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. 2017 Nov 20;8(1):1621.
doi: 10.1038/s41467-017-01716-9.

A Programmed Cell Division Delay Preserves Genome Integrity During Natural Genetic Transformation in Streptococcus Pneumoniae

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Free PMC article

A Programmed Cell Division Delay Preserves Genome Integrity During Natural Genetic Transformation in Streptococcus Pneumoniae

Matthieu J Bergé et al. Nat Commun. .
Free PMC article

Abstract

Competence for genetic transformation is a differentiation program during which exogenous DNA is imported into the cell and integrated into the chromosome. In Streptococcus pneumoniae, competence develops transiently and synchronously in all cells during exponential phase, and is accompanied by a pause in growth. Here, we reveal that this pause is linked to the cell cycle. At least two parallel pathways impair peptidoglycan synthesis in competent cells. Single-cell analyses demonstrate that ComM, a membrane protein induced during competence, inhibits both initiation of cell division and final constriction of the cytokinetic ring. Competence also interferes with the activity of the serine/threonine kinase StkP, the central regulator of pneumococcal cell division. We further present evidence that the ComM-mediated delay in division preserves genomic integrity during transformation. We propose that cell division arrest is programmed in competent pneumococcal cells to ensure that transformation is complete before resumption of cell division, to provide this pathogen with the maximum potential for genetic diversity and adaptation.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
Initiation of constriction of the cytokinetic ring and completion of cell division are delayed in competent cells. “Wild type” cells (strain R3956) were incubated at 37 °C with or without CSP for 30 min. a Schematic representation of pneumococcal cells classified into two groups and six different classes according to the progression in their cell cycle. b Representative fields of non-competent (upper panels) and competent (lower panels) cultures are shown. Left panels: phase contrast images; right panels: same images with cells from group I and group II false-colored white and black, respectively. c Histograms quantifying the percent of cells of non-competent (upper) and competent (lower) cultures in the cell-type categories presented in a. Note that in non-competent cultures, the proportion of cells in categories A, B and C is similar and that cells from category F represent a minority. Values and standard deviations are based on data from six independent experiments. n number of cells analyzed. d Pie chart representations of results shown in c. The distribution of non-dividing (white) and dividing (black) cells in non-competent (upper chart) or competent (lower chart) cultures is shown. e Still images from fluorescence time-lapse microscopy of R3702 cells producing a functional FtsZ-GFP fusion at 37 °C. Cells were induced (+CSP) or not (−CSP) to develop competence by the addition of synthetic CSP and spotted on a microscope slide containing a pad of agarose and C + Y growth medium. Average doubling times of 30 ± 4 min and 42 ± 5 min were measured for a total of 137 non-competent cells and 117 competent cells, respectively, collected over three independent experiments. Overlays between phase contrast (gray) and GFP (green) are shown. The time-lapse starting point corresponds to 5 min of CSP induction. Scale bar, 1 µm
Fig. 2
Fig. 2
Late com genes are not required for the cell division delay observed in competent cells. comX cells (strain R2002) were CSP-induced to develop the competence program, incubated at 37 °C for 30 min and analyzed by phase contrast microscopy. Pie charts show the distribution of non-dividing (white) and dividing (black) cells in non-competent cultures (indicated as “uninduced”, left) or CSP-induced cultures (right). The histogram representations indicate the percentage of cells in the cell-type categories presented in Fig. 1a. n number of cells analyzed
Fig. 3
Fig. 3
ComM is required to delay cell division in competent cells. a Diagram of the comM-spr1761-spr1760-lytR chromosomal region. The ComE-binding site (ComE-box, upstream of comM), the extended −10 σA-dependent promoter (Pc, upstream spr1761) and the σx-dependent promoter (ComX-box, in front of cinA) are indicated. The T1T2 transcription terminator (red hairpin) inserted downstream of comM and upstream of Pc in strain R3048 (comM + lytR ind−) and the position of the mariner insertion (red flag) in the comM gene of strain R1887 (comM ) are also shown. For details of strain constructions, see Supplementary Methods. Cells were induced to develop competence (indicated as “CSP-induced”) or not (“uninduced”), incubated at 37 °C for 30 min and analyzed by phase contrast microscopy. b Histograms quantifying the percentage of cells in the cell-type categories presented in Fig. 1a. n number of cells analyzed. The data are from a single representative of three biological experiments. c Pie chart representations of results shown in b. The distribution of non-dividing (white) and dividing (black) cells is shown. Strains used: “wild-type” (WT, R3047), comM + lytR ind (R3048), ΔcomM (R3049), comM (R1887)
Fig. 4
Fig. 4
ComM is a cell division inhibitor. a ComM is sufficient to delay cell constriction in non-competent cells. Pie charts indicate the percentage of non-dividing (white) and dividing (black) cells in cultures of strain R3957 (comM + PBIP:comM) harboring the endogenous copy of comM under the control of CSP induction during competence and a second copy expressed independently of competence, under the control of BIP induction; and strain R2524 (comM + PBIP:luc) containing the gene encoding luciferase (luc) under the control of BIP induction. Cells were grown in C + Y medium to OD550 nm 0.08 and induced with or without CSP or BIP as indicated, incubated at 37 °C for 30 min before phase contrast microscopy analysis. n number of cells analyzed. The data are from a single representative of four biological experiments. b ComM localizes at midcell. Cells containing a gfp-comM translational fusion at the endogenous chromosomal locus (strain R3983) were analyzed by fluorescence microscopy 15 min after CSP addition. Overlays between phase contrast (gray) and GFP (green) are shown. Scale bar, 1 µm. The data are representative of three biological replicates
Fig. 5
Fig. 5
Competence interferes with peptidoglycan synthesis and StkP kinase activity. a Localization of peptidoglycan synthesis in the presence (ComM + ) or absence (ΔComM) of ComM. Competent and non-competent cells were incubated for 15 min with the blue fluorescent derivative of D-alanine HADA. Phase contrast (left), fluorescent (middle), and false-colored overlay images (phase contrast, gray; HADA, blue) are shown. Scale bars, 1 µm. Images are representative of seven biological replicates. b Fraction of cells harboring polar HADA staining and cells with a single thin HADA band at midcell in ComM+ (R3966) and ΔComM (R3967) cultures. The total number of cells analyzed is indicated. Values are indicated with s.d. for three independent experiments. c, d Western blot of cell lysates probed with anti-phosphothreonine antibodies. Samples were prepared from competent (+) or non competent (−) cultures. Control strains harboring a deletion of the divIVA gene (R4134), a Jag-SPA fusion (R4143), and phosphoablative mutants of DivIVA (DivIVA*, R4144) and MapZ (MapZ*, mapZ-2TA) were used to distinguish the phosphorylation signals for StkP, MapZ, Jag, and DivIVA. c Strain used: WT (R1501). Data are representative of seven biological replicates and three technical replicates. Full blot is shown in Supplementary Fig. 12a. d Strains used: ΔComX (R2002), ΔComX ΔComM (R2132). Data are representative of two biological replicates and one technical replicate. Full blot is shown in Supplementary Fig. 12b
Fig. 6
Fig. 6
ComM facilitates the resolution of chromosome dimers generated during transformation. Reduction in merodiploid frequency in comM strains compared with that in wild-type and cbpD C75A cells. Comparison of transformation efficiency using as donor a mixture of PCR fragments that promote merodiploid formation and enables their selection. Values and standard deviations are based on data from three independent experiments. Recipient strains used: wild-type (WT, R1501), cbpD (R3966), cbpD comM (R3967), cbpD recO xerS (R3970), cbpD comM recO xerS (R3973)
Fig. 7
Fig. 7
ComM plays a role in the resolution of chromosome dimers generated during transformation by postponing cell division. In wild-type cells, ComM delays the formation of daughter cells by inhibiting initiation of septum constriction in pre-divisional cells as well as its closure. This delay allows time for completion of transformation, replication, and chromosome segregation. It benefits transformation events leading to genome rearrangements with the creation of a chromosome dimer as an intermediate. Resolution of chromosome dimers occurs when the termini of the future sister chromosomes are aligned in the septal region. In the absence of ComM, progressive septum closure could entrap DNA resulting in loss of genome integrity. A donor transforming DNA (red) pairing with the two copies of a partially replicated recipient chromosome is shown. The origin (Ori1 and Ori2) and the terminus (Ter1 and Ter2) regions of the sister chromosomes are indicated. For simplicity, only one replication round is shown

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