In the identification of 20 genes that are known to be involved in PG metabolism. Importantly, the search identified orthologs of all the genes necessary for the de novo synthesis of PG in V. spinosum (Table 1).Kinetic properties of the MurE ligase from V. spinosumThe optimal pH, temperature and magnesium concentration for MurEVs were found to be 9.6, 44?6uC, and 30 mM, respectively. In vitro assays were thus performed at pH 9.6 and with 30 mM MgCl2, the usual temperature of 37uC being used. With meso-A2pm and UDP-MurNAc-L-Ala-D-Glu as substrates, the maximum velocity was 3662 mmol?min21?mg21. The Km values for the substrates were: ATP, 290670 mM; UDP-MurNAc-L-AlaD-Glu, 2466 mM; and meso-A2pm, 1763 mM. The enzyme Calcitonin (salmon) supplier proved to be stereospecific for meso-A2pm, since the rate of incorporation of the L,L or D,D isomer was ,1 that of the meso isomer. No incorporation of L-lysine or L-ornithine could be detected, evenIdentification of the MurE ortholog from V. spinosumThe orthologous MurE Lixisenatide protein from V. spinosum was initially identified using the MurE protein sequence from C. trachomatis (NP_219774) as a query. The BlastP algorithm from the Integrated Microbial Genomes (IMG) database was employed. The search resulted in the identification of a putative MurE from V. spinosum annotated by the locus tag VspiD_010100019130, which is 37 identical to the C. trachomatis MurE [10].Table 1. List of genes involved in PG metabolism of V. spinosum DSM 4136T.Locus Tag VspiD_010100024635 VspiD_010100022270 VspiD_010100006740 VspiD_010100020475 VspiD_010100007940 VspiD_010100017450 VspiD_010100019135 VspiD_010100018680 VspiD_010100019120 VspiD_010100011745 VspiD_010100019100 VspiD_010100019125 VspiD_010100018175 VspiD_010100019115 VspiD_010100019130 VspiD_010100026230 VspiD_010100018130 VspiD_010100018130 VspiD_010100000100 VspiD_Protein Symbol Annotated Gene Product Name PBP PBP PBP PBP PBP PBP PBP PBP MraY MurA MurG MurF Ddl MurD MurE UppP MurB MurC AlaR MurID-Alanyl-D-alanineEC Number 3.4.16.4 2.4.1.129 2.4.1.129 2.4.1.129 2.4.1.129 2.4.1.129 2.4.1.129 2.4.1.129 2.7.8.13 2.5.1.7 2.4.1.227 6.3.2.10 6.3.2.4 6.3.2.9 6.3.2.13 3.6.1.27 1.1.1.158 6.3.2.8 5.1.1.1 5.1.1.carboxypeptidase-class CMultimodular transpeptidase-transglycosylase-class A Penicillin-binding protein 1C- class A Penicillin-binding protein 2-class A Peptidoglycan transpeptidase-class B Peptidoglycan transpeptidase-class B Peptidoglycan synthetase FtsI-class B Cell elongation specific D,D-transpeptidase- class B Phospho-N-acetylmuramoyl-pentapeptide-transferase UDP-N-acetylglucosamine 1-carboxyvinyltransferase UDP-N-acetylglucosamine-N-acetylmuramoyl-(pentapeptide) pyrophosphorylundecaprenol N-acetylglucosamine transferase UDP-N-acetylmuramoyl-tripeptide:D-alanyl-D-alanine ligaseD-Alanine:D-alanineligaseUDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-2,6-diaminopimelate ligase Undecaprenyl pyrophosphate phosphatase UDP-N-acetylenolpyruvoylglucosamine reductase UDP-N-acetylmuramate:L-alanine ligase Alanine racemase Glutamate racemaseThe annotated gene product names are from NCBI (www.ncbi.nlm.nih.gov/protein/) queried of 23977191 February 28, 2013. The pencillin-binding proteins (PBP) class designations are denoted by activity based on protein family (pfam) domains. Class A and class B PBPs are high-molecular mass PBPs while class C PBPs are lowmolecular mass PBPs. Class A PBPs are predicted to have both transglycosylase and transpeptidase activities; c.In the identification of 20 genes that are known to be involved in PG metabolism. Importantly, the search identified orthologs of all the genes necessary for the de novo synthesis of PG in V. spinosum (Table 1).Kinetic properties of the MurE ligase from V. spinosumThe optimal pH, temperature and magnesium concentration for MurEVs were found to be 9.6, 44?6uC, and 30 mM, respectively. In vitro assays were thus performed at pH 9.6 and with 30 mM MgCl2, the usual temperature of 37uC being used. With meso-A2pm and UDP-MurNAc-L-Ala-D-Glu as substrates, the maximum velocity was 3662 mmol?min21?mg21. The Km values for the substrates were: ATP, 290670 mM; UDP-MurNAc-L-AlaD-Glu, 2466 mM; and meso-A2pm, 1763 mM. The enzyme proved to be stereospecific for meso-A2pm, since the rate of incorporation of the L,L or D,D isomer was ,1 that of the meso isomer. No incorporation of L-lysine or L-ornithine could be detected, evenIdentification of the MurE ortholog from V. spinosumThe orthologous MurE protein from V. spinosum was initially identified using the MurE protein sequence from C. trachomatis (NP_219774) as a query. The BlastP algorithm from the Integrated Microbial Genomes (IMG) database was employed. The search resulted in the identification of a putative MurE from V. spinosum annotated by the locus tag VspiD_010100019130, which is 37 identical to the C. trachomatis MurE [10].Table 1. List of genes involved in PG metabolism of V. spinosum DSM 4136T.Locus Tag VspiD_010100024635 VspiD_010100022270 VspiD_010100006740 VspiD_010100020475 VspiD_010100007940 VspiD_010100017450 VspiD_010100019135 VspiD_010100018680 VspiD_010100019120 VspiD_010100011745 VspiD_010100019100 VspiD_010100019125 VspiD_010100018175 VspiD_010100019115 VspiD_010100019130 VspiD_010100026230 VspiD_010100018130 VspiD_010100018130 VspiD_010100000100 VspiD_Protein Symbol Annotated Gene Product Name PBP PBP PBP PBP PBP PBP PBP PBP MraY MurA MurG MurF Ddl MurD MurE UppP MurB MurC AlaR MurID-Alanyl-D-alanineEC Number 3.4.16.4 2.4.1.129 2.4.1.129 2.4.1.129 2.4.1.129 2.4.1.129 2.4.1.129 2.4.1.129 2.7.8.13 2.5.1.7 2.4.1.227 6.3.2.10 6.3.2.4 6.3.2.9 6.3.2.13 3.6.1.27 1.1.1.158 6.3.2.8 5.1.1.1 5.1.1.carboxypeptidase-class CMultimodular transpeptidase-transglycosylase-class A Penicillin-binding protein 1C- class A Penicillin-binding protein 2-class A Peptidoglycan transpeptidase-class B Peptidoglycan transpeptidase-class B Peptidoglycan synthetase FtsI-class B Cell elongation specific D,D-transpeptidase- class B Phospho-N-acetylmuramoyl-pentapeptide-transferase UDP-N-acetylglucosamine 1-carboxyvinyltransferase UDP-N-acetylglucosamine-N-acetylmuramoyl-(pentapeptide) pyrophosphorylundecaprenol N-acetylglucosamine transferase UDP-N-acetylmuramoyl-tripeptide:D-alanyl-D-alanine ligaseD-Alanine:D-alanineligaseUDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-2,6-diaminopimelate ligase Undecaprenyl pyrophosphate phosphatase UDP-N-acetylenolpyruvoylglucosamine reductase UDP-N-acetylmuramate:L-alanine ligase Alanine racemase Glutamate racemaseThe annotated gene product names are from NCBI (www.ncbi.nlm.nih.gov/protein/) queried of 23977191 February 28, 2013. The pencillin-binding proteins (PBP) class designations are denoted by activity based on protein family (pfam) domains. Class A and class B PBPs are high-molecular mass PBPs while class C PBPs are lowmolecular mass PBPs. Class A PBPs are predicted to have both transglycosylase and transpeptidase activities; c.
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