This thesis describes the structural and biochemical characterization of the β-lactamase BlaC from Mycobacterium tuberculosis (Mtb), and the Alr and YlmE proteins from Streptomyces coelicolor A3(2)... Show moreThis thesis describes the structural and biochemical characterization of the β-lactamase BlaC from Mycobacterium tuberculosis (Mtb), and the Alr and YlmE proteins from Streptomyces coelicolor A3(2).Mtb is the main cause of tuberculosis. The inherent production of BlaC by Mtb makes the antibiotic treatment of tuberculosis particularly difficult because BlaC renders Mtb naturally resistant to β-lactam antibiotics. One possible way to circumvent this BlaC-mediated resistance is the co-administration of β-lactamase inhibitors, thus preventing antibiotics’ hydrolysis. The crystal structure of BlaC was determined in complex with the β-lactamase inhibitors clavulanic acid, sulbactam, tazobactam, and avibactam, and new BlaC-inhibitors covalent adducts were visualized. The affinity of BlaC for the inhibitors was further studied using catalytically inactive mutants of the enzyme.In parallel, the Alr and YlmE proteins from S. coelicolor A3(2) were studied. Alr and YlmE are putatively involved in the racemization of L-Ala into D-Ala. The latter is an essential peptidoglycan building block, and ensures cell wall compaction and bacterial survival. The structural and biochemical characterization of the heterologous, purified Alr and YlmE proteins showed that while Alr is indeed involved in Ala racemization, YlmE is not. Our findings revealed a possible new, surprising role for YlmE in nucleic acid binding. Show less
During developmental cell division in sporulation-committed aerial hyphae of streptomycetes, up to a hundred septa are simultaneously produced, in close harmony with synchromous chromosome... Show moreDuring developmental cell division in sporulation-committed aerial hyphae of streptomycetes, up to a hundred septa are simultaneously produced, in close harmony with synchromous chromosome condensation and segregation. Several unique protein families are involved in the control of this process, including that of the SsgA-like proteins (SALPs). While SsgA and SsgB are essential for sporulation-specific cell division in S. coelicolor, SsgC-G are responsible for correct DNA segregation/condensation, spore wall synthesis, autolytic spore separation, or exact septum localisation. The SALPs are a novel protein family that acts through timing and localisation of the activity of penicillin-binding proteins and autolysins, thus controlling important steps during the initiation and the completion of sporulation. The formation of septa is initiated by the formation of a ring of the tubulin-like protein FtsZ (the Z-ring), functioning as a scaffold for the construction of septa. Subsequently, other cell division proteins are recruited to the Z-ring, forming the divisome. In S. coelicolor, the cell division proteins FtsE and FtsX participate during autolytic spore separation, and most likely function by re-importing peptidoglycan subunits for recycling. The cytoskeletal protein MreB is involved in cell shape determination and chromosome segregation in many rod-shaped bacteria. In S. coelicolor, the actin-like proteins MreB and Mbl are not essential for vegetative growth but exert their function in the formation of environmentally stable spores, thereby primarily influencing the assembly of the spore wall. Show less
