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

Model of ParA anchorage of the <i>oriC</i>/ParB complex at the tips of extending hyphae.

<p>Following chromosome replication, the tip-proximal of one of the two daughter <i>oriC</i>s is captured by the polarisome associated ParA which maintains its constant distance to the tip. The other daughter <i>oriC</i> remains associated with the replisome and is left behind by the extending tip. During branching the <i>oriC</i>/ParB complex proximal to newly established hyphal tip is captured by ParA and targeted into the branch.</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

ParB-EGFP complexes co-localize with all <i>oriC</i>s in multigenomic <i>S</i>. <i>coelicolor</i> vegetative hyphae.

<p>(A) Scheme of FROS cassette localization in the <i>S</i>. <i>coelicolor</i> chromosome. (B) Images of ParB-EGFP (green) and FROS (red) foci in vegetative hyphae of FROS <i>parB-egfp</i> strain (AK113). The hyphal tips are marked with an asterisk, scale bar—1 μm. (C) Co-localization of FROS and ParB-EGFP foci in AK113 strain along the vegetative hyphae and at the tips of hyphae; the percentage of the foci localizing within the given distance is indicated. (D) Correlation between the distance from ParB-EGFP to the tip of hyphae and distance from FROS signal to tip of FROS <i>parB-egfp</i> (AK113) strain hyphae. The scatterplot with a fitted linear model shows data from 16 hyphae measured at 10 minute time intervals. Data were analyzed using a mixed effects model which can compensate for an individual hypha effect and the standard linear model. Results of both models were similar, comparison of Log-likelihoods of both models showed that random effects of individual hyphae were not significant. Correlation was calculated using the Pearson method.</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 anchorage is required for chromosome migration to germ tubes and hyphal branches.

<p>(A) Time-lapse snapshots of FROS complexes during germination (top panels) and branch formation (bottom panels) in “wild type” FROS (DJ-NL102) and Δ<i>parA</i> FROS (AK115) strains. The images are the overlay of TetR-mCherry (red) fluorescence and DIC image (for separate images of TetR-mCherry fluorescence and DIC image see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006488#pgen.1006488.s011" target="_blank">S11 Fig</a>). The asterisks indicate the tip of outlined hyphae, scale bar—1 μm. (B) Germ tube length at the time of the first <i>oriC</i> appearance in “wild type” FROS (DJ-NL102), Δ<i>parA</i> FROS (AK115) and Δ<i>parB</i> FROS (AK114) strains (analyzed for 41 DJ-NL102, 31 AK115 and 30 AK114 germ tubes). (C) Branch length at the time of the first <i>oriC</i> appearance in “wild type” FROS (DJ-NL102), Δ<i>parA</i> FROS (AK115) and Δ<i>parB</i> FROS (AK114) strains. 95 hyphae of DJ-NL102, 106 of AK115 and 85 of AK114 strain were analyzed. In B and C, red crossbars show means with 95% confidence intervals. (D) Percentage and length of stalled branches with and without the FROS signal in “wild type” FROS (DJ-NL102), Δ<i>parA</i> FROS (AK115) and Δ<i>parB</i> FROS (AK114) strains. Hyphae were classified as stalled if no re-initiation of growth could be observed until the end of the experiment or for at least one hour (whichever was longer). 99 hyphal branches were analyzed of the DJ-NL102, 128 of AK115 and 104 of AK114 strain.</p

The tip-proximal chromosome follows the extending vegetative hyphae tip.

<p>(A) Time-lapse snapshots of the FROS strain (DJ-NL102) germinating spore (top panel) and vegetative hypha (bottom panel). The images are the overlay of TetR-mCherry fluorescence (red) and DIC image (gray) (for separate images of TetR-mCherry fluorescence and DIC see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006488#pgen.1006488.s003" target="_blank">S3D Fig</a>). The arrows indicate: red—<i>oriC</i>1 (closest to the tip of the hypha), yellow—<i>oriC</i>2, purple—<i>oriC</i>3, asterisks indicate the tip of outlined hyphae, scale bar—1 μm. (B) Positions of the FROS complexes in the extending hyphae of FROS strain (DJ-NL102). Grey bars are representations of the extending hyphae with 95% confidence interval for hyphal length and semitransparent colored dots represent <i>oriC</i> positions (red–<i>oriC</i> 1, yellow–<i>oriC</i> 2, purple–<i>oriC</i> 3, as shown in the schematic drawing at the right), colored lines indicate 95% mean confidence intervals (analyzed for 41 hyphae). Inset: Distribution (shown as probability density function) of the distances between the hyphal tip and the <i>oriC</i> 1 (red), <i>oriC</i> 2 (yellow) and <i>oriC</i> 3 (purple). (C) Correlation of hyphal extension rate and FROS complex movement calculated for 8 subsequent <i>oriC</i>s from the tip. Scatterplots with fitted linear models show data from 20 hyphae measured at 10 minute time intervals, grey area indicates 95% confidence interval for the model. Minus sign means that the distance between the chromosome and the tip is increasing and a plus sign that it is decreasing. Data were analyzed using a mixed effects model, which can compensate for the effect of individual hyphae and a standard linear model. Results of both models were very similar, comparison of Log-likelihoods of both models showed that random effects of individual hyphae were not significant. Inset: the calculated correlation in relation to the <i>oriC</i> position in the hyphae with a fitted linear model. Correlations were calculated using Pearson method with 95% confidence intervals.</p

Localization of ParB complex is dependent on ParA.

<p>(A) Images of ParB-EGFP (green) complexes in the hyphae of”wild type” <i>parB-egfp</i> (J3310) Δ<i>parA parB-egfp</i> (J3318), <i>parA</i>_{<i>overexp</i>} <i>parB-egfp</i> (DJ532), <i>parA</i>_<i>mut</i> <i>parB-egfp</i> (DJ598) strains, merged with cell wall staining (gray). (B) Co-localization of ParB-EGFP (green) with immunostained ParA (blue) in “wild type” <i>parB-egfp</i> (J3310) and <i>parA</i>_overexp <i>parB-egfp</i> (DJ532). Top panel shows the ParA immunofluorescence (blue) merged with ParB-EGFP fluorescence (green). The bottom panel shows ParB-EGFP fluorescence (green) merged cell wall staining (grey). (C) Localization of ParB-EGFP (green) within the nucleoid (DNA staining—red) in”wild type” <i>parB-egfp</i> (J3310) and in Δ<i>parA parB-egfp</i> (J3318). In panels A, B and C asterisks indicate the tip of hyphae and scale bars—1 μm. (D) The distribution (shown as probability density function) of the distances between the hyphal tip and the tip-proximal ParB-EGFP complex in „wild type” <i>parB-egfp</i> (J3310), Δ<i>parA parB-egfp</i> (J3318), <i>parA</i>_{<i>overexp</i>} <i>parB-egfp</i> (DJ532), <i>parA</i>_<i>mut</i> <i>parB-egfp</i> (DJ598) (analyzed for 170–300 hyphae). (E) Fluorescence intensity of ParB-EGFP and DNA stain measured from the hyphal tip in “wild type” <i>parB-egfp</i> (J3310) and Δ<i>parA parB-egfp</i> (J3318) (15 and 21 hyphae analyzed). For each hypha, the fluorescence signal was normalized so that the maximum signal was 100%. Lines are models fitted using a Loess algorithm implemented in the R program, grey area indicates 95% confidence interval. Dashed line shows maximum ParB fluorescence intensity as calculated by the model.</p

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

The constant distance between <i>oriC</i> and the hyphal tip is dependent on ParAB.

<p>(A) Images of FROS in the “wild type” FROS strain (DJ-NL102), Δ<i>parA</i> FROS (AK115) and Δ<i>parB</i> FROS (AK114) strains. The images are the overlay of TetR-mCherry fluorescence (red) and DIC image (grey), asterisks indicate the tip of hyphae, scale bar—1 μm. (B) Distribution (shown as probability density function) of the distances between the hyphal tip and tip-proximal FROS signal in “wild type” FROS (DJ-NL102), Δ<i>parA</i> FROS (AK115) and Δ<i>parB</i> FROS (AK114) strains. (C) Correlation between hyphal extension rate and the tip-proximal <i>oriC</i> movement velocity in “wild type” FROS (DJ-NL102), Δ<i>parA</i> FROS (AK115) and Δ<i>parB</i> FROS (AK114) strains (analyzed for 41 of DJ-NL102, 31 AK115 and 30 AK114 hyphae). Scatterplots with fitted linear models, grey area indicates 95% confidence interval for the model.</p