Additional file 6: of Characterising resuscitation promoting factor fluorescent-fusions in mycobacteria

Table S2. Primers used in this study. (PDF 14 kb

Additional file 1: of Characterising resuscitation promoting factor fluorescent-fusions in mycobacteria

Figure S1. Growth of M. smegmatis Rpf-EGFP and Rpf-mCherry producing strains in 7H9. (a) Growth curve of strains overproducing Rpf-EGFP proteins and a control strain overproducing EGFP. (b) Growth curve of strains overproducing Rpf-mCherry proteins and a control strain overproducing mCherry. Growth curves were made with data collected from 3 biological replicates. Error bars indicate standard deviation. (PDF 16 kb

Additional file 5: of Characterising resuscitation promoting factor fluorescent-fusions in mycobacteria

Table S1. Bacterial strains and plasmids used in this study. (PDF 27 kb

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 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

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

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