Genomic Sequences of the Ethanol Producing Bacteria Thermoanaerobacter ethanolicus 39E and Thermoana

1
Genomic Sequences of the Ethanol Producing
Bacteria Thermoanaerobacter ethanolicus 39E and
Thermoanaerobacter ethanolicus X514 C. L.
Hemme1,2, M. W. Fields3, A. Copeland4, S. Lucas4,
K. Barry4, J. C. Detter4,5, T. Glavina4, N.
Hammon4, S. Israni4, S. Pitluck4, M. Land1, E.
H. Saunders5, D. C. Bruce5, A. Lapidus4, C. S.
Han5, F. Larimar1, P. Richardson4, E. Rubin4 and
J. Zhou2Environmental Sciences Division, Oak
Ridge National Laboratory, Oak Ridge, TN1
Institute for Environmental Genomics, University
of Oklahoma, Norman, OK2 Department of Soil
Crop Sciences , Department of Microbiology, Miami
University, Oxford, OH3 DOE Joint Genome
Institute, Walnut Creek, CA4 Los Alamos National
Laboratory, Los Alamos, NM5
Introduction

• 3 Thermoanaerobacter species
• Tten 595
• F0F1-ATPase complex
• Hydrogenase maturation factors
• Kdp high-affinity potassium transport system
• X514 207
• 39E 128
• 2 T. ethanolicus strains
• X514 283
• a-galactosidase
• glycerate kinase
• cobalamin biosynthesis genes
• multiple phage proteins/integrase
• 39E 271
• KDPG permease
• 1-phosphofructokinase

Recent instability in the global energy markets
due to increased demand coupled with disruption
of supply has sparked a renewed interest in the
development of alternative energy sources. One
such alternative energy source that has been
singled out for particular study is biogenerated
ethanol derived from plant cell wall material.
Clostridia bacteria are often employed in such
efforts due to their general ability to degrade
complex carbohydrates (cellulose, hemicellulose,
chitin, etc.) to monomeric sugars (glucose,
galactose, xylose, etc.) which can in turn be
fermented to commercially valuable end products
(ethanol, etc.). The genomic sequences of two
thermophilic ethanol-producing bacteria,
Thermoanaerobacter ethanolicus 39E and T.
ethanolicus X514, have recently been sequenced
and are undergoing finishing. 39E was originally
isolated from a thermal spring in Yellowstone
National Park that is capable of producing
ethanol from a variety of polysaccharides
including hemicellulose, xylan,
cyclomaltodextrins and starch. X514 is a deep
subsurface, metal-reducing strain of the species
that has been geologically separated from 39E for
200 Mya. The partially-completed genome
sequences of these two strains are compared with
each other and with the completed sequence of T.
tengcongensis MB4T.
A)
B)
Genome Statistics
T. ethanolicus 39E Genome Size (Mb) 2.3
Contigs 88 (20) Predicted CDS 2254
GC 34.5 T. ethanolicus X514 Genome Size
(Mb) 2.2 Contigs 94 (18) Predicted
CDS 2230 GC 34.4 T. tengcongensis
Genome Size (Mb) 2.7 Contigs 1
Predicted CDS 2588 GC 37.6 Genome
sequenced independently from this project
C)
Fig 5. Unique genes present in each
Thermoanaerobacter when compared by genus and
species. Gene families were determined using the
method of Lerat et al., 2005. Genes from
families consisting of only the self-to-self hit
were considered to be unique for that species.
Fig 6. Artemis comparison of T. tengcongensis
(A), T. ethanolicus 39E (B) and T. ethanolicus
X514 (C). Only hits gt500 bp are shown.
Putative strain-Specific Gene Expansions

• T. ethanolicus 39E
• Fe-containing alcohol dehydrogenase
• ABC sugar transporters
• KDPG and KHG aldolase
• Altronate dehydratase
• Auxin efflux carrier
• T. ethanolicus X514
• Heavy metal-transloating P-type ATPase
• Glycosidase
• Ferric iron uptake regulator
• Fe-containing alcohol dehydrogenase
• ABC sugar transporters
• NADHflavin oxidoreductase
• Xylulokinase
• Transketolase
• Acetate Kinase

16S rRNA Phylogeny
Fig 2. Central carbon metabolism and feeder
pathways of the three Thermoanaerobacter strains.
The presence of the enzymes for a given step are
indicated for each organism T. tengcongensis
(magenta), 39E (cyan) and X514 (yellow). The
bold red arrows indicate the direction of carbon
flow during fermentation of glucose. Ethanol
production is predominantly carried out by the
bifunctional secondary alcohol dehydrogenase/acety
l-CoA thioesterase, with the primary alcohol
dehydrogenase serving to recycle nicotinamide.
Fig 7. Sampling of putative gene
duplications/expansions in T. ethanolicus strains
Future Work
94 ANI Species Cutoff (70 DNA-DNA Reassociation)

• Close both T. ethanolicus genomes
• Test metal reduction capabilities of 39E and
  compare to X514
• Test ethanol production capabilities of X514
  and compare to 39E
• Design and build microarray for 39E to test
  gene expression of cultures grown on a variety of
  carbon sources with the intent to clarify carbon
  flow from the carbon source to ethanol

Fig 1. 16S rRNA phylogeny of Thermoanaerobacter,
Thermoanaerobacterium and Clostridium. Organisms
listed in blue have been or are being sequenced
independently of this project. The two
Thermoanaerobacter ethanolicus strains sequenced
as part of this project are listed in red.
Fig 3. Genome organization for T. ethanolicus
39E as reported in the literature (A) and as
observed from the genomic sequence (B).
Sequencing and assembly of the two T. ethanolicus
strains was performed at the JGI Production
Genomics Facility (Walnut Creek, CA) Closure of
the genomes is being conducted at JGI-PGF (X514)
and JGI-Los Alamos (39E) Automatic annotation of
the sequences was performed JGI-ORNL All work
was conducted under the auspices of the US
Department of Energy Microbial Genome Program