Biology Reference
In-Depth Information
tion between its predicted capabilities and those of its close relatives,
A. aromaticum
str EbN1 and
Azoarcus
BH72.
Dechloromonas aromatica
strain RCB is a gram negative Betaproteobacterium
found in soil environments [1]. Other members of the Betaproteobacteria class are
found in environmental samples (such as soil and sludge) or are pathogens (such as
Ralstonia solanacearum
in plants and
Neisseria meningitidis
in humans) and in gen-
eral the genus
Dechloromonas
has been found to be ubiquitous in the environment.
A facultative anaerobe,
D. aromatica
was initially isolated from Potomac River
sludge contaminated with benzene, toluene, ethylbenzene, and xylene compounds
(BTEX) based on its ability to anaerobically degrade chlorobenzoate [1]. This mi-
crobe is capable of aromatic hydrocarbon degradation and perchlorate reduction, and
can oxidize Fe(II) and H
2
S [2]. Although several members of the Rhodocyclales group
of Betaproteobacteria are of interest to the scientifi c community due to their ability to
anaerobically degrade derivatives of benzene,
D. aromatica
is the fi rst pure culture
capable of anaerobic degradation of the stable underivitized benzene molecule to be
isolated. This, along with its ability to reduce perchlorate (a teratogenic contaminant
introduced into the environment by man) and inquiry into its use in biocells [3] has led
to interest in using this organism for bioremediation and energy production. Since the
isolation of
D. aromatica
, other species of
Azoarcus
have been found to possess the
ability to anaerobically degrade benzene, but have not been genomically sequenced
[4].
The pathway for anaerobic benzene degradation has been partially deduced [5],
but the enzymes responsible for this process have yet to be identifi ed, and remain elu-
sive even after the intensive annotation efforts described here-in. Conversely, central
anaerobic pathways for aromatic compounds described in various other species were
not found to be present in this genome [6].
MATERIALS AND METHODS
Sequencing
Three libraries (3, 8, and 30 kb) were generated by controlled shearing (Hydroshear,
Genomic Solutions, Ann Arbor, MI) of spooled genomic DNA isolated from
D. ar-
omatica
strain RCB and inserted into pUC18, pCUGIblu21, and pcc1Fos vectors,
respectively. Clonal DNA was amplified using rolling circular amplification [http://
www.jgi.doe.gov/webcite] and sequenced on ABI 3700 capillary DNA sequenc-
ers (Applied Biosystems, Foster City, CA) using BigDye technology (Perkin Elmer
Corporation, Waltham, MA). Paired end-reads [7] were used to aid in assembly, and
proved particularly useful in areas of repeats.
The Phrap algorithm [8, 9] was used for initial assembly. Finishing and manual
curation was conducted on CONSED v14 software [10], supplemented with a suite of
fi nishing analysis tools provided by the Joint Genome Institute.
In silico
cross-over er-
rors were corrected by manual creation of fake reads to guide the assembly by forcing
the consensus to follow the correct path.
Gaps were closed through a combination of primer walks on the gap-spanning
clones from the 3 and 8 kb libraries (identifi ed by paired-end analysis in the CONSED
