Cell shape and PG structure evolution among the <i>Moraxellaceae</i> family.

<p>Schematic phylogeny of the <i>Moraxellaceae</i> family, based on 16s analysis, along with scanning electronic microscopy images of representative species. The mean ratio (GM4+GM4_GM4)/(GM5+GM5_GM4) is presented (with standard deviation) for the different lineages (*** p≤0.001; ** p≤0.01; * p≤0.05). Each dote, represents an independent isolate (tested for <i>pbpX</i> presence) from the CNRM collection that have been classified as <i>M</i>. <i>catharrhalis</i> (3), <i>M</i>. <i>sp LNP20863</i> (1), <i>M</i>. <i>bovis (gift from Dr</i>. <i>S</i>. <i>Higlander—1) M</i>. <i>osloensis</i> (1), <i>M</i>. <i>sp</i>. LNP 26500 (1), <i>A</i>. <i>lwoffi</i> (2). Finally, the right part displays the deletion detected at the node of evolution by presenting the genomic organization of species that diverged before node 1 (assessed from the genome sequence of <i>M</i>. <i>bovis</i> and <i>A</i>. <i>lwoffi</i>) and after node 1 (from the <i>M</i>. <i>catharrhalis</i> genome).</p

Deleterious effects of <i>yacF</i> deletion can be fixed by inhibiting cell elongation.

<p>A) Scanning electron microscopy images showing similar morphology (coccus) of <i>N</i>. <i>elongata</i> Δ<i>mreBCD</i>,<i>pbpX</i>,<i>rodA</i> and the double mutant Δ<i>yacF</i> Δ<i>mreBCD</i>,<i>pbpX</i>,<i>rodA</i>. B) Same reverse-phase HPLC profile of muropeptides after mutanolysin digestion of purified insoluble PG of <i>N</i>. <i>elongata</i> Δ<i>mreBCD</i>,<i>pbpX</i>,<i>rodA</i> and the double mutant Δ<i>yacF</i> Δ<i>mreBCD</i>,<i>pbpX</i>,<i>rodA</i>. C) Similar ratio of GM4 quantity over GM5, GM4-GM3 over GM5-GM3 and finally GM4-GM4 over GM5-GM4 in the PG of Δ<i>mreBCD</i>,<i>pbpX</i>,<i>rodA</i> and the double mutant Δ<i>yacF</i> Δ<i>mreBCD</i>,<i>pbpX</i>,<i>rodA</i>. The raw quantification can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005338#pgen.1005338.s002" target="_blank">S2 Fig</a> for <i>N</i>. <i>elongata</i> and the corresponding mutants. The ratio of wild type bacteria, the single mutant Δ<i>yacF</i> and the complemented strain Δ<i>yacF+yacF</i> are also presented. The increased proportion of GM5 in the PG of Δ<i>yacF</i> (as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005338#pgen.1005338.g003" target="_blank">Fig 3</a>) can be again observed but this increase is less important than when the <i>mreBCD</i>,<i>pbpX</i>,<i>rodA</i> locus, encoding for the elongation machinery, is deleted. (*** p≤0.001; ** p≤0.01; * p≤0.05 compare to wild-type).</p

Different properties of coccus cells.

<p>A) NF-κB luciferase expression in response to PG from <i>N</i>. <i>elongata</i> rod (wild-type) or cocci (Δ<i>mreBCD</i>,<i>pbpX</i>,<i>rodA</i> Δ<i>yacF</i>) of HEK-293 cells transfected with human Nod1 (grey), human Nod2 (black) and murin Nod1 (white). The two controls measured the luciferase in absence of stimulation or in presence of purified specific agonist (MurTriDap for hNod1, MDP for hNod2, and MurTetraDap for mNod1). This is representative result of two independent experiments. All the results are statistically significant (p<0.05 rod vs cocci) except those noted ns (for non statistically significant). B) Estimated ratio surface/volume extracted from SEM images of around 20 cells (*** p≤0.001) and C) TEM image showing pili (red arrow) from <i>N</i>. <i>elongata</i> wild type (bacillus) and <i>N</i>. <i>elongata</i> Δ<i>yacF</i> Δ<i>mreBCD</i>,<i>pbpX</i>,<i>rodA</i> (coccus).</p

Deleterious effects of <i>yacF</i> deletion can be fixed by inhibiting cell division.

<p>A) Scanning electron microscopy images showing similar morphology (filaments) of <i>N</i>. <i>elongata</i> wild type and Δ<i>yacF</i> grown in presence of sub-inhibitory concentrations of penicillin G. B) Similar reverse-phase HPLC profile of muropeptides after mutanolysin digestion of purified insoluble PG of <i>N</i>. <i>elongata</i> wild type and Δ<i>yacF</i> grown in presence of penicillin G. C) Similar ratio of GM4 over GM5, GM4-GM3 over GM5-GM3 and finally GM4-GM4 over GM5-GM4. (*** p≤0.001; ** p≤0.01; * p≤0.05 compare to wild-type).</p

Distribution of <i>yacF</i> and other key components of the elongation machinery in proteobacteria.

<p>Representation of the proteobacteria taxonomy associated with a table presenting the information of the presence in all (white), absence in all (black). The presence/absence was assessed using the STRING database as previously described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005338#pgen.1005338.ref045" target="_blank">45</a>]. A grey box indicates that the distribution was not uniform (presence of outliers) in the specific lineage. Finally, the cell morphology is also presented only for <i>yacF</i>-positive lineages to emphasize that the majority of <i>yacF</i>-positive strains are bacillus. A grey cell-shape indicates the presence of outliers in the lineage (herein <i>Methylococcus capsulatus</i>). Red asterisks indicate situation where the distribution is described more in details in the text. (e.g. the case of <i>Pseudomonadeles</i> is described more in detail in the text and in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005338#pgen.1005338.g009" target="_blank">Fig 9</a>).</p

Cellular adaptation to artificial coccoïd transition.

<p>A) A graphical representation of the <i>N</i>. <i>elongata</i> genome sequenced by PacBio is illustrated here using CIRCOS software. The first circle represents the different <i>orf</i> and their orientation. The second circle represents the coverage values (orange) of the Illumina reads obtain by sequencing <i>N</i>. <i>elongata</i> Δ<i>mreBCD</i>,<i>pbpX</i>,<i>rodA</i> and mapped along the genome of <i>N</i>. <i>elongata</i> wild-type. One can notice the increased coverage by two fold of the DNA region from 0.202 to 0.558 Mbp. Finally, the third internal circle represent the fold change expression, in function of the position, of genes up-regulated (green) and down-regulated (red) of Δ<i>mreBCD</i>,<i>pbpX</i>,<i>rodA</i> compare to wild type <i>N</i>. <i>elongata</i>. Only genes with a p-value below 0.01 and fold change over 3 are represented. All the fold change values and statistics are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005338#pgen.1005338.s006" target="_blank">S3 Table</a>. B) Verification of transcriptional changes of selected genes, in the different mutants, using rt-PCR and calculated using ΔΔCT (*** p≤0.001; ** p≤0.01; * p≤0.05).</p

Effect of <i>yacF</i> deletion in <i>N</i>. <i>elongata</i> and <i>N</i>. <i>baciliformis</i>.

<p>Scanning electron microscopy images of A) <i>N</i>. <i>elongata</i> wild type, Δ<i>yacF</i> and ∆<i>yacF</i> complemented with <i>yacF</i> in a heterologous locus (Δ<i>yacF+yacF</i>) and B) <i>N</i>. <i>bacilliformis</i> wild type and Δ<i>yacF</i>.</p

GM5 detection on polar and lateral PG.

<p>A) Transmission electronic microscopy image of immuno-gold detection of GM5 using vancomycin labelling of <i>N</i>. <i>bacilliformis</i> saculli. B) Estimation of the mean numbers, with standard deviation, of gold beads by 10μm² of sacculi surface of both polar/septal and lateral PG measured on around 20 cells (*** p≤0.001).</p