PykF time-series fitness effects

Results of competitions to measure the effect of six of the evolved pykF mutations at up to six time points along their evolutionary trajector

PykF fitness effects: ancestor and 20K

Colony counts of competitions measuring the effect of pykF mutations in the ancestral strain (REL606) and in the 20,000 generation evolved strains each mutation was found in

PykF allele transfer fitness effects

Results of fitness competitions measuring the effect of pykF alleles transferred across different 20,000 generation evolved strains

PykF fitness analysis: ancestor and 20K

R script to analyze and plot pykF fitness effects in ancestor and 20,000 generation evolved strain

Specificity of MurE_Vs for the amino acid substrate.

a<p>Determined as described in Materials and Methods with fixed concentrations of ATP (5 mM), UDP-MurNAc-l-Ala-d-Glu (0.15 mM) and amino acid (0.15 mM).</p>b<p>ND, no activity detected after 30 minutes with 11 µg of enzyme.</p

pykF allele transfer analysis

R script to analyze and plot fitness of pykF mutations across different 20,000 generation evolved strain

Scanning electron microscopy of <i>V. spinosum</i> DSM 4136^T.

<p>The white arrows show the wart-like prosthecae (WLP) and the white bar depicts a tube-like prosthecae (TLP). The picture was taken at 25 K magnification. The scale bar is 1 µm.</p

Biochemical Characterization of UDP-<i>N</i>-acetylmuramoyl-L-alanyl-D-glutamate: <i>meso</i>-2,6-diaminopimelate ligase (MurE) from <i>Verrucomicrobium spinosum</i> DSM 4136^T

<div><p><i>Verrucomicrobium spinosum</i> is a Gram-negative bacterium that is related to bacteria from the genus <i>Chlamydia</i>. The bacterium is pathogenic towards <i>Drosophila melanogaster</i> and <i>Caenorhabditis elegans</i>, using a type III secretion system to facilitate pathogenicity. <i>V. spinosum</i> employs the recently discovered l,l-diaminopimelate aminotransferase biosynthetic pathway to generate the bacterial cell wall and protein precursors diaminopimelate and lysine. A survey of the <i>V. spinosum</i> genome provides evidence that the bacterium should be able to synthesize peptidoglycan <i>de novo</i>, since all of the necessary genes are present. The enzyme UDP-<i>N</i>-acetylmuramoyl-l-alanyl-d-glutamate: <i>meso</i>-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.15) catalyzes a reaction in the cytoplasmic step of peptidoglycan biosynthesis by adding the third amino acid residue to the peptide stem. The <i>murE</i> ortholog from <i>V. spinosum</i> (<i>murE</i>_Vs) was cloned and was shown to possess UDP-MurNAc-l-Ala-d-Glu:<i>meso</i>-2,6-diaminopimelate ligase activity <i>in vivo</i> using functional complementation. <i>In vitro</i> analysis using the purified recombinant enzyme demonstrated that MurE_Vs has a pH optimum of 9.6 and a magnesium optimum of 30 mM. <i>meso</i>-Diaminopimelate was the preferred substrate with a <i>K</i>_m of 17 µM, when compared to other substrates that are structurally related. Sequence alignment and structural analysis using homology modeling suggest that key residues that make up the active site of the enzyme are conserved in MurE_Vs. Our kinetic analysis and structural model of MurE_Vs is consistent with other MurE enzymes from Gram-negative bacteria that have been characterized. To verify that <i>V. spinosum</i> incorporates diaminopimelate into its cell wall, we purified peptidoglycan from a <i>V. spinosum</i> culture; analysis revealed the presence of diaminopimelate, consistent with that of a bona fide peptidoglycan from Gram-negative bacteria.</p></div

Homolgy model of MurE_Vs.

<p>(a) The homology model of MurE_Vs highlighting domains A (grey), B (violet) and C (pink). (b) Shows the structure model of MurE_Vs bound to UDP-MurNAc-tripeptide (UMT) product (yellow). (c) Active site residue hypothesized to bind to UMT product is shown in red. The structure has been rotated 90° on the right panel for the better viewing of the binding pocket. (d) Cross eye stereo view showing the interaction between amino acid residues of the binding site and UMT product.</p

Analysis of crude and purified PG from <i>V. spinosum</i> DSM 4136^T.

a<p>Crude and purified PG designate the macromolecule before and after, respectively, treatment with pancreatin, pronase and trypsin (see Materials and Methods).</p