Graduation Year

2009

Document Type

Thesis

Degree

M.S.

Degree Granting Department

Biology (Integrative Biology)

Major Professor

Kathleen M. Scott, Ph.D.

Committee Member

Jim Garey, Ph.D.

Committee Member

Degeng Wang, Ph.D.

Keywords

Thiomicrospira crunogena, central carbon metabolism, citric acid cycle, Proteobacteria, chemolithoautotroph

Abstract

Thiomicrospira crunogena,

a deep-sea hydrothermal vent chemolithoautotroph,

uses the Calvin-Bensen-Bassham cycle to fix carbon. To meet its biosynthetic needs for

oxaloacetate, oxoglutarate, and succinyl-coA, one would expect that this obligately

autotrophic

Gammaproteobacterium would use a ‘wishbone’ version of the citric acid

cycle (CAC) to synthesize the intermediates necessary for biosynthesis, instead of the

fully oxidative version to minimize carbon loss as carbon dioxide. However, upon

examination of its complete genome sequence, it became apparent that this organism did

not fulfill this expectation.

Instead of a wishbone pathway,

T. crunogena appears to run a fully oxidative

CAC. The cycle is ‘locked’ in the oxidative direction by replacement of the reversible

enzyme malate dehydrogenase with malate: quinone oxidoreductase, which is capable

only of operation in the oxidative direction. Furthermore, oxoglutarate decarboxylation is

catalyzed by oxoglutarate: acceptor oxidoreductase. The presence of both

oxidoreductases was confirmed via assays on

T. crunogena cell extracts.

To determine whether this peculiar CAC was novel, complete genome sequences

of ~340 Proteobacteria were examined via BLAST and COG searches in the Integrated

Microbial Genome database. Genes catalyzing steps in the CAC were collected from

each organism and vetted for paralogs that had adopted an alternative, ‘non-CAC’

function through genome context and cluster analysis. Alignments were made with the

remaining sequences and were verified by comparing them to curated alignments at Pfam

database and examination of active site residues. Phylogenetic trees were constructed

from these alignments, and instances of horizontal gene transfer were determined by

comparison to a 16S tree.

These analyses verified that the CAC in

T. crunogena is indeed unique, as it does

not resemble any of the canonical cycles of the six classes of proteobacteria.

Furthermore, three steps of the nine in its CAC appear to be catalyzed by enzymes

encoded by genes that are likely to have been acquired via horizontal gene transfer. The

gene encoding citrate synthase, and perhaps aconitase, are most closely affiliated with

those present in the

Cyanobacteria, while those encoding oxoglutarate: acceptor

oxidoreductase cluster among the

Firmicutes, and malate: quinone oxidoreductase

clusters with the

Epsilonproteobacteria.

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