Bacterial cell shape is of fundamental and medical importance, contributing to survival and virulence by influencing nutrient uptake, cell-to-surface attachment, motility, differentiation, and resistance to predation or host immunity. Furthermore, morphological studies are illuminating previously unrecognized interactions among diverse biochemical events that drive bacterial growth. In short, morphology plays crucial roles in bacterial physiology, ecology and pathogenesis, and serves as a window into cell biology. Most bacteria determine their shapes by making a peptidoglycan cell wall, and interfering with this structure is one of the most important targets for anti-bacterial therapy. Despite decades of work, there remain large gaps in understanding how peptidoglycan synthesis is integrated with related metabolic pathways. One integration point is the carrier molecule undecaprenyl-phosphate (Und-P), which helps synthesize peptidoglycan and other oligosaccharides. It has become clear that Und-P and its derivatives form a vital nexus that must be characterized in detail to help combat increasing antibiotic resistance. To this end we propose the following Aims.
Aim 1 ] Determine how Und-P management alters bacterial physiology and biochemistry. Und-P and its homologues transport numerous compounds and polymers across the cytoplasmic membranes of cells in all biological kingdoms, including archaea and eukaryotes. Because the end-products are synthesized by diverse routes, the carrier must be shared among several biochemical pathways, but how this is accomplished is unknown. It is, however, vital ? if Und-P is sequestered in one pathway, less is available for peptidoglycan synthesis and cells grow poorly or die. We will develop tools to monitor the amounts of Und-P in living cells and will determine how competition for Und-P affects bacterial growth and the operation of each pathway.
Aim 2 ] Identify and characterize new and little-known Und-P-utilizing pathways. Although several Und-P- dependent pathways are known, there are many for which little information is available and, undoubtedly, others we know nothing about at all. We will characterize those pathways projected to exist in Escherichia coli and will initiate genetic screens to look for others.
Aim 3 ] Identify and characterize cell shape mutants. Historically, most morphological mutants were discovered while studying something else. Here, we will continue to use flow cytometry to isolate such mutants, to identify new mechanisms that affect bacterial shape. We will also characterize more fully the mutants we already have in hand. In summary, these tools and approaches will enable us to investigate, faster and in greater depth, the mysteries surrounding Und-P utilization and its interactions with basic metabolic pathways. In addition, the work will create a well- developed bacterial model that will inform and enhance similar investigations in many other organisms.
The rigid peptidoglycan cell wall is a vital component of nearly all bacteria, making it a premium target for antimicrobial therapy. However, there remain important gaps in understanding how this macromolecule is synthesized, modified and shaped, which makes it difficult to treat bacteria that are becoming increasingly resistant to current antibiotics. This project seeks to fill these gaps by devising new genetic and biochemical tools and by studying important metabolic relationships, eventually leading to novel ways for controlling pathogenic bacteria.
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