Biology Reference
In-Depth Information
proteins were used as seed sequences for Flower Power and internal tree-viewing tools
or SCI-PHY analyses. These two approaches employed neighbor-joining trees using
the Scoredist correction setting in the Belvu alignment editor, or the SCI-PHY util-
ity and tree viewer. In either case resulting phylogenomic tree builds were reviewed,
and contiguous protein alignments of two or more proteins from
D. aromatica
were
considered to be candidates for a gene duplication event, either in the
D. aromatica
genome or in a predecessor species.
Resequencing to Verify Absence of Plasmid Structure
After finishing the
D. aromatica
genome, analysis of the annotated gene set revealed
the notable absence of several anaerobic aromatic degradation pathways that were
expected to be present, due to their presence in
A. aromaticum
EbN1 (an evolutionary
near-neighbor, as determined by 16sRNA phylogeny). Because many catabolic path-
ways are encoded on plasmid DNA, we felt it was important to preclude this possibil-
ity. We re-isolated DNA from a clonal preparation of
D. aromatica
that experimentally
supported anaerobic benzene degradation, using three different plasmid purification
protocols, each based on different physical parameters. All three generated a single
band of DNA. The protocol that generated the highest yield of DNA was used to cre-
ate a complete, new library of 2 kb inserts, and the library was submitted to sequence
analysis using the protocols previously cited.
DISCUSSION
Discussion of results and analyses concerning aromatic degradation, various predicted
metabolic cycles, secretion, signaling, quorum-sensing, and gene family expansion are
included in the relevant sections.
RESULTS
Overview of Gene and Protein Features
The finished sequence for
D. aromatica
reveals a single circular, closed chromosome
of 4,501,104 nucleotides created from 130,636 screened reads, with an average G + C
content of 60% and an extremely high level of sequence coverage (average depth of 24
reads/base ). Specific probing for plasmids confirmed no plasmid structure was pres-
ent in the clonal species sequenced, which supports anaerobic benzene degradation.
It is noted however that the presence of two Tra clusters (putative conjugal transfer
genes; VIMSS582582-582597 and VIMSS582865-582880), as well as plasmid par-
titioning proteins, indicates this microbial species is likely to be transformationally
competent and thus likely to be able to support plasmid DNA structures.
The VIMSS, [http://www.microbesonline.org] and the Joint Genome Institute
[http://genome.jgi-psf.org/finished_microbes/decar/decar.home.html] report 4,170
and 4,204 protein coding genes, respectively. Cross-database comparisons were done
to assure the highest probability of capturing candidate orfs for analysis. The majority
of proteins are shared between data sets. Variations in N-termini start sites were noted,
both between JGI and VIMSS datasets and between initial and later annotation runs
(approximately 200 N-termini differences between four runs of orf predictions were
