4.Lateral
Gene Transfer from Archaeans?
§Huber et al.’s discovery of glycerol diethers in A. degensii’s lipid bilayer[3] provided the first suggestion of lateral gene transfer (LGT) from archaeans.
§A. degensii’s genome produced 76 putative protein hits from archaeans (Fig. 4), some of which are known to be frequent
agents of LGT.[4]
§A. degensii’s genome contains several
important metabolic proteins that may have been acquired via LGT including: Mo ABC transporter (Table 1), periplasmic binding proteins (data not shown), and Fe (III) ABC transporter (data not shown), the latter two of which are essential for
iron uptake.[13]
Introduction
The therrmophilic bacterium Ammonifex degensii (Fig. 1) hails from a terrestrial volcanic solfataric hot spring in Indonesia. A. degensii is a strictly anaerobic,
chemolithoautotroph that thrives on H2 and CO2, deriving its energy from nitrate ammonification. Other bacterial and archaeal nitrate
ammonifiers have since been discovered in
geothermal hot spring, deep-sea hydrothermal vents, and marine sedimentary layers.[1-2] A. degensii empirically stains
Gram-negative, but is phylogenetically related to the low G+C subgroup of Gram-positive bacteria based on its 16S rRNA sequence.[3] A degensii contains ~ 85% glycerol diethers in its lipid
bilayer, a hallmark feature of archaens.[3-4]
The UCLA Undergraduate Genomics Research Initiative (UGRI) with assistance
from DOE/Joint Genome Institute is sequencing A. degensii’s genome. UGRI is precedent-setting undergraduate education in which students conduct cutting-edge research in genomic
biology and biotechnology. UGRI is a collaborative, cross-disciplinary effort among undergraduates in
lower and upper division life science courses (Fig. 2). Students participating in the
UGRI intend to be the first group of undergraduates to produce an
annotated microbial genome.
Materials
and Methods
§ Whole genome shotgun sequencing of A. degensii donated by Karl Stetter
§ Genomic library clones distributed to
collaborating courses for plasmid purification
§ Sequencing completed by LS 187 student
researchers (Fig. 2)
§ PCR-based bi-directional sequencing
analyzed on a LICOR 4200 DNA Analyzer
§ Scaffolding and preliminary contigs
assembled using DNAStar’s Seqman program
§ Sequence data annotated using BLASTx
against the NCBI and IMG databases
§ Trace files deposited in the NCBI database
(Fig.
3)
Fig. 1 | A. degensii, courtesy of Karl Stetter
References
1.Cowen et al. (2003) Fluids from aging ocean
crust that support microbial life, Science.
299:5603, 120-123
2.Vetriani
et al. (2004) Thermovibrio ammonificans sp.
nov.,a thermophilic, chemolithotrophic, nitrate-ammonifying bacterium from
deep-sea hydrothermal vents. Intnl
Journ Syst Evol Microb. 54: 175-181
3.Huber et
al. (1996) Formation of ammonium from nitrate during chemolithoautotrophic
growth of the extremely thermophillic bacterium Ammonifex
degensii gen. nov. sp. nov. Syst Appl Microbiol. 19,
40-49
4.Makarova et al. (2003) Comparative genomics of
Archaea: how much have we learned in six years, and what’s next? Genome
Biology, 4, 115-131
5.Ljungdahl, L. G. (1994) The acetyl-CoA pathway
and the chemiosmotic generation of ATP during acetogenesis, p. 63-87. In H. L.
Drake (ed.), Acetogenesis. Chapman & Hall, New York,
NY
6.Diekert et al. (1994) Metabolism of
homoacetogens. Ant. Leeuwenhoek Int. J. Gen. Microb. 66:
209-221
7.Drake et al. (2004) Physiology of the
thermophilic acetogen Moorella thermoacetica. Research
in Microb. 155:6, 422-436
8.Müller; Volker. (2003) Energy Conservation in
Acetogenic Bacteria. Applied and Environmental Microbiology. 69:6345-6353
9.Integrated Microbial Genomes (IMG) System.
(2006) Joint Genome Institute, Department of Energy. V1.4
10.Merrick et al. (1995) Nitrogen Control in
Bacteria. Microbiological Reviews, 59.4,
604-622
11.Grunden et al. (1997) Shanmugam. Molybdate
transport and regulation in bacteria. Arch Microbiol 168:
345-354
12.Crawford
et al. (2000) The stringent response in Myxococcus xanthus is
regulated by SocE and the CsgA C-signaling protein. Genes & Dev. 14: 483-492
13.Danese et al. (2004) The Ton System, an ABC
Transporter, and a Universally Conserved GTPase Are Involved in Iron
Utilization by Brucella melitensis 16M. Infect
Immun. 72(10): 5783-5790
14.Homma et
al. (1990) FlgB, FlgC, FlgF and FlgG. A Family of Structurally Related
Proteins in the Flagellar Basal Body of Salmonella typhimurium. J. Mol.
Biol. 211: 465-77
15.Gunenfelder
et al. (2002) Role of the Cytoplasmic C terminus of the FliF Motor Protein in
Flagellar Assembly and Rotation. J. Bacteriology. 185:1624-1633
Fig. 8 | Paralogous glnB (nitrogen
regulatory protein) genes (red) in the bacterium Rhodospeudomonas palustris.[9] Neighboring R. palustris genes are involved in amino
acid transport and metabolism, including a glutamine amidotransferase (GATase)
domain of a multisubunit synthase. A. degensii’s genome also appears to encode a
GATase.
Results
Overview
The ~1.7 Mb of A. degensii’s genome sequenced thus far has
yielded 813 putative protein hits (E-value cutoff < 9E-4), of which 90% are bacterial and 9% archaean (Fig. 4). Roughly 33% of A. degensii’s protein hits (Fig. 5) are from three thermophilic
acetogens that are phylogenetically related to A. degensii based on their 16S rRNA
sequences (data not shown). A. degensii’s genomic sequence data thus far allows us to speculate about some features of its metabolism and
evolution:
Table 1 | Nitrogen metabolism proteins for A. degensii. Align column: numerator (query sequence) and
denominator (gene length).
Fig. 3 | UGRI’s listing at NCBI
Fig. 7 | One of
several orthologous gene clusters in M. thermoacetica’s genome associated with the Wood-Ljungdahl pathway; CODH (red) is a diagnostic enzyme of that pathway. A. degensii’s genome also contains orthologs for eight enzymes (blue arrows) flanking the CODH gene. Similar
results (not shown) were obtained in other Wood-Ljungdahl pathway gene clusters (e.g., formate dehydrogenase) found within M. thermoacetica. Alignments for that pathway (and associated) enzymes were
substantiated using reciprocal BLASTx of A. degensii’s enzyme sequence data to M. thermoacetica.[9]
Fig. 2 | UGRI’s collaborative effort: http://www.lsic.ucla.edu/ugri/
Fig. 6 | Wood-Ljungdahl pathway. Reductants can be
hydrogen (shown) or other organic substrates.[8]
Table 2 | Protein function of sporulation proteins
determined experimentally. [7,9,12]
Align column: numerator (query
sequence) and denominator (gene
length). BLASTx against IMG, DOE/JGI’s curated database.
C. Kirshner, K. Houck, L. Idylle, C. Shin,
D. Taylor, T. Nguyen, M. Kelley, R. Simons, S. Thai, C. Kerfeld
Sequencing and Preliminary Analysis of
Ammonifex degensii Genome
Spore inducing
cofactor
190/486
50%
5E-20
SocE
Spore cortex
formation
92/282
40%
2E-4
Cell wall
hydrolyses
Spore cortex
formation
291/492
60%
1E-100
Stage IV
sporulation A
Function
Align
Identities
E-Value
Homologous
Protein
3.
Sporulation
A. degensii produced no spores when
cultured.[3]
Interestingly, orthologous proteins (three strongest hits summarized in Table 2) used by sporulating microbes
were found in A. degensii’s sequence data.
1. Carbon Fixation
§
Acetogens fix CO2 using a modified citric acid cycle (a
reductive acetyl-CoA pathway) called the Wood-Ljungdahl pathway.[5-8] (Fig. 6)
§ Three signature enzymes used in the Wood-Ljungdahl pathway have been identified in A. degensii’s sequence data: CO dehydrogenase/acetyl-CoA synthase (CODH) (shown in red, Fig. 7), formate dehydrogenase (not shown), and formyltetrahydrofolate synthetase (not shown).
§
Strong homology exists between these three enzymes found in A. degensii with those used by Moorella thermoacetica, a well-studied model acetogen[7] that is
phylogenetically related to A. degensii based on their 16S rRNA sequences (data not shown).
§ A. degensii’s genome also produced hits for additional enzymes found within orthologous
gene clusters in M. thermoacetica’s genome that are associated with the
Wood-Ljungdahl pathway.
The UCLA Undergraduate Genomics Research
Initiative gratefully acknowledges the generous
support of:
2.Nitrogen
Metabolism
§Studies suggest the highly
conserved nitrogen regulatory protein P-II is used for the biosynthesis of N-containing compounds and may be essential for nitrate ammonification.[3,10]
§
§P-II is the most frequent protein hit in our database (84 hits) (Table 1)
§A. degensii’s genome likely contains three paralogs of P-II (Fig. 8)
Ammonification
239/276
94%
3E-116
Solute-binding protein, Mo ABC transporter
Ammonification
112/112
100%
2E-55
N-reg protein
P-II
Putative Function
Align
Identities
E-Value
Homologous
Protein
§A contig of seven DNA fragments
in A. degensii’s genome likely encodes the solute-binding protein of the molybdate ABC
transporter, an enzyme that activates the
molybdoenzyme nitrate reductase, an essential enzyme in the ammonification
process.[11]
§Two related protein hits include molybdenum cofactor
biosynthesis protein (activates molybdoenzymes) and molybdopterin biosynthesis
protein (constituent of molybdoenzymes).
[11]
§Interestingly, the highest scoring alignment for the Mo ABC transporter was
found in Methanosarcina acetivorans, an anaerobic archaen (Table 1)
Fig. 10 | An orthologous gene cluster found in M.
thermoacetica coding for flagella. FliF (red) is a principal bacterial flagellar protein. A. degensii’s genome contains orthologs for
three other flagellar proteins flanking FliF: (1) Flagellar hook-basal body complex protein, FliE; (2) flagellar motor switch protein, FliG; and (3) flagellar biosynthesis protein, FlhA.[9,15]
Fig. 9 | Simplified scheme of principal bacterial flagellar proteins.[14,15]
1 2
3
5. Flagella
§ A. degensii’s sequence data encodes for principal proteins used in bacterial flagella: FliF (shown in red, Fig. 9), FliE, FliG, and FlhA.[14-15]
§ A. degensii’s FliF bears homology to that found in M. thermoacetica (61% identities; E-value 7e-36)
(Fig.
10)
Moorella
Thermoacetica
Fig. 4 | Tally of A. degensii putative orthologs by superkingdom.
Fig. 5 | Tally of A. degensii putative orthologs (cutoff E-value <
9E-4) by species (top 16 represented). Archaeans highlighted in violet.
