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.
| Identifer | oai:union.ndltd.org:USF/oai:scholarcommons.usf.edu:etd-4898 |
| Date | 02 November 2009 |
| Creators | Quasem, Ishtiaque |
| Publisher | Scholar Commons |
| Source Sets | University of South Flordia |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Graduate Theses and Dissertations |
| Rights | default |
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