><i>155ΔparB</i> strain. Are shown Differential Intereference Contrast image (DIC), GFP fluorescence (GFP) and a merged image of DIC and GFP fluorescence (Merge). Localization of ParB isoforms are shown in three different panels: the upper panel shows wild-type ParB (ParB), the middle panel shows phosphoablative ParB (ParB_Ala) and the lower panel shows phosphomimetic ParB (ParB_Asp). Scalebar 2μm <b>(B)</b> Immunoblotting of ParB-GFP derivatives in <i>M</i>.<i>smegmatis mc</i>^<i>2</i><i>155ΔparB</i> complemented strains. Crude extracts of <i>M</i>. <i>smegmatis mc</i>^<i>2</i><i>155ΔparB</i> complemented with pVV16_<i>egfp_parB</i>, pVV16_<i>egfp_parB_Ala</i> or pVV16_<i>egfp_parB_Asp</i> were electrophoresed on SDS-PAGE gel, ParB-GFP derivatives were then detected by immoblotting using anti-GFP antibody according to the manufacturer’s instructions (Santa Cruz) and revealed with secondary antibodies labeled with IRDye infrared dyes (Odyssey, LiCOR).</p

Phosphorylation of ParB in mycobacteria.

<p><i>E</i>. <i>coli</i> harboring pETPhos_<i>parB</i> was used as a source of non phosphorylated ParB (ParB), and the strain harboring pDuet_<i>parB</i> coexpressing PknB and ParB provided the phosphorylated ParB isoform (ParB-P). ParB and ParB_Ala were produced in <i>M</i>. <i>smegmatis mc</i>^<i>2</i><i>155</i>Δ<i>parB</i> strains harboring pVV16_<i>parB</i> or pVV16_<i>parB</i>_<i>Ala</i>, respectively. Three μg of purified His-tagged ParB derivatives were migrated and detected on independent SDS-PAGE gels by immunoblotting using anti-phosphothreonine (middle panel) or anti-phosphoserine (lower panel) antibodies according to the manufacturer’s instructions (Invitrogen), and revealed with secondary antibodies labeled with IRDye infrared dyes (Odyssey, LiCOR). <i>M</i>, molecular mass markers.</p

DNA-binding activity of ParB derivatives.

<p>Gel electrophoretic mobility shift analysis (EMSA) of ParB binding to the <i>parS</i> sequence. The <i>parS</i> region was amplified by PCR, radioactively labeled, and incubated with 0.5, 1, 1.5, and 2.5μM of purified ParB, resolved by non-denaturing PAGE and visualized by autoradiography after overnight exposure to a film. <b>(A)</b> Binding of the unphosphorylated ParB (ParB), <b>(B)</b> ParB phosphoablative mutant (ParB_Ala), <b>(C)</b> ParB phosphomimetic mutant (ParB_Asp), and <b>(D)</b> phosphorylated ParB (ParB-P), to the <i>parS</i> region.</p

ParA interaction with ParB derivatives.

<p>Interactions of <i>M</i>. <i>tuberculosis</i> ParA with ParBWT <b>(A)</b> ParBAla <b>(B)</b> and ParBAsp <b>(C)</b> in bacterial two hybrid system. The red rectangles indicate differences in ParA-ParB interaction between WT (A), phosphoablative (B) and phosphophomimetic (C) ParB mutant proteins.</p

Phosphoacceptors identified after purification of <i>M</i>. <i>tuberculosis</i> ParB from the <i>E</i>. <i>coli BL21(DE3)star</i> strain co-expressing <i>M</i>. <i>tuberculosis</i> PknB.

<p>Sequences of the phosphorylated peptides identified in ParB as determined by mass spectrometry following tryptic digestion are indicated, and phosphorylated residues (pT or pS) are shown in bold.</p><p>Phosphoacceptors identified after purification of <i>M</i>. <i>tuberculosis</i> ParB from the <i>E</i>. <i>coli BL21(DE3)star</i> strain co-expressing <i>M</i>. <i>tuberculosis</i> PknB.</p

<i>In vitro</i> phosphorylation of ParB and mutant derivatives.

<p><b>(A)</b><i>in vitro</i> phosphorylation of <i>M</i>. <i>tb</i> ParB by PknB. The soluble domains of seven recombinant <i>M</i>. <i>tb</i> STPKs (PknA to PknL) were expressed and purified as GST-tagged fusions and incubated with purified His-tagged ParB and [γ-³³P]ATP. The amount of the STPKs used varied from 0,3 to 2 μg to obtain the optimal autophosphorylation activity for each kinase. Samples were separated by SDS-PAGE, stained with Coomassie Blue (upper panel), and visualized by autoradiography after overnight exposure to a film (lower panel). Upper bands reflect the phosphorylation signal of ParB, and the lower bands correspond to the autophosphorylation activity of each kinase. <i>M</i>, molecular mass markers. <b>(B)</b><i>in vitro</i> phosphorylation of the ParB_Ala mutant. Purified ParB and phosphoablative ParB (ParB_Ala) were incubated with PknB and [γ³³-P]ATP. Samples were separated by SDS-PAGE, stained with Coomassie Blue (upper panel), and visualized by autoradiography (lower panel) after overnight exposure to a film.</p

Antagonistic regulations by distinct RR domains shape Frz-dependent responses to stimulations.

<p>At low input levels, intramolecular phosphotransfer from the FrzE kinase domain (FrzE^kinase) to the FrzE response regulator domain (FrzE^RR) quenches the signal and prevents unwanted activation of reversals in absence of physiological signals. In the presence of activating signals (dotted line), the kinase activity of FrzE^kinase overcomes the phosphatase activity of FrzE^RR and the signal can be transduced downstream, presumably by the direct phosphorylation of RomR. In absence of FrzZ, signal transmission to the downstream motility machineries is not amplified (blue curve), only allowing S-motility-dependent regulations. In the presence of FrzZ, interactions between FrzE, FrzZ and RomR amplify the signaling activity allowing a steep and high amplitude response required to regulate both A- and S-motility (purple curve). Although the exact amplification mechanism remains to be determined the epistastic interactions between FrzE, FrzZ and RomR suggest that the FrzE kinase might activate reversals both by acting directly or indirectly on RomR and by direct phosphorylation of FrzZ, which would then interact with RomR to further activate it. The blue and purple curves are drawn from data shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005460#pgen.1005460.g007" target="_blank">Fig 7B</a>.</p

Genetic control of the <i>Myxococcus</i> A- and S-motility machineries.

<p><b>(A)</b> Dynamics of the motility machineries and their associated regulators during the reversal cycle. MglA (Red circle) is recruited by RomR (Blue circle) to the leading cell pole where it activates A- motility (Agl/Glt, T-shapes) and S-motility (Type-IV pili) at least partially through AglZ and FrzS, respectively (Crimson and Brown circles). At the lagging cell pole, MglB (green circle) co-localizes with RomR and FrzS where it prevents MglA accumulation by activating GTP hydrolysis by MglA. Frz signaling provokes the concerted polarity switching of MglA and MglB to opposite poles, allowing A- and S-motility activation at the new leading and movement in the opposite direction. <b>(B)</b> Genetic control of the reversal cycle. Schematic of the regulation cascade compiled from previous works. Frz signaling is thought to promote a phosphorylation cascade that activates reversals. In more details, activation of the FrzCD receptor by unknown signals activates the auto-phosphorylation of the FrzE kinase through the FrzA CheW-like protein. The exact function of FrzB, another CheW-like protein, is unknown, contrarily to FrzA it is not absolutely essential for the activation of FrzE. FrzE then transfers a phosphoryl group to up to three response regulator proteins, its cognate receiver domain (FrzE^RR), the FrzZ protein, a fusion of two RR domains, and the N-terminal RomR receiver domain. The outcome of these phosphorylation events is unknown but the phosphorylated output(s) is thought to interact directly with the polarity proteins and provoke their re-localization to opposite poles. Plain arrows indicate established interactions and dotted arrows indicate suspected interactions. The protein color code applies throughout the manuscript.</p

Increased Frz signaling activity is required for the regulation of A- and S-motility, which depends on FrzZ.

<p><b>(A)</b> Weak Frz protein expression supports S-motility-dependent aggregation but not A- and S-motility-dependent aggregation. The ability of a strain in which the <i>frz</i> promoter has been replaced by an IPTG-inducible promoter to form aggregates is tested on soft (0.5%) or hard (1.5%) CF agar plates in the presence or in the absence of IPTG. A western blot analysis (bottom) of FrzCD expression in this strain reveals that promoter leakage is sufficient to support aggregation on the soft surface. <b>(B)</b> Extracellular complementation of the <i>frzZ</i> mutant developmental aggregation phenotype by IAA. Multicellular development on hard (1.5% agar) starvation CF medium containing increasing IAA concentrations by the WT strain and the <i>frzE</i> and <i>frzZ</i> mutants. Note that the <i>frzE</i> and <i>frzZ</i> mutants are indistinguishable in absence of IAA but that aggregation is only restored for the <i>frzZ</i> mutant in the presence of IAA</p