10 research outputs found
Unique function of the bacterial chromosome segregation machinery in apically growing streptomyces - targeting the chromosome to new hyphal tubes and its anchorage at the tips
The coordination of chromosome segregation with cell growth is fundamental to the proliferation of any organism. In most unicellular bacteria, chromosome segregation is strictly coordinated with cell division and involves ParA that moves the ParB nucleoprotein complexes bi- or unidirectionally toward the cell pole(s). However, the chromosome organization in multiploid, apically extending and branching Streptomyces hyphae challenges the known mechanisms of bacterial chromosome segregation. The complex Streptomyces life cycle involves two stages: vegetative growth and sporulation. In the latter stage, multiple cell divisions accompanied by chromosome compaction and ParAB assisted segregation turn multigenomic hyphal cell into a chain of unigenomic spores. However, the requirement for active chromosome segregation is unclear in the absence of canonical cell division during vegetative growth except in the process of branch formation. The mechanism by which chromosomes are targeted to new hyphae in streptomycete vegetative growth has remained unknown until now. Here, we address the question of whether active chromosome segregation occurs at this stage. Applied for the first time in Streptomyces, labelling of the chromosomal replication initiation region (oriC) and time-lapse microscopy, revealed that in vegetative hyphae every copy of the chromosome is complexed with ParB, whereas ParA, through interaction with the apical protein complex (polarisome), tightly anchors only one chromosome at the hyphal tip. The anchor is maintained during replication, when ParA captures one of the daughter oriCs. During spore germination and branching, ParA targets one of the multiple chromosomal copies to the new hyphal tip, enabling efficient elongation of hyphal tube. Thus, our studies reveal a novel role for ParAB proteins during hyphal tip establishment and extension
Localization of ParB complex is dependent on ParA.
<p>(A) Images of ParB-EGFP (green) complexes in the hyphae ofâwild typeâ <i>parB-egfp</i> (J3310) Î<i>parA parB-egfp</i> (J3318), <i>parA</i><sub><i>overexp</i></sub> <i>parB-egfp</i> (DJ532), <i>parA</i><sub><i>mut</i></sub> <i>parB-egfp</i> (DJ598) strains, merged with cell wall staining (gray). (B) Co-localization of ParB-EGFP (green) with immunostained ParA (blue) in âwild typeâ <i>parB-egfp</i> (J3310) and <i>parA</i><sub>overexp</sub> <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 stainingâred) inâwild typeâ <i>parB-egfp</i> (J3310) and in Î<i>parA parB-egfp</i> (J3318). In panels A, B and C asterisks indicate the tip of hyphae and scale barsâ1 ÎŒm. (D) The distribution (shown as probability density function) of the distances between the hyphal tip and the tip-proximal ParB-EGFP complex in âwild typeâ <i>parB-egfp</i> (J3310), Î<i>parA parB-egfp</i> (J3318), <i>parA</i><sub><i>overexp</i></sub> <i>parB-egfp</i> (DJ532), <i>parA</i><sub><i>mut</i></sub> <i>parB-egfp</i> (DJ598) (analyzed for 170â300 hyphae). (E) Fluorescence intensity of ParB-EGFP and DNA stain measured from the hyphal tip in âwild typeâ <i>parB-egfp</i> (J3310) and Î<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
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
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 âwild typeâ FROS (DJ-NL102) and Î<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 barâ1 ÎŒm. (B) Germ tube length at the time of the first <i>oriC</i> appearance in âwild typeâ FROS (DJ-NL102), Î<i>parA</i> FROS (AK115) and Î<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 âwild typeâ FROS (DJ-NL102), Î<i>parA</i> FROS (AK115) and Î<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 âwild typeâ FROS (DJ-NL102), Î<i>parA</i> FROS (AK115) and Î<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: redâ<i>oriC</i>1 (closest to the tip of the hypha), yellowâ<i>oriC</i>2, purpleâ<i>oriC</i>3, asterisks indicate the tip of outlined hyphae, scale barâ1 ÎŒm. (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 (redâ<i>oriC</i> 1, yellowâ<i>oriC</i> 2, purpleâ<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
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 barâ1 ÎŒm. (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
<i>oriC</i> is captured at the tip soon after replication.
<p>(A) Time-lapse snapshots of FROS (TetR-mCherry fluorescence, red) and DnaN-EGFP foci (green) in the extending hyphae of âwild typeâ FROS <i>dnaN-egfp</i> (AK122) (top panel) and Î<i>parA</i> FROS <i>dnaN-egfp</i> (AK123) (bottom panel) strains. The fluorescence images are merged with the DIC images (grey) (for separate images of TetR-mCherry overlaid with DnaN-EGFP fluorescence and DIC see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006488#pgen.1006488.s008" target="_blank">S8 Fig</a>). Asterisks indicate the tip of the outlined hyphae, green arrows point to the replisome complex, red arrows point to the tip-proximal <i>oriC</i>, yellow arrows point to tip-distal <i>oriC</i>, scale barâ1 ÎŒm. (B) Position of the <i>oriC</i> and DnaN-EGFP complexes in relation to the tips of extending hyphae in âwild typeâ FROS <i>dnaN-egfp</i> (AK122) (left panel) and Î<i>parA</i> FROS <i>dnaN-egfp</i> (AK123) (right panel) strains (analyzed for 32 AK122 hyphae and 29 AK123 hyphae). Grey bars are representations of the extending hyphae with 95% confidence interval for hyphae length and semitransparent colored dots represent <i>oriC</i> positions (red: tip-proximal <i>oriC</i> 1, yellow: tip-distal <i>oriC</i> 2, green: replisome, as shown on the schematic drawings), colored lines indicate 95% mean confidence intervals. (C) The distance between the tip and the tip-proximal <i>oriC</i> 10â20 minutes before and 10â20 minutes after <i>oriC</i> duplication in âwild typeâ FROS <i>dnaN-egfp</i> (AK122) and Î<i>parA</i> FROS <i>dnaN-egfp</i> (AK123) strains. (D) Distance between duplicated <i>oriC</i>s at the indicated time after replisome appearance in âwild typeâ FROS <i>dnaN-egfp</i> (AK122) and Î<i>parA</i> FROS <i>dnaN-egfp</i> (AK123) strains. In C and D panel crossbars show the mean with 95% confidence intervals. (E) Percentage of hyphae in which the duplicated <i>oriCs</i> could be detected at the indicated time after replisome appearance. Error bars show 95% confidence intervals.</p
The constant distance between <i>oriC</i> and the hyphal tip is dependent on ParAB.
<p>(A) Images of FROS in the âwild typeâ FROS strain (DJ-NL102), Î<i>parA</i> FROS (AK115) and Î<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 barâ1 ÎŒm. (B) Distribution (shown as probability density function) of the distances between the hyphal tip and tip-proximal FROS signal in âwild typeâ FROS (DJ-NL102), Î<i>parA</i> FROS (AK115) and Î<i>parB</i> FROS (AK114) strains. (C) Correlation between hyphal extension rate and the tip-proximal <i>oriC</i> movement velocity in âwild typeâ FROS (DJ-NL102), Î<i>parA</i> FROS (AK115) and Î<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