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During amino acid deprivation, the amount of meso-diaminopimelic acid (Dap) incorporated into peptidoglycan by dap lys amino acid auxotrophs of Escherichia coli was found to be dependent on the activity of the relA gene product. In relA⁺ bacteria, the- incorporation was substantially reduced, whereas the incorporation in relA⁻ bacteria was essentially equal to that in the unstarved control. The inhibition of Dap incorporation in relA⁺ bacteria was readily overcome by restoration of the required amino acid or by addition of chloramphenicol (CAM). Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) is the product of the reaction between the relA gene product and idling ribosomes in stringent cells. In vitro experiments indicated that physiological levels of ppGpp inhibited at least two steps in peptidoglycan biosynthesis. One was the phospho-N-acetylmuramoyl-pentapeptide transferase (EC2.7.1.13) reaction and the other inhibition was probably at the transfer of peptidoglycan precursors from the glycosyl carrier lipid (GCL) to the nascent peptidoglycan. Quantitation of the peptidoglycan precursors and the net peptidoglycan in relA⁺ control and amino acid-deprived bacteria indicated that peptidoglycan accumulation was inhibited. There was as much UDP-MurNAc-pentapeptide and GCL-linked intermediates in the amino acid-deprived bacteria as in the control bacteria. This suggests that the transfer of lipid-linked precursors to nascent acceptor is the site of inhibition of Dap incorporation. In addition, the pool of soluble nucleotide-linked precursors was found to accumulate when relA⁻ bacteria were deprived of required amino acids. This suggests that the size of the precursor pool is also regulated by the activity of the relA gene product. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

O bacillus cereus είναι ένα παθογόνο, θετικό κατά Gram βακτήριο, ομόλογο με τον Bacillus anthracis. Ο στόχος της παρούσας μελέτης είναι να διαλευκάνει το ρόλο που παίζει η πεπτιδογλυκάνη, και συγκεκριμένα οι απακετυλάσες της, και με βάση τις εκτεταμένες ομολογίες τους να συνεισφέρπυν στη κατανόηση της φυσιολογίας του B. anthracis. / Bacillus cereus is an opportunistic pathogenic, Gram positive bacterium, closely related to Bacillus anthracis. The goal of this study is to shed light on the role of bacterial peptidoglycan deacetylases and furthermore based on the extensive homologies to contribute to our understanding of the physiology of B. anthracis.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 3 uppsatser.

<i>Clostridioides difficile</i> is a highly antibiotic resistant and infectious endospore-forming bacterium, responsible for an estimated 450,000 cases per year. The formation of an endospore is necessary for the survival of the bacterium while in-transit between hosts and while passing through the toxic environment of the host's stomach. Essential to the endospore's resistance is a thick layer of highly modified peptidoglycan called the cortex. While the endospore cortex is forming, the enzymes CwlD and PdaA convert N-acetylmuramic acid (NAM) into muramic--lactam (MAL). MAL serves as a recognition element for germination-specific lytic enzymes that degrade cortex peptidoglycan layers during germination. Without the MAL residues the endospore cannot complete germination. Unique to the Peptostreptococcaceae family, which includes C. difficile, is the lipoprotein GerS, which is required for the function of CwlD. The interaction between these two proteins is poorly understood. In this work, attempts to complement a Bacillus subtilis cwlD mutant using C. difficile gerS and/or cwlD were unsuccessful. No MAL residues were produced, and spores produced were incapable of completing germination. In vitro assays of CwlD activity on purified peptidoglycan revealed binding and activity of C. difficile CwlD, which were significantly increased when in complex with GerS. The ability of C. difficile CwlD+GerS to function in vitro but not to complement in B. subtilis suggests that in vivo activity is blocked by some factor in this heterologous system. Such a factor might be the in vivo ionic environment or a failure to properly localize within the forespore in B. subtilis cells. Furthering the understanding of C. difficile's germination machinery will potentially provide new targets for therapies. / Master of Science / Antibiotics have saved countless lives since their initial discovery and subsequent use to kill harmful bacteria. However, they have also led to the rise of antibiotic associated diarrhea (AAD), which can be fatal. AAD is caused by antibiotic resistant bacteria that can infect the gut after a large number of bacteria, which exist normally in a healthy gut, are killed by antibiotics. The number one cause of AAD is Clostridioides difficile, which accounts for approximately 450,000 cases a year in the United States, and millions of cases worldwide. C. difficile is highly antibiotic resistant and can exist in the environment for decades as an endospore, protected from many types of disinfectants. This bacterium is commonly spread in hospital settings where it can survive many of the cleaning regiments to infect vulnerable patients. Our work focused on how one of the structures of the endospore is modified in C. difficile to better understand a part of the machinery necessary for causing infection. Studying how the bacteria produces an endospore can shed light on targets for new treatments.

Bacteria in the environment face constant stress, due to lack of nutrients or presence of growth inhibiting compounds. As a result, they have developed several strategies to evade unfavourable growth conditions. These range from entering into dormant or quiescent states, through to motility, and biofilm formation. Using the model organism Streptomyces, we investigated how the bacterial cell surface regulates dormancy, biofilm formation, and motility. Dormancy via spore formation allows cells to shut down metabolism in response to poor nutrient conditions. Spores can then be dispersed throughout the environment to encounter favourable conditions. This is an incredibly resilient survival strategy, so long as the spores resuscitate from dormancy and resume growth once favourable conditions are sensed. We established that peptidoglycan remodeling by resuscitation promoting factors is critical for rapid germination of dormant Streptomyces spores, which likely provides a competitive advantage over slower growing microbes in the same environment. Previously it was thought that these proteins produce a signal to stimulate germination in neighbouring cells. We determined that the resuscitation promoting factors are lytic transglycosylases, and were not capable of producing a germination signal on their own. Instead, they function by cleaving the peptidoglycan to make room for new cell growth. This work highlights the importance of peptidoglycan remodeling to the germination process. Biofilms are multicellular communities of microorganisms which are adhered to each other using a protective matrix. Formation of biofilms is thought to be inversely correlated with motility. We established that Streptomyces forms biofilms during the exploratory growth identifying potential extracellular matrix components. These biofilms use sliding motility to expand rapidly across their environment. Components of the biofilm matrix effect colony expansion, suggesting that biofilm formation and motility are intricately linked in Streptomyces. These works demonstrate the importance of surface polymers to the growth and development of Streptomyces. / Thesis / Doctor of Philosophy (PhD) / Bacteria are all around us. In these different environments, whether in the soil, or inside our guts, or in a body of water, they will encounter stress. This can take the shape of nutrient stress, or the presence of growth inhibiting compounds. In response, bacteria can evade these poor conditions by entering into dormancy, analogous to hibernation, by building a biofilm, analogous to building a bunker, or by moving away. The surface of bacterial cells becomes decorated with different polymers as it transitions into one of these three modes of stress evasion. The cell wall holds the cell together and supports its shape, making it the most important surface polymer. I examined how rapid remodeling of the cell wall provides a competitive advantage to cells waking up from dormancy. I also examined the importance of additional polymers to the formation of biofilms that slide across surfaces, away from stressors. These works establish how important the surface of the bacterium is for surviving stressful conditions.

Borrelia burgdorferi — the causative agent of Lyme borreliosis — accounts for ~500,000 infections in the United States per year. Relative to other bacteria, B. burgdorferi is highly unusual in many regards. For instance, the synthesis and composition of B. burgdorferi cell wall is extremely unique and plays a critical role in Lyme pathogenesis. The cell wall is made up of peptidoglycan (PG) - a mesh-like structure, composed of long rigid glycan strands of repeating sugars GlcNAc and MurNAc, and flexible peptide stems, interlinked by amino acid cross-bridges. PG is an essential component for survival of the bacterial cell, protecting it from the osmotic stress and environmental threats, as well as defining the shape of the bacterium and aiding in the motility. One unique feature of the B. burgdorferi PG is the chemical composition of stem peptide, which involves the atypical cross-link between Ornithine and Glycine. We identified gene bb0586 as a femX homologue in borrelial genome and hypothesize that it encodes a glycyl transferase enzyme responsible for synthesis of glycine cross-bridges, that hold together glycan strands in the peptidoglycan cell wall. Here, we predicted the structure of FemXBb, identified and characterized the substrate-binding site, and proposed a novel mechanism for substrate recognition and recruitment, involving previously uncharacterized elements of the structure. We have also determined the ability of recombinant FemXBb to add Glycine bridges to mDAP in E. coli and investigated the effect that femX knock-out can have on the B. burgdorferi. In addition, we have investigated the steps of PG biosynthesis in B. burgdorferi. The results of our research suggest the existence of a highly unusual mechanism of PG synthesis in Lyme disease spirochete, which has a potential to be used for development of targeted antibacterial therapies. / Master of Science in Life Sciences / Borrelia burgdorferi — the causative agent of Lyme borreliosis — accounts for ~500,000 infections in the United States per year. Relative to other bacteria, B. burgdorferi is highly unusual in many regards. For instance, the synthesis and composition of B. burgdorferi cell wall is extremely unique and plays a critical role in Lyme pathogenesis. The cell wall is a mesh-like structure, a sacculus, enclosing the vulnerable inside contents of a bacterial cell. It is composed of long rigid glycan strands of repeating sugars, and flexible peptide stems, interlinked by cross-bridges, holding the whole structure together. PG is an essential component for survival of the bacterial cell, protecting it from the outside stress and environmental threats, as well as defining the shape of the bacterium and aiding in the motility. B. burgdorferi PG is known to be highly atypical compared to other bacteria. One of its features is the unusual cross-link between peptides, made up of single Glycine amino acid. We identified a gene encoding a protein responsible for the addition of this amino acid during the cell wall biosynthesis. Here, we predicted the structure of this protein, its substrate-binding site, and proposed a novel mechanism for substrate recognition and recruitment. We have also expressed the borrelial protein in E. coli and confirmed its activity and the impact it has on the bacterium and investigated the effect that gene knock-out can have on the B. burgdorferi. In addition, we have investigated the steps of PG biosynthesis in B. burgdorferi. The results of our research suggest the existence of a highly unusual mechanism of PG synthesis in Lyme disease spirochete, which has a potential to be used for development of targeted antibacterial therapies.

Peptidoglycan (PG) is an essential cell-wall biopolymer in virtually all bacteria. It is composed of glycan strands of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) crosslinked by peptide chains of alternating D- and L- amino acids and diamines. PG plays an important role in 1) cell elongation and division, 2) cell strength and morphology, 3) antibiotic susceptibility, and 4) host immune detection and modulation. While differences in peptide chains are common, deviations in glycan strand composition were not previously known to occur. Here, we provide characterization of the first known deviation to bacterial glycan strand composition — GlcNAc-GlcNAc-anhMurNAc (G-G- anhM) in Borrelia burgdorferi, the causative agent of Lyme disease. B. burgdorferi with less G-G-anhM were found to be significantly less motile, flexible, and stress-tolerant while possessing gross morphological defects and less overall PG. Our studies also characterized the muropeptide profile of Borrelia afzelii, Borrelia garinii, and Borrelia hermsii — species of Borrelia associated with causing different disease manifestations of Lyme disease, and relapsing fever, respectively. These species were found to incorporate appreciable amounts of G-G-anhM into their PG, suggesting an evolutionary adaptation to life inside a tick that predates the differentiation of Lyme disease and relapsing fever Borrelia. Finally, we provide partial characterization of a putative penicillin-binding protein in B. burgdorferi — a class of highly conserved PG synthesis enzymes present in the vast majority of bacteria. Collectively, the work in this thesis furthers our understanding of the structure, function, and synthesis of PG in Borrelia. / Master of Science in Life Sciences / Peptidoglycan (PG) is the main cell-wall component in the vast majority of bacteria. PG is composed of strong, rigid sugars linked together by short, flexible amino acid chains, and resembles a mesh-like bag that surrounds the cell. In nearly all bacteria that have PG, it plays an important role in how 1) the cell grows and divides, 2) the cell dictates its shape, 3) antibiotics treat bacterial infections, and 4) the human body detects and responds to a bacterial infection. While the amino acids that make up PG are known to differ between bacterial species, deviations in sugar organization are not known to occur. Here, we characterize the first known deviation to sugar organization in bacterial PG in Borrelia burgdorferi — the bacteria that causes Lyme disease. B. burgdorferi with less of this deviation possess defects absent in their normal counterparts. In addition, we show that other Borrelia species that cause a variety of different diseases around the world mimic this sugar deviation, suggesting the majority, if not all, of Borrelia may do so. Finally, we provide partial characterization of the function of an enzyme thought to synthesize PG in B. burgdorferi. Collectively, the work in this thesis furthers our understanding of the structure, function, and synthesis of PG in Borrelia.