Amino Acid Metabolism In Maternal Bacteriocytes Of The Pea Aphid, Acyrthosiphon Pisum
The pea aphid contains symbiotic bacteria, Buchnera aphidicola, in specialized cells called bacteriocytes. These bacteria have been implicated with essential amino acid provisioning to the aphid. The host relies on their symbiotic bacteria for the 10 essential amino acids (EAAs) yet Buchnera lacks key genes in the biosynthesis of five of the ten essential amino acids (isoleucine, leucine, valine, phenylalanine, and methionine). I tested the hypothesis, derived from genome annotation, that the missing Buchnera reactions are mediated by host enzymes. To lay the metabolic framework for the bacteriocytes, we performed large-scale, high accuracy tandem mass spectrometry (nanoLC-LTQ-Orbtrap) to identify aphid and Buchnera proteins in the whole aphid body, purified bacteriocytes, isolated Buchnera cells and the residual bacteriocyte fraction (host fraction, HF). This study, in addition to being the first proteomic study of a bacteriocyte symbiosis, showed the enrichment (relative to the whole body) of candidate enzymes that could mediate the missing Buchnera reactions in isoleucine, leucine, valine, and phenylalanine synthesis in the bacteriocytes. I found an enrichment for a branched chain aminotransferase (BCAT) and an aspartate aminotransferase (GOT2) that are believed to mediate the terminal reaction of the branched chain amino acid (BCAA - isoleucine, leucine, valine) and phenylalanine synthesis, respectively. I also found enrichment for cystathionine-[gamma]-lyase, which is believed to synthesize the carbon skeleton for isoleucine. To validate the candidate host enzymes identified from proteomics, the bacteriocytes were separated into a Buchnera-fraction and a Buchnera-free host cell fraction (HF). Addition of HF to isolated Buchnera preparations significantly increased the production of leucine and phenylalanine; and recombinant enzymes mediating the final reactions in branched chain amino acid and phenylalanine synthesis rescued the production of these EAAs by Buchnera preparations without HF. The likely precursors for the missing proximal reactions in isoleucine and methionine synthesis were identified, and differed from predictions based on genome annotations: the carbon skeleton for isoleucine synthesis was stimulated by homoserine and production of the homocysteine precursor of methionine was driven by cystathionine. The coevolution of shared metabolic pathways in this symbiosis can be attributed to host compensation for genomic deterioration in the symbiont, involving changes in host gene expression networks to recruit specific enzymes to the host cell.
aphid; buchnera; endosymbiosis
Searle, Angela E.
Jander, Georg; Scott, Jeffrey Graham
Ph. D., Entomology
Doctor of Philosophy
dissertation or thesis