Characterization of citB in the methylcitric acid cycle of Bacillus subtilis 168 ; and, characterization of antimicrobial activity in the mucosal epithelial layer and gill tissue of largemouth bass (Micropterus salmoides)

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Sherona R. Sirkisoon (Creator)
The University of North Carolina at Greensboro (UNCG )
Web Site:
Jason Reddick

Abstract: Bacillus subtilis 168 is a gram-positive, aerobic, rod shaped bacteria that has the ability to undergo sporulation when under nutritional stress. There is an incomplete understanding of the metabolic pathways in B. subtilis 168 during sporulation. Characterizing the metabolic pathways of B. subtilis is important for understanding the sporulation process in pathogenic bacteria including B. anthraces (anthrax), for understanding cell differentiation of stem cells, and for maximizing biotechnological applications of B. subtilis 168. In this study, we hypothesize that citB, the only know aconitase in B. subtilis 168, can convert 2-methylaconitate to 2-methylisocitrate in the methylcitric acid cycle. This is an important step missing from the methylcitric acid cycle encoded by the mother cell metabolic gene (mmg) operon. In this study we utilized an overall approach involving purified citB protein, HPLC coupled to UV-VIS, and LC-MS to show that the citB protein can convert 2-methylaconitate to 2-methylisocitrate. We have also shown that a previously uncharacterized 2-methylisocitrate lyase (yqiQ) from B. subtilis 168 can convert the citB product 2-methylisocitrate to succinate and pyruvate, thus completing the methylcitric acid cycle. This study confirmed our hypotheses regarding citB and yqiQ and also achieved the first in vitro reconstitution of a complete methylcitric acid cycle from B. subtilis. There is little known about the biochemistry of largemouth bass (Micropterus psalmodies), especially the antimicrobial properties that may be present in the outer mucosal layer of the skin and gills as an initial defense against bacteria, protozoa and fungi. Understanding the antimicrobial properties of the small antimicrobial peptides in largemouth bass is important for the development of new antibiotics for potential use in humans and for decreasing the mortality rates of largemouth bass handled in recreational and tournament fishing, as well as those captured and released for studies by state wildlife management agencies. It is hypothesized that the skin secretions and gills of largemouth bass contain a small, cationic, amphipathic peptide that prevents initial infection from microorganisms present in the environment. In this study, we have tested the hypothesis with in vitro experiments through the use of disc diffusion assays on Escherichia coli K12 and Bacillus subtilis 168 bacteria and liquid growth assays using Staphylococcus aureus. Results of this study show that there is antimicrobial activity in the gills of largemouth bass. We will also report ongoing efforts toward isolating and characterizing the components responsible for this antimicrobial activity.

Additional Information

Language: English
Date: 2014
Antimicrobial peptide, Bacillus subtilis 168, CitB, Fish Gills, Largemouth Bass, Methyl Citric Acid Pathway
Bacillus subtilis
Microbial metabolism
Largemouth bass $x Microbiology

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