Cterial cell wall is involved in PG cross-linking, the lack of or UKI 1 web incorrect substrateincorporation into the PG macromolecule can lead to improperly constructed PG and ultimately to cell death via lysis due to inability of the bacterium to maintain osmotic pressure [14,15]. Here we report the first characterization of a Mur ligase from the genus Verrucomicrobium, namely MurE from V. spinosum (MurEVs). In vivo analysis demonstrates that the enzyme is able to functionally complement an Escherichia coli strain that harbors a mutation in the murE gene. Using in vitro analyses, 10457188 we show that MurEVs is a meso-A2pm-adding enzyme. Furthermore, we present a structural analysis of the enzyme using protein sequence alignment and homology modeling, which shows that key amino acids for substrate binding and/or catalysis are conserved in MurEVs. Together, these experiments contribute to the further understanding of the kinetic, physical and structural properties of the Mur ligase involved in the synthesis of PG from the organism V. spinosum. Finally, V. spinosum PG was purified and analyzed; its composition in which A2pm is one of the main constituents is similar to that of most Gram-negative bacteria.Materials and Methods V. spinosum growth conditionsV. spinosum DSM 4136T was cultured in R2A medium at 26uC [10].PCR amplification and cloning of the V. spinosum murE open reading frame (ORF) for protein expression and purificationThe open reading frame annotated by the locus tag (VspiD_010100019130) UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-2,6-diaminopimelate ligase was amplified by PCR. The following forward and reverse primers were used: murEVs-forward 59-CACCATGACCATTTTGCGCGATCTTATCGAGGGT-39 and murEVs-reverse 59-GTCGACTCACTGACGGTCATCCCTCCTTTGGCGTGC-39 (the underlined sequence represents the restriction enzyme site used to facilitate sub-cloning of the ORF while the bold and italicized sequences represent initiation and termination codons). The PCR reaction contained 12 pmol of forward and reverse primers, 1 mM MgSO4, 0.5 mM of each of the four deoxynucleotide triphosphates, 0.5 ng ofMurE from Verrucomicrobium spinosum DSM 4136Tgenomic DNA and 1 unit of Platinum Pfx DNA polymerase (Invitrogen Corporation, Carlsbad, CA, USA). PCR conditions were: 1 cycle at 94uC for 2 min, followed by 30 cycles of 94uC for 15 s, 60uC for 30 s and 72uC for 2 min. The murE PCR fragment was ligated into the plasmid pET100D/topo (Invitrogen Corporation, Carlsbad, CA, USA) to produce the plasmid pET100D::murEVs. The recombinant protein encoded by this plasmid carries a MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDHPFT sequence containing a hexa-histidine tag derived from the pET100D plasmids at the amino terminus. To confirm the fidelity of the PCR reaction, the murE ORF was sequenced from pET100D using the T7 promoter primer, 59TAATACGACTCACTATAGGG-39 and the T7 reverse primer, 59-TAGTTATTGCTCAGCGGTGG-39. The cloned murE ORF was 100 identical to the sequences deposited in the Integrated Microbial Genomes public database (http://img.jgi.doe.gov/cgibin/w/main.cgi).the labeled substrate; in that case, radioactive substrate and product were separated by thin-layer chromatography on silica gel plates LK6D (Whatman) using 1-propanol/ammonium hydroxide/water (6:3:1; v/v) as the mobile phase, and the radioactive spots were located and quantified with a radioactivity scanner (Rita Star, Raytest Isotopenmebgerate GmbH). ?Calcitonin (salmon) web determination of the kinetic constantsFor the determination of the k.Cterial cell wall is involved in PG cross-linking, the lack of or incorrect substrateincorporation into the PG macromolecule can lead to improperly constructed PG and ultimately to cell death via lysis due to inability of the bacterium to maintain osmotic pressure [14,15]. Here we report the first characterization of a Mur ligase from the genus Verrucomicrobium, namely MurE from V. spinosum (MurEVs). In vivo analysis demonstrates that the enzyme is able to functionally complement an Escherichia coli strain that harbors a mutation in the murE gene. Using in vitro analyses, 10457188 we show that MurEVs is a meso-A2pm-adding enzyme. Furthermore, we present a structural analysis of the enzyme using protein sequence alignment and homology modeling, which shows that key amino acids for substrate binding and/or catalysis are conserved in MurEVs. Together, these experiments contribute to the further understanding of the kinetic, physical and structural properties of the Mur ligase involved in the synthesis of PG from the organism V. spinosum. Finally, V. spinosum PG was purified and analyzed; its composition in which A2pm is one of the main constituents is similar to that of most Gram-negative bacteria.Materials and Methods V. spinosum growth conditionsV. spinosum DSM 4136T was cultured in R2A medium at 26uC [10].PCR amplification and cloning of the V. spinosum murE open reading frame (ORF) for protein expression and purificationThe open reading frame annotated by the locus tag (VspiD_010100019130) UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-2,6-diaminopimelate ligase was amplified by PCR. The following forward and reverse primers were used: murEVs-forward 59-CACCATGACCATTTTGCGCGATCTTATCGAGGGT-39 and murEVs-reverse 59-GTCGACTCACTGACGGTCATCCCTCCTTTGGCGTGC-39 (the underlined sequence represents the restriction enzyme site used to facilitate sub-cloning of the ORF while the bold and italicized sequences represent initiation and termination codons). The PCR reaction contained 12 pmol of forward and reverse primers, 1 mM MgSO4, 0.5 mM of each of the four deoxynucleotide triphosphates, 0.5 ng ofMurE from Verrucomicrobium spinosum DSM 4136Tgenomic DNA and 1 unit of Platinum Pfx DNA polymerase (Invitrogen Corporation, Carlsbad, CA, USA). PCR conditions were: 1 cycle at 94uC for 2 min, followed by 30 cycles of 94uC for 15 s, 60uC for 30 s and 72uC for 2 min. The murE PCR fragment was ligated into the plasmid pET100D/topo (Invitrogen Corporation, Carlsbad, CA, USA) to produce the plasmid pET100D::murEVs. The recombinant protein encoded by this plasmid carries a MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDHPFT sequence containing a hexa-histidine tag derived from the pET100D plasmids at the amino terminus. To confirm the fidelity of the PCR reaction, the murE ORF was sequenced from pET100D using the T7 promoter primer, 59TAATACGACTCACTATAGGG-39 and the T7 reverse primer, 59-TAGTTATTGCTCAGCGGTGG-39. The cloned murE ORF was 100 identical to the sequences deposited in the Integrated Microbial Genomes public database (http://img.jgi.doe.gov/cgibin/w/main.cgi).the labeled substrate; in that case, radioactive substrate and product were separated by thin-layer chromatography on silica gel plates LK6D (Whatman) using 1-propanol/ammonium hydroxide/water (6:3:1; v/v) as the mobile phase, and the radioactive spots were located and quantified with a radioactivity scanner (Rita Star, Raytest Isotopenmebgerate GmbH). ?Determination of the kinetic constantsFor the determination of the k.
