Biotechnology for the Future / Jens Nielsen.

Scholarly and ProfessionalXII, 229 pages

Spontaneous competence development in pneumococci relies on a time-growth dependent mechanism.

<p>The 4 strains used R895, TD82, TG55 and TCP1251 harboring the <i>ssbB</i>::<i>luc</i> fusion to monitor competence development throughout growth have been renamed by the lineage they belong to, R800, D39, G54 and CP1250, respectively. Pre-culture stocks were inoculated at 50, 100, 500, 1500 and 3000 fold dilutions, depicted on the graph as closed dark brown circles, closed brown circles, closed dark orange circles, closed orange circles and closed yellow circles, respectively as described in M&M. RLU and OD readings were recorded as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006113#pgen.1006113.g001" target="_blank">Fig 1A</a>. Experiments were reproduced at least three times independently, and led to identical results to the one presented. (A) Competence development versus incubation time. (B) Competence development versus cell density. (C) OD at competence shift versus OD of the inoculum. Dashed red line represents a theoretical competence shift relying on a cell density-dependent initiation mechanism (QS). Dashed blue line represents a theoretical competence shift relying on a GTD mechanism. To calculate these two theoretical representations, the fixed OD value considered to set up the theoretical dashed line for each lineage is the one obtained for the highest cell density inoculum. Closed colored circles on the black curve represent data extracted for each strain derivative with color respecting the dilution ratio of inoculum (for data extraction, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006113#sec016" target="_blank">M&M</a>).</p

Spontaneous pneumococcal competence development.

<p>(A) The four steps of spontaneous competence development at the population level in the pneumococcus. A pre-culture stock of the R895 strain (which harbors the <i>luc</i> transcriptional fusion under the control of the <i>ssbB</i> late competence gene promoter) was inoculated at a 50 fold dilution as described in M&M. Open diamonds represent cell growth, closed diamonds the competence specific activity expressed in RLU.OD⁻¹. Black diamonds correspond to the non competent state of the cell population (pre-competence), green diamonds correspond to the competence shift taken as the point of intersection between the non competence and the competence development phase, blue diamonds represent the period of competence development from which the rate can be calculated. Red diamonds correspond to competence shut off. RLU and OD reading were recorded in a Varioskan flash luminometer at 3-min intervals. (B) Schematic representation of pneumococcal competence regulation. <i>comC</i> encodes pre-CSP (depicted in red), which is exported as mature CSP (red triangles) by the <i>comAB</i> gene product. <i>comDE</i> encodes a two-component system made of ComD that senses CSP and, in turn, by phosphorylating the transcription regulator ComE. ComE~P activates a set of genes called early competence genes, pictured in green, with the exception of <i>comC</i> that is depicted in red. The products of the <i>com</i>AB and <i>com</i>CDE operons form a core sensor, named “ComABCDE”, which constitutes a positive feedback loop controlling competence induction (as depicted in C). The early genes also include <i>comX</i>, which encodes an alternative sigma factor (ComX) that directs the RNA polymerase core to a second set of competence genes, named the late competence genes. Amongst these, <i>dprA</i> shuts off competence, thus generating a negative feedback loop of competence. A transcriptional fusion between any early or late competence gene promotor with the luciferase gene (<i>luc</i>) will report on competence expression throughout the growth, as shown in A. (C) The ComABCDE core sensor of pneumococcal competence. In the pre-competence state, components of the ComABCDE core sensor are expressed at a basal level [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006113#pgen.1006113.ref016" target="_blank">16</a>], insufficient to produce enough CSP for competence induction. This state is referred to as an idle mode shown by dotted arrows on the circular representation of the ComABCDE core sensor. Several external or internal inputs can negatively or positively impact the rate of synthesis and the stability of the components of the ComABCDE (for review see: [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006113#pgen.1006113.ref011" target="_blank">11</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006113#pgen.1006113.ref017" target="_blank">17</a>]). When positive inputs dominate, the core sensor is activated and the CSP reaches a threshold value that shifts the cell into a competence development state. This activated mode of the core sensor is represented by full arrows.</p

Competence development rate of the cell population is limited by CSP availability.

<p>Competence was monitored for the R895 strain inoculated at 50 fold (brown circles, 40: wt) or 2000 fold (orange circles, 1: wt) dilutions of the same pre-culture. The 2000-fold dilution was repeated with the addition of 100 ng/ml of synthetic CSP at the time is pointed by a green arrow (green squares, 1: wt + CSP). RLU and OD readings were recorded in a LucyI luminometer every 6 min (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006113#sec016" target="_blank">M.&M</a>). Competence development rate and correlation coefficient R² are presented for each assay.</p

Programmed Protection of Foreign DNA from Restriction Allows Pathogenicity Island Exchange during Pneumococcal Transformation

<div><p>In bacteria, transformation and restriction-modification (R-M) systems play potentially antagonistic roles. While the former, proposed as a form of sexuality, relies on internalized foreign DNA to create genetic diversity, the latter degrade foreign DNA to protect from bacteriophage attack. The human pathogen <em>Streptococcus pneumoniae</em> is transformable and possesses either of two R-M systems, DpnI and DpnII, which respectively restrict methylated or unmethylated double-stranded (ds) DNA. <em>S. pneumoniae</em> DpnII strains possess DpnM, which methylates dsDNA to protect it from <em>Dpn</em>II restriction, and a second methylase, DpnA, which is induced during competence for genetic transformation and is unusual in that it methylates single-stranded (ss) DNA. DpnA was tentatively ascribed the role of protecting internalized plasmids from <em>Dpn</em>II restriction, but this seems unlikely in light of recent results establishing that pneumococcal transformation was not evolved to favor plasmid exchange. Here we validate an alternative hypothesis, showing that DpnA plays a crucial role in the protection of internalized foreign DNA, enabling exchange of pathogenicity islands and more generally of variable regions between pneumococcal isolates. We show that transformation of a 21.7 kb heterologous region is reduced by more than 4 logs in <em>dpnA</em> mutant cells and provide evidence that the specific induction of <em>dpnA</em> during competence is critical for full protection. We suggest that the integration of a restrictase/ssDNA-methylase couplet into the competence regulon maintains protection from bacteriophage attack whilst simultaneously enabling exchange of pathogenicicy islands. This protective role of DpnA is likely to be of particular importance for pneumococcal virulence by allowing free variation of capsule serotype in DpnII strains via integration of DpnI capsule loci, contributing to the documented escape of pneumococci from capsule-based vaccines. Generally, this finding is the first evidence for a mechanism that actively promotes genetic diversity of <em>S. pneumoniae</em> through programmed protection and incorporation of foreign DNA.</p> </div

Pneumococcal competence is induced by a cell fraction and propagated by cell contact throughout the population.

<p>On the classical competence profile of a growing planktonic cell population described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006113#pgen.1006113.g001" target="_blank">Fig 1A</a>, the X_A and X_B values correspond to the duration of the pre-competence and the competence development periods respectively, separated by competence shift. On the bottom part of the graph, each column represents how the ComABCDE core sensor module (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006113#pgen.1006113.g001" target="_blank">Fig 1C</a>) evolves in individual cells during these 3 distinct stages. During the pre-competence period (left column), distinct and various stresses (lightning arrows) are sensed by individual cells either as positive input (green) or negative input (black) modifying the state of their core sensor accordingly (symbolized by measuring cylinder cartoon). The core sensor could be turned on in some cells <i>via</i> an autocrine mode in response to competence inducing stresses. Upon reaching a threshold value at X_A this subpopulation triggers competence propagation throughout the population at the shift point (medium column). This propagation proceeds by CSP transmission <i>via</i> cell-to-cell contact during the X_B period (right column). During competence propagation, the receptor cells switch into competence by turning on their ComABCDE core sensor <i>via</i> a paracrine mode (core sensor colored in blue).</p

Competence development rate of the cell population depends on cell density.

<p>For both graphs: R800 lineage, brown squares, D39 lineage, green triangles, G54 lineage, purple circles and CP1250 lineage, blue diamonds. Experiments were reproduced at least three times and always gave the same profile (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006113#sec016" target="_blank">M&M</a> for the mode of calculation). (A) Competence shift time is presented for each strain versus cell density of each inoculum (B) Competence development rate plotted versus fold dilution at each inoculum. Strains used as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006113#pgen.1006113.g002" target="_blank">Fig 2</a>.</p

Importance of DpnA-mediated protection from restriction is dependent on the number of GATC sites present in the heterologous cassette.

<p>(A) Relationship between heterology length and <i>dpnA⁻</i> to <i>dpnA</i>⁺ ratio. Ratios represent transformation efficiency in <i>dpnA⁻</i> compared to <i>dpnA⁺</i>. me⁺ donor DNA, closed diamonds. me⁰ DNA, open squares. Error bars are calculated from triplicate repeats of experiments (N = 3). Cassettes transferred and donor strains can be found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003178#ppat-1003178-t001" target="_blank">Table 1</a>. Recipient strains: R3087 (DpnII); R3088 (DpnII, <i>dpnA⁻</i>); R3163 (DpnII) and R3164 (DpnII, <i>dpnA⁻</i>) were used for transfer of <i>fcsR</i>::<i>ermAM1</i>^C to overcome antibiotic incompatibilities. To create the 21.7 kb heterology, the following recipient strains were used, which lacked the fully capsule locus: R3148 (DpnII, <i>cps</i>::<i>kan</i>) and R3149 (DpnII, <i>dpnA⁻</i>, <i>cps</i>::<i>kan</i>). (B) Relationship between number of GATC sites in donor heterologous cassette and <i>dpnA⁻</i> to <i>dpnA⁺</i> ratio. Same experiment as in panel A, but with ratios plotted against GATC sites in the heterology. (C) Comparison of transformation efficiency of <i>glnR</i>::<i>kan22</i>^C cassettes with varying number of GATC sites. me⁺ donor DNA was not tested. Error bars as in panel A legend. Strains used: donor, R3154 (DpnI, <i>glnR</i>::<i>kan22</i>^{C (8)}, <i>rpsL41</i>), R3238 (DpnI, <i>glnR</i>::<i>kan22</i>^{C (3)}, <i>rpsL41</i>) and R3239 (DpnI, <i>glnR</i>::<i>kan22</i>^{C (6)}, <i>rpsL41</i>); recipient, R3087 (DpnII) and R3088 (DpnII, <i>dpnA⁻</i>).</p

Diagrammatic representation of the impact of R-M systems on transformation.

<p>(A) Integration of a point mutation carried by me⁰ donor DNA is not affected by restriction. Color code: green, transforming (donor) DNA; black, host DNA; grey, displaced recipient strand; red, newly replicated DNA. me⁺ DNA shown as closed circles and me⁰ DNA as open circles; point mutation as R in donor and s for recipient. (B) Integrated me⁺ heterologous donor DNA is resistant to <i>Dpn</i>II, which is unable to cleave me^+/0 dsDNA produced by replication. Color code as above; thick red, neosynthesized complement to heterology. (C) Integrated me⁰ heterologous donor DNA is sensitive to restrictase (e.g., <i>Dpn</i>II) because me⁰ dsDNA is produced by replication. We hypothesize that DpnA, which, unlike DpnM, acts on ssDNA, protects the transformation intermediate by ensuring production of me^+/0 dsDNA after replication (as in panel B).</p

Information on heterology cassettes used in this study.

*<p>^C and ^A indicate, respectively, the co-transcribed and reverse orientation of an inserted mini-transposon antibiotic resistance cassette with respect to the target gene.</p>†<p>See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003178#ppat.1003178.s002" target="_blank">Table S2</a>.</p>‡<p><i>cps2E</i>::<i>spc7</i>^C cassette transformed into R6-derived strains R3087 and R3088 (with 8 kb deletion in capsule locus).</p>§<p><i>cps2E</i>::<i>spc7</i>^C cassette transformed into <i>cps</i>::<i>kan</i> mutant strains R3148 and R3149 (with 21 kb capsule locus deleted).</p