Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureus

The peptidoglycan of Staphylococcus aureus is characterized by a high degree of crosslinking and almost completely lacks free carboxyl groups, due to amidation of the D-glutamic acid in the stem peptide. Amidation of peptidoglycan has been proposed to play a decisive role in polymerization of cell wall building blocks, correlating with the crosslinking of neighboring peptidoglycan stem peptides. Mutants with a reduced degree of amidation are less viable and show increased susceptibility to methicillin. We identified the enzymes catalyzing the formation of D-glutamine in position 2 of the stem peptide. We provide biochemical evidence that the reaction is catalyzed by a glutamine amidotransferase-like protein and a Mur ligase homologue, encoded by SA1707 and SA1708, respectively. Both proteins, for which we propose the designation GatD and MurT, are required for amidation and appear to form a physically stable bi-enzyme complex. To investigate the reaction in vitro we purified recombinant GatD and MurT His-tag fusion proteins and their potential substrates, i.e. UDP-MurNAc-pentapeptide, as well as the membrane-bound cell wall precursors lipid I, lipid II and lipid II-Gly5. In vitro amidation occurred with all bactoprenol-bound intermediates, suggesting that in vivo lipid II and/or lipid II-Gly5 may be substrates for GatD/MurT. Inactivation of the GatD active site abolished lipid II amidation. Both, murT and gatD are organized in an operon and are essential genes of S. aureus. BLAST analysis revealed the presence of homologous transcriptional units in a number of gram-positive pathogens, e.g. Mycobacterium tuberculosis, Streptococcus pneumonia and Clostridium perfringens, all known to have a D-iso-glutamine containing PG. A less negatively charged PG reduces susceptibility towards defensins and may play a general role in innate immune signaling

It's All in the Interpretation

We often see disparate interpretations in language and politics, but the same is true in science expriments. This is particularly true in chemistry where one cannot see the molecules directly. Laren Tolbert of the Georgia Tech School of Chemistry & Biochemistry joins us to discusses challenges in interpreting such experiments

Ethics of Cloning

Dr. Roberta Berry, Director of the Law, Science and Technology Program at Georgia Tech will join us to discuss the ethics of clonin

Counterfeit Drugs

Dr. Facundo Fernandez will be discussing the conterfeiting of pharmaceuticals and his recent work on antimalarial drug

Cloning and characterization of argC gene of Streptomyces clavuligerus

Comunicación oral A 49 presentada al citado simposio, celebrado del 12-18, agosto, 1990, en Estrasburgo, Francia.S. clavuligerus is a producer of the beta-lactam antibiotic cephamycin C and the beta-lactamase inhibitor clavulanic acid. Arginine and/or ornithine are precursors of the clavulanic acid molecule and stimulate the production of clavulanic acid (Romero et al. Appl. Env. Microbiol. 52. 892. 1986). We are interested in elucidating the relationship between the arginine biosynthetic pathway and antibiotic production in S. clavuligerus.Peer reviewe

Role of murF in Cell Wall Biosynthesis: Isolation and Characterization of a murF Conditional Mutant of Staphylococcus aureus

The Staphylococcus aureus murF gene was placed under the control of a promoter inducible by IPTG (isopropyl-β-d-thiogalactopyranoside). It was demonstrated that murF is an essential gene; it is cotranscribed with ddlA and growth rate, level of beta-lactam antibiotic resistance, and rates of transcription of the mecA and pbpB genes paralleled the rates of transcription of murF. At suboptimal concentrations of the inducer, a UDP-linked muramyl tripeptide accumulated in the cytoplasm in parallel with the decline in the amounts of the normal pentapeptide cell wall precursor. The abnormal tripeptide component incorporated into the cell wall as a monomeric muropeptide, accompanied by a decrease in the oligomerization degree of the peptidoglycan. However, incorporation of the tripeptide into the cell wall was limited to a relatively low threshold value. Further reduction of the amounts of pentapeptide cell wall precursor caused a gradual decrease in the cellular amounts of peptidoglycan, the production of a thinner peripheral cell wall, aberrant septae, and an overall increase in the diameter of the cells. The observations suggest that the role of murF exceeds its primary function in peptidoglycan biosynthesis and may also be involved in the control of cell division