Model of ParA and ParB dynamics.

<p>(a) Model describing dynamics in an individual (non-conjoined) cell 1: A single ParB focus is situated usually at midcell (it can also be shifted towards one of the poles). 2: The ParB focus splits into two. One of the new foci moves towards or stays close to the region with the highest concentration of ParA (situated near the new pole in at least 69% of cells). 3: The ParB focus near the old pole moves towards it, perhaps triggered by DNA-cell envelope interactions. 4: The septum is formed and the two daughter cells divide. 5: ParA accumulates at the new poles created by the septum. A new round of segregation can begin. (b) Model describing dynamics in conjoined cells. 1: In a cell already containing two ParB foci, the ParA gradient situated near the new pole moves or stays close to the area of septum formation (that will become the new pole of the daughter cells). 2: One of the ParB foci splits and one of the new foci moves towards/stays close to the septum area whereas the other new focus moves towards the pole. In the figure, only the conjoined sister cell on the left experiences this second round of segregation, but both conjoined sisters can undergo this second round of replication (resulting in 4 ParB foci before cytokinesis). 3: The sister cells split (one inherits two ParB foci).</p

Analysis of ParAB dynamics in mycobacteria shows active movement of ParB and differential inheritance of ParA

<div><p>Correct chromosomal segregation, coordinated with cell division, is crucial for bacterial survival, but despite extensive studies, the mechanisms underlying this remain incompletely understood in mycobacteria. We report a detailed investigation of the dynamic interactions between ParA and ParB partitioning proteins in <i>Mycobacterium smegmatis</i> using microfluidics and time-lapse fluorescence microscopy to observe both proteins simultaneously. During growth and division, ParB presents as a focused fluorescent spot that subsequently splits in two. One focus moves towards a higher concentration of ParA at the new pole, while the other moves towards the old pole. We show ParB movement is in part an active process that does not rely on passive movement associated with cell growth. In some cells, another round of ParB segregation starts before cell division is complete, consistent with initiation of a second round of chromosome replication. ParA fluorescence distribution correlates with cell size, and in sister cells, the larger cell inherits a local peak of concentrated ParA, while the smaller sister inherits more homogeneously distributed protein. Cells which inherit more ParA grow faster than their sister cell, raising the question of whether inheritance of a local concentration of ParA provides a growth advantage. Alterations in levels of ParA and ParB were also found to disturb cell growth.</p></div

Analysis of ParA-mCherry and ParB-EGFP dynamics in <i>M</i>. <i>smegmatis</i> mc²155 Δ<i>parAB</i> [pMEND-AB] single cells.

<p>(a, c, d) ParA-mCherry dynamics are represented as a concentration gradient covering the entire cell. Black: minimum ParA concentration; white: maximum ParA concentration. ParB-EGFP foci dynamics are represented as gray lines with black markers. Each line represents the movement of a single ParB-EGFP focus. Cells are drawn such that the new pole of each cell is always situated at the bottom of the graph. (a) One ParB-EGFP focus splits into two foci. This figure represents a single cell in which a single ParB-EGFP focus splits, and one of the foci moves towards the new pole of the cell, towards an area where the ParA concentration is simultaneously increasing, whereas the other focus moves towards the old pole. The cell divides into two daughters at the end of the period shown. A subset of the time-lapse images of the cell represented in cartoon (a) are shown in panel (b). The ParA-mCherry maximum and ParB-EGFP foci are denoted with white triangles. The ParB-EGFP intensity trace of the cell is depicted in the fourth row, with the raw intensity in gray, the smoothed intensity in black, and the assigned ParB-EGFP foci denoted with white circles. The new pole of the cell is denoted by a yellow star. (c, d) Three ParB-EGFP foci per cell. Two independent cells with two ParB-EGFP foci at the start of the visualisation period, in which one of the foci splits and moves towards the midcell area, where the ParA-mCherry concentration is higher. (c) The focus closer to the old pole splits. (d) The focus closer to the new pole splits. Both cells in (c) and (d) divide into two daughters at the end of the period shown.</p

Example of ParA-mCherry and ParB-EGFP dynamics in <i>M</i>. <i>smegmatis</i> mc²155 Δ<i>parAB</i> [pMEND-AB].

<p>A selection of images from a 9 h time-lapse experiment is depicted. The dynamics of ParA-mCherry and ParB-EGFP are shown separately. Just before cell division, ParA accumulates in the midcell area, which will become the new pole of the daughter cells. At this point the two ParB foci are situated at symmetrical subpolar positions. After the division of the cell, a ParB focus situated near midcell in the newborn cell splits in two. One of the new ParB foci moves towards the new pole of the cell (where there is a higher concentration of ParA) whereas the other moves towards the old pole. White arrows indicate the area of maximum ParA concentration and the localisation of the ParB foci. Numbers in the top right corner of the pictures indicate time elapsed (in minutes) since the first frame.</p

Analysis of ParA-mCherry and ParB-EGFP dynamics in a <i>M</i>. <i>smegmatis</i> mc²155 Δ<i>parAB</i> [pMEND-AB] lineage of cells. Two ParB-EGFP foci per cell.

<p>Dynamics are depicted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199316#pone.0199316.g003" target="_blank">Fig 3a</a>. This figure represents a lineage of cells starting with a single cell that divides twice to result in four daughter cells. In this lineage, all cells whose birth can be tracked are born with a single ParB-EGFP focus that splits into two.</p

Free Glucosylglycerate Is a Novel Marker of Nitrogen Stress in <i>Mycobacterium smegmatis</i>

Nitrogen is an essential element for bacterial growth, and as such, bacteria have evolved several pathways to assimilate nitrogen and adapt to situations of nitrogen limitation. However, the adaptation of mycobacteria to nitrogen stress and the regulation of the stress response pathways is unknown. Identification of key metabolites produced by mycobacteria during nitrogen stress could therefore provide important insights into mycobacterial survival strategies. Here we used NMR-based metabolomics to monitor and quantify intracellular and extracellular metabolite levels (metabolic footprinting) in <i>Mycobacterium smegmatis</i> grown under nitrogen-limiting and nitrogen-rich conditions. There were several metabolic differences between the two conditions: following nitrogen run-out, there was an increase in intracellular α-ketoglutarate and a decrease in intracellular glutamine and glutamate levels. In addition, a sugar-derived compound accumulated in nitrogen-starved cells that was subsequently assigned as glucosylglycerate (GGA). Free GGA production was responsive to nitrogen stress in <i>M. smegmatis</i> but not to oxidative or osmotic stress; lack of a functional GGA synthesis pathway slightly reduced growth and decreased ammonium uptake rates under nitrogen-limiting conditions. Hence, GGA could contribute to the fitness of mycobacteria under nitrogen limitation

Movement of ParB-EGFP foci in <i>M</i>. <i>smegmatis</i> mc²155 Δ<i>parAB</i> [pMEND-AB].

<p>(a-c) Mean squared displacement of 219 ParB-EGFP foci relative to the ParA maximum position over time. For each ParB foci, squared change in distance (Δ<i>x</i>²) from the ParA maximum over increasing time windows (Δ<i>t</i>) is determined, with values binned according to the initial distance of ParB from the ParA maximum at <i>t</i> = 0. The mean squared displacement (〈Δ<i>x</i>²〉) is plotted for each time window (Δ<i>t</i>) with error bars showing the 95% confidence interval of each mean. ParB foci originating less than 1.5 μm from the ParA maximum (a) have low mean squared displacement, and are sub-linear, indicating passive and limited sub-diffusion. ParB foci between 1.5 and 3 μm from the ParA maximum (b) retain this sub-diffusive pattern, but ParB foci between 3 and 4.5 μm (c) show a distinct non-linear increase in mean squared displacement with time, indicating non-passive movement. The direction of the movement of ParB-EGFP foci was measured in relation to the ParA maximum with foci classified as moving toward, with, or away from the maximum according to their relative velocity, and the numbers of each type of movement are depicted in (d).</p

ParA-mCherry inheritance in <i>M</i>. <i>smegmatis</i> mc²155 Δ<i>parAB</i> [pMEND-AB].

<p>Whereas there is a direct relationship between cell area and total ParA-mCherry intensity at birth within the population (a), there is no relationship between cell area and the maximum intensity of ParA-mCherry at birth within the population (b). <i>n</i> = number of cells analysed; <i>r</i>² = coefficient of determination. The least squares linear regression line is depicted as a solid line, and the 95% confidence of this fit is represented by the shaded region. (c-e) Sister cells that inherit a higher level of total ParA-mCherry (high inheritor; blue) have a greater area (c) and grow at a faster rate (d) than low inheritors (green). When analysed independently of ParA-mCherry inheritance, we do not observe a statistically significant difference in growth rate between larger and smaller sibling cells (e). (f) An example cell division where a peak of ParA-mCherry (denoted by filled triangles) is inherited by the larger sister cell (blue). (g, h) Despite a lack of relationship between cell area and maximum ParA-mCherry intensity in the population, sister cells that inherit the local region of maximum ParA-mCherry intensity (high inheritor; blue) are larger (g), and grow at a faster rate (h) than their sisters (low inheritor; green). Mean values are depicted with a red line, and P-values were calculated using Welch’s t-test.</p

Differences in cell size in <i>M</i>. <i>smegmatis</i> mc²155 cells overproducing ParA-mCherry and/or ParB-EGFP.

<p>In the cells harbouring plasmid pMEND-AB, both ParA-mCherry and ParB-EGFP are induced except in the cases indicated. Differences in median cell size values were calculated using the Kruskal-Wallis One Way Analysis of Variance on Ranks. To isolate the groups that differ from the others, All Pairwise Multiple Comparison Procedures (Dunn’s Method) were used. Box plots are shown representing the cell size of each of the strains. From bottom to top, 10^th and 25^th percentiles, median, 75^th and 90^th percentiles are plotted. Outliers are indicated by black crosses. The strains are clustered in three different groups (A, B, and C). Within each group, there are non-significant differences in median size between strains. The strains within each group have a statistically significant difference in median cell size from any of the strains represented in either of the two other groups (P<0.05). The median cell size is largest in A>B>C. The number of cells analysed for each strain were: (1) 2,865; (2) 2,812; (3) 2,780; (4) 2,067; (5) 2,654; (6) 2,798; (7) 2,744; and (8) 3,044.</p

PBP1a-mCherry localizes centrally at septa that form future cell division sites, independently of cell length.

<p>PBP1a-mCherry expression in <i>M. smegmatis</i> mc²155 was induced with 20 ng/ml Tc for 3.5 hr cells and images were captured every 10 mins as described. Panels <b>A</b> and <b>B</b> are time series of images showing diffuse patches of staining at variable locations between the poles (white arrow). These eventually condensed into a central septal spot (white asterisk) around mid-cell. Cell envelope invagination and separation was not seen in the bright field images until 160 minutes after this condensation event (n = 10; yellow arrow). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044582#pone.0044582.s001" target="_blank">File S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044582#pone.0044582.s002" target="_blank">File S2</a> for complete movie sequences. The septal spots of PBP1a-mCherry form within the central 20% of the cell (Panel <b>C</b>; n = 23), without the outliers seen with Vancomycin staining. Panel D shows a control strain expressing mCherry alone. Septal spots are present in cells of various lengths (panels <b>E</b> and <b>F</b>), ranging between 4 and 12 µm (<b>G</b>), showing that cell division occurs at a wide range of cell lengths, indicating it is not a cue for placement of the new septum. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044582#pone.0044582.s003" target="_blank">File S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044582#pone.0044582.s004" target="_blank">File S4</a> for complete movie sequences.</p