Graduation Term

2016

Degree Name

Doctor of Philosophy (PhD)

Department

School of Biological Sciences

Committee Chair

Brian J. Wilkinson

Abstract

Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a disease characterized by gastroenteritis, meningitis, spontaneous miscarriages and high mortality rate among infected individuals. L. monocytogenes is a major concern in the food industry, due to its ability to grow at refrigeration temperatures and the zero tolerance policy of the FDA, resulting in expensive food recalls. Growth at low temperatures is aided in part by the high membrane content of branched-chain fatty acids (BCFAs) which imparts greater fluidity to the membrane. Mutants of L. monocytogenes impaired in BCFA biosynthesis display diminished growth at normal and low temperatures, exhibit lower tolerance to acidity and alkalinity, and demonstrate lower virulence. Addition of 2-methylbutyrate, the source of the membrane BCFA anteiso C15:0, restores the membrane BCFA content and virulence factor expression. Supplementation with unnatural branched-chain carboxylic acids (BCCAs) such as 2- ethylbutyrate and 2-methylpentanoate results in the incorporation of novel BCFAs in the listerial membrane. Incorporation of supplemented carboxylic acids is evidence of their entry into the fatty acid biosynthesis pathway and thus a Bkd-independent pathway which catalyzes the conversion of these compounds into their activated CoA derivatives must exist in L. monocytogenes. We hypothesize that Ptb and Buk, the products of the first two genes of the bkd operon, are involved in the sequential conversion of the supplemented BCCAs into their acyl CoA derivatives, which then presumably enter the fatty acid biosynthesis pathway for elongation. Ptb catalyzes the reversible conversion of acyl CoAs into acyl phosphates and Buk catalyzes the reversible phosphorylation of carboxylic acids. Ptb and Buk were heterologously expressed in E. coli, purified by affinity chromatography and utilized for analysis of their kinetic properties to determine their role in the activation of such carboxylic acids. Ptb and Buk both demonstrate broad substrate specificity and do not use acetate or hexanoate efficiently as substrates. Ptb and Buk exhibit a strong preference for substrates which have a chain length of C3-C5 thus indicating that they are not involved either in acetate metabolism or in the activation of long chain fatty acids. Ptb shows a strong preference for branched-chain substrates while Buk appears to demonstrate preference for BCCAs only with respect to C3 and C4 substrates. Both Ptb and Buk from L. monocytogenes demonstrate significant activity with unnatural BCCAs such as 2-ethylbutyrate and 2-methylpentanoate. Additionally, Buk exhibits substantial phosphorylation activity at low temperatures and appears to prefer BCCAs thus demonstrating a switch in substrate specificities at low temperatures. Ptb catalysis involves the formation of a ternary complex with acyl CoA and phosphate before release of the products. Similarly, Buk also forms a ternary complex with carboxylic acid and ATP before catalysis and release of products. Our work here indicates that Ptb and Buk likely act in a sequential manner in the activation and subsequent assimilation of exogenous straight chain carboxylic acids (SCCAs) and BCCAs.

Access Type

Dissertation-Open Access

DOI

http://doi.org/10.30707/ETD2016.Sirobhushanam.S

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