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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.

Sporulation is a process of differentiation that allows capable cells to go into a dormant and resistant stage of life. To become active again, the spores must germinate into vegetative cells. One key process in spore germination is hydrolysis of the cortex peptidoglycan. This process has been studied in a variety of sporulating species; however, it has not been studied in <i>Bacillus anthracis</i>. A clear understanding of cortex degradation may provide information that will allow for better cleanup of spore contaminated sites. The structure of cortex peptidoglycan of <i>Bacillus anthracis</i> was characterized. The peptidoglycan of the dormant spores was extracted, digested with Mutanolysin, and analyzed using HPLC to determine the structure. The analyses revealed that the cortex peptidoglycan of <i>B. anthracis</i> was very similar to other <i>Bacillus sp.</i>. Spores were stimulated to germinate and cortex peptidoglycan was extracted and analyzed at various times. <i>Bacillus anthracis</i> appeared to hydrolyze its cortex more rapidly than other <i>Bacillus </i>species. While the spores of three species release the spore solute dipicolinic acid and resume metabolism at similar rates, the <i>B. anthracis </i> spores released 75% their cortex material within 10 minutes while the other species released only 20% in the same time frame. This suggests that the <i>B. anthracis</i> spore coats are more permeable to cortex fragments than those of the other species, or that <i>B. anthracis</i> rapidly cleaves the cortex into smaller fragments. Novel cortex fragments analyzed during <i>B. anthracis</i> germination were produced by a glucosaminidase; however, additional studies need to be performed for confirmation. / Master of Science

CwlD has sequence similarities to N-acetyl muramoyl-L-alanine amidases, a class of enzymes known to cleave the bond between the peptide side chain and the N-acetyl muramic acid residue in cortex peptidoglycan formation during sporulation. A major difference between vegetative peptidoglycan and spore peptidoglycan is the presence of muramic-<FONT FACE="Symbol">d</FONT> -lactam (MAL) in spore peptidoglycan. It was previously determined that a <I>cwlD</I> null mutant does not contain muramic-<FONT FACE="Symbol">d</FONT> -lactam in the spore cortex peptidoglycan and the mutant spores were unable to complete germination. Therefore, it is believed that CwlD plays a role in MAL formation during sporulation. However, the specific role of the protein had not been demonstrated. It was also previously found that <I>cwlD</I> is in a two-gene operon with <I>orf1</I>. Orf1 is produced within the forespore with CwlD. The hypothesized role of Orf1 is to inhibit CwlD activity from within the forespore. Muramoyl-L-alanine amidase activity was demonstrated by CwlD <I>in vivo</I>. Therefore, CwlD is carrying out the first step of MAL synthesis, cleaving the peptide side chain while other enzymes are needed to complete MAL formation. Two different forms of CwlD were over-expressed, with and without the protein's signal peptide sequence. Both forms of the protein were purified and in both cases activity was undetectable. Antibodies specific for CwlD were obtained which can be used in future research as a tool to further characterize CwlD activity. A series of <I>B. subtilis</I> <I>cwlD</I> operon mutants were constructed altering the expression patterns of Orf1 and CwlD within the mother cell and forespore compartments. Various resistance properties and the germination ability of the mutant dormant spores were analyzed. It was determined that the absence of just Orf1 or Orf1 and CwlD from within the forespore has no effect on the phenotypes tested. Peptidoglycan from developing mutant forespores was extracted and analyzed throughout sporulation. Evidence was obtained demonstrating that the role of Orf1 is not to inhibit CwlD from within the forespore as hypothesized. / Master of Science

<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.