Phylogenetic distribution of peptidoglycan synthesis enzymes across selected bacterial species.

<p><b>(A)</b> The blue, stacked bars represent the total number of proteins present in each species that have a transpeptidase (dark blue) or transglycosylase (light blue) domain. The total number of proteins that have at least one of the two domains is displayed numerically. Strains highlighted in pink are bacteria reported not to possess cell wall, although they may produce small but functional amounts of peptidoglycan. The maximum likelihood species tree was calculated using PhyML [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004891#ppat.1004891.ref053" target="_blank">53</a>] and concatenated bacterial marker genes identified with the AMPHORA2 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004891#ppat.1004891.ref052" target="_blank">52</a>] software. <b>(B)</b> Table showing the peptidoglycan synthesis proteins from <i>S</i>. <i>aureus</i> and their established or hypothetical activities.</p

<i>S</i>. <i>aureus</i> minimal mutant strain COL MIN displays normal growth and requires PBP1 and PBP2 for survival.

<p><b>(A)</b> Growth of the parental strain COL and the minimal mutant strain COL MIN was followed in rich liquid medium by monitoring the absorbance at OD_600nm. The mutant strain COL MIN (doubling time 40 min) showed similar growth to the parental strain COL (doubling time 36 min). <b>(B)</b> Growth of COL and COL MIN was followed in minimal medium by monitoring the absorbance at OD_600nm. The mutant strain COL MIN (doubling time 67 min) showed similar growth to the parental strain COL (doubling time 61 min). <b>(C)</b> Depletion of PBP1 from COL PBP1i and COL MIN PBP1i, in which PBP1 expression is under the control of the IPTG inducible P_<i>spac</i> promoter, by growing cells in the absence of IPTG, led to a halt in cell growth and subsequent drop in optical density indicating PBP1 is essential for survival of both the parental and mutant strains. <b>(D)</b> In the absence of PBP2, strain COL PBP2i (parental strain COL with PBP2 expression under the control of the IPTG inducible P_<i>spac</i> promoter) continues to grow. However, depletion of PBP2 from COL MIN PBP2i causes arrest in growth indicating PBP2 is essential for growth of COL MIN. Averages of three independent replicates are shown and error bars show standard deviations.</p

<i>S</i>. <i>aureus</i> COL MIN showed attenuated virulence in a <i>Drosophila</i> infection model and increased susceptibility to lysozyme.

<p><b>(A, B)</b> Estimated survival curves for wild type (WT) and PGRP-SA mutant (<i>seml</i>) flies infected with COL and COL MIN <i>S</i>. <i>aureus</i> strains or PBS (to monitor the physical effects of the injection <i>per se</i>). WT flies strongly succumbed to infection with COL by 96 hours whereas 88% of WT flies infected with COL MIN survived. Curves were statistically separable, log-rank test P<0.05. At least 90% of the PGRP-SA-deficient flies were killed by WT bacteria (within 60 hours) and by COL MIN mutant strain (within 96 hours). Curves were statistically separable, log-rank test P<0.05. (<b>C)</b> Bacterial cell lysis monitored through the decrease of OD₆₀₀ was determined for COL and COL MIN strains in the presence (+) or absence (-) of lysozyme (300 μg/ml). The minimal strain showed increased cell lysis in the presence of lysozyme. Data shows mean with 95% confidence intervals of three independent biological repeats.</p

<i>S</i>. <i>aureus</i> strain COL MIN displays normal morphology and correct localization of PBP1 and PBP2.

<p><b>(A)</b> Structured illumination microscopy images of cells incubated with (i) Van-FL, and (ii) Hoechst 33342 to label the cell wall and DNA, respectively, show no difference between the parental strain COL and COL MIN, (iii) overlay of Van-FL and Hoechst labeled cells. Scale bars represent 1μm. (<b>B</b>) Percentage of cells with complete septa (i), partial septa (ii) and no septa (iii) in COL (n = 333) and COL MIN (n = 223) strains. <b>(C)</b> Representative electron microscopy images of COL and COL MIN show that cells retain a normal shape and septum placement in the absence of seven PG synthesis enzymes. <b>(D)</b> Localization of PBP1 (i), PBP2 (ii) and FtsZ (iii), by immunofluorescence, in COLΔ<i>spa</i> and COL MIN Δ<i>spa</i> cells shows that the three proteins localize to the septum in the COL MIN strain, similarly to the parental strain COL. FtsZ was used as a control for septal localization. Strains lacking the <i>spa</i> gene were used for immunofluorescence experiments as the <i>spa</i> gene product Protein A binds with high affinity to IgG molecules. Scale bar represents 1μm.</p