Project Report

Alginic acids, or alginates, are unbranched polymers that were first discovered from seaweeds. They are composed of repeating units of β-D-mannuronic acid and α-L-guluronic acid. Currently they are used in several industries that include food, material sciences, cosmetic, medical and pharmaceutical applications. But some researchers found that alginate can be produce by bacteria like Pseudomonas aeruginosa (Ohman and Chakrabarty, 1981). The isolate was from a cystic fibrosis patient and showed a mucoid appearance in the colonies formed. That morphology was caused due to alginate production and it is used as a diagnosis of the mentioned disease. Thus, alginate production is a virulence factor of P. aeruginosa. As complex a polymer is, its biosynthetic pathway will also be. Alginate biosynthesis is regulated by several proteins that are involved in different reactions (polymerization reactions, acetylations, isomerizations; protease and regulatory functions). One of the regulators found by Hay and co-workers (2009; program hosts) is mucR. Its product is MucR, which is an alginate-specific synthesizing enzyme. The researchers created mutants of MucR (PA1727) and they found that mucoid morphology was hindered plus a decrease in alginate production was quantified. Also, transfer of that gene coding for MucR into a non-mucoid strain, PDO300, caused an increase in alginate production and mucoid morphology of the colonies. In general, this molecule regulates alginate production by increasing and localization of signal molecules in the vicinity of another protein (Alg44). The principal objective of this project was to create mutations in MucR and study the medical relevance by quantifying their alginate production. This mutations were performed by site-directed ligase-independent mutagenesis (SLIM; Chiu et al., 2004 and 2008). One of the mutants will include a mutation as H122A (MHYT 2). Other mutants (MHYT 1 and EAL) were previously created and the alginate quantification was done to see if there is any change when comparing with the wild type PA300. Some MucR regions are suspected to play a role in quorum sensing functions, perceiving changes in signals for oxygen, NO or CO2. Other regions are involved in the conversion of energy-rich molecules (GTP) to cyclic molecules, which then are sensed by Alg44 to produced alginate. If a decrease in alginate production from these mutations is observed, their applicability to combat cystic fibrosis, for example, will be more probable. This is the foundation to create a gene delivery system that may cause mutations on mucoid P. aeruginosa and help the antibiotics penetrate the exopolymeric substance easily. However, if an increase in alginate production is observed from the mutants created, then the application could be focused on developing mass production processes for the food and pharmaceutical industries. Having the opportunity to participate in the EAPSI Program have giving me a chance to experiment in another different area within microbiology. Back in Montana State University, my doctoral research is focused on environmental microbiology (siderophore production and characterization), using chemistry as my fundamentals for my experiments. Here at Massey University, working under the supervision of Dr. Rehm personnel and graduate students showed me the great diversity that exists in microbiology. Their work can not only have an impact in the medical field but also the pharmaceutical/industrial. The new method, site-directed ligase-independent mutagenesis (SLIM), could be applied for any molecular work that requires plasmids and creation of mutations for the study of certain traits within the microorganism of study. One of the possible applications of SLIM to my project back at Montana State University could be the introduction of mutations in siderophore genes or proteins involved in siderophore transport. This could help to identify if there is any important regions of the proteins, or proteins that could be attacked for the treatment of bacterial infections due to the importance of iron uptake for them. Collaboration could potentially exist between one of my committee members, Dr. Michael Franklin, because his research interests are in P. aeruginosa biopolymer production and biofilm formation. Sincerely, I thank the Program for this important opportunity of doing cutting-edge research abroad and have also the experience to interact with people from other nationalities and cultures.

National Science Foundation (NSF)
Office of International and Integrative Activities (IIA)
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Carter Kimsey
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Serrano-Figueroa Luis O
United States
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