Download FEMS ML 00 CODH cooF

FEMS Microbiology Letters 191 (2000) 243^247
www.fems-microbiology.org
Genetic analysis of Carboxydothermus hydrogenoformans
carbon monoxide dehydrogenase genes cooF and cooS1
Juan M. Gonzälez, Frank T. Robb *
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 E. Pratt St., Baltimore, MD 21202, USA
Received 27 April 2000; received in revised form 10 June 2000; accepted 18 August 2000
Abstract
Carboxydothermus hydrogenoformans is an extremely thermophilic, Gram-positive bacterium growing on carbon monoxide (CO) as single
carbon and energy source and producing only H2 and CO2 . Carbon monoxide dehydrogenase is a key enzyme for CO metabolism. The
carbon monoxide dehydrogenase genes cooF and cooS from C. hydrogenoformans were cloned and sequenced. These genes showed the
highest similarity to the cooF genes from the archaeon Archaeoglobus fulgidus and the cooS gene from the bacterium Rhodospirillum rubrum,
respectively. The cooS gene was identified immediately downstream of cooF, however, the cooF and cooS genes from C. hydrogenoformans
have substantially different codon usage, and the cooF gene Arg codon usage pattern, dominated by AGA and AGG, resembles the archaeal
pattern. The data therefore suggest lateral transfer of these genes, possibly from different donor species. ß 2000 Federation of European
Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : CO-utilizing bacterium; Phylogeny; Carbon monoxide dehydrogenase ; cooS; cooF; Carboxydothermus hydrogenoformans
1. Introduction
Carbon monoxide (CO) is metabolized by numerous
prokaryotic genera from both the bacteria and archaea
domains. The CO dehydrogenases (CODHs) are the key
enzymes in CO metabolism (reviewed in [1]). Apparently,
the mechanisms of CO utilization in aerobic and anaerobic
prokaryotes are fundamentally di¡erent [1,2]. CODHs
from aerobic bacteria (e.g. Oligotropha carboxydovorans)
are not related to CODHs from anaerobic microorganisms
[2,3]. Among anaerobic bacteria, CODHs from Rhodospirillum rubrum [4] and Moorella thermoacetica [5] have been
investigated thoroughly. CODHs are also key enzymes for
methanogenic archaea; among the best studied species are
Methanothrix soehngenii, Methanosarcina thermophila and
Methanobacterium thermoautotrophicum [1,6]. Archaeoglobus fulgidus is a sulfate-reducing archaeon with CODH
activity [7].
* Corresponding author. Tel. : +1 (410) 234-8870;
Fax: +1 (410) 234-8896; E-mail: [email protected]
1
Accession number of the sequences from C. hydrogenoformans:
AF249899.
Carboxydothermus hydrogenoformans [8] was the ¢rst
extremely thermophilic, strictly anaerobic, chemolithotrophic, CO-utilizing bacterium to be described. It grows
rapidly with CO as the sole energy and carbon source,
and produces equimolar quantities of H2 and CO2 according to the global equation : CO+H2 OCH2 +CO2
(vGo = 320 kJ mol31 ). C. hydrogenoformans grows at
78³C and neutral pH. Growth is inhibited by penicillin,
chloramphenicol and streptomycin. Recently, high levels
of CODH activity have been found in this microorganism
(T. Sokolova, personal communication). In this study, we
report on the sequences of the CODH genes cooF and
cooS from C. hydrogenoformans and speculate on their
origin.
2. Materials and methods
DNA was extracted from cultures of C. hydrogenoformans strain Z-2901 (DSM 6008) grown as previously described [8]. A genomic library was prepared as described
elsewhere (Gonzälez and Robb, submitted) by using lambda-Zip Lox, EcoRI arms (Gibco BRL). Gene walking using the Vectorette II system (Genosys) and the cooS speci¢c primer 5P-CCA TCG ATA CTT TCA AAC GGC
0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 3 9 7 - 9
FEMSLE 9619 27-9-00
244
J.M. Gonzälez, F.T. Robb / FEMS Microbiology Letters 191 (2000) 243^247
FEMSLE 9619 27-9-00
J.M. Gonzälez, F.T. Robb / FEMS Microbiology Letters 191 (2000) 243^247
245
Fig. 1. Multiple sequence alignment of carbon monoxide dehydrogenase subunits CooF (A) and CooS (B) from C. hydrogenoformans (CH), A. fulgidus
(AF), R. rubrum (RR) and M. jannaschii (MJ1 and MJ2).
6
GC-3P was performed in order to complete the sequence of
the cooS gene from C. hydrogenoformans. The short gene
upstream of cooS was identi¢ed as cooF based on the
homology search using the BLASTx algorithm [9]. Multiple sequence alignments were performed by the program
CLUSTALW v1.7 [10] using a 10.0 gap opening penalty
and BLOSUM62 protein weight matrix. Codon usage was
analyzed by the program CodonFrequency (Genetic Computer Group, Inc.).
3. Results
3.1. CooF
The cooF open reading frame spans 434 nucleotides (144
amino acids) with a G+C content of 41.7%. This sequence
is preceded by a putative ribosome-binding site (GGAG).
The closest homolog of C. hydrogenoformans cooF sequence identi¢ed by similarity search was the CooF subunit of CODH from the archaeon A. fulgidus (39% identity); the similarity to the CooF of R. rubrum and iron^
sulfur proteins from other bacteria was much less. Codon
usage analysis of the C. hydrogenoformans cooF sequence
also supports a similarity to archaeal cooF genes. A preference for the Arg codons AGA and AGG in cooF gene
(84%) from C. hydrogenoformans is found only in archaeal
cooF sequences. Preference for the codons GAA (Glu,
91%), ATT (Ile, 58%) and GTT (Val, 50%) is also reminiscent of archaeal cooF genes. An absolute preference for
the codons TAT (Tyr) and CAA (Gln) relates to Methanococcus jannaschii cooF genes. Since cysteines are highly
conserved residues in CooF subunits, the pattern of codon
usage for Cys should also be of importance when looking
for related genes and their species ; C. hydrogenoformans
cooF use of Cys-encoding codons relates to M. jannaschii
cooF genes.
Alignment and comparison to other anaerobic bacterial
(R. rubrum) and archaeal species (A. fulgidus, M. jannaschii) showed the presence in CooF of four 4-cysteine motifs typical of iron^sulfur proteins (Fig. 1A). Oxidoreductases (e.g. in Escherichia coli, Pyrobaculum aerophilum, A.
fulgidus), electron transport proteins (e.g. in E. coli, Pyrococcus abyssi), formate dehydrogenases (e.g. in P. abyssi,
M. thermoautotrophicum), sulfur-related reductases (e.g. in
Salmonella typhimurium, Wolinella succinogenes, Aeropyrum pernix), nitrate or nitrite reductases (e.g. in Clostridium perfringens, Haemophilus in£uenzae) and other iron^
sulfur cluster-binding proteins (e.g. in Thermotoga maritima) are all among the close relatives of cooF genes identi¢ed in BLAST searches.
3.2. CooS
The cooS sequence spans 1911 nucleotides (637 amino
acids) with a G+C content of 50.4% and is preceded by a
putative ribosome-binding site (GAAG) (Fig. 1B). C. hydrogenoformans CooS sequence is most closely related to
the L-subunits of CODHs from R. rubrum (55% identity)
and M. thermoacetica (48% identity). Codon usage in the
C. hydrogenoformans cooS sequence also points to similarity with anaerobic bacterial sequences, although it is also
indicative of proximity to Methanococcus/Archaeoglobus
cooS. Some of the similarities with anaerobic bacteria
and archaea are a preference for ATT and ATC (Ile,
87%), GCC (Ala, 48%), TCC (Ser, 37%), CGC (Arg,
53%), ACC (Thr, 52%), CCC (Pro, 50%), and scarcely
used codons like CCA (Pro, 0%), CTA (Leu, 6%), ACA
(Thr, 3%). The C. hydrogenoformans cooS gene shows no
occurrences of AGG and AGA encoding Arg which contrasts sharply with the codon usage in the C. hydrogenoformans cooF gene. Preference for the codons ATT and ATC
(Ile, 87%), TGT (Cys, 71%), discrimination against CTA
(Leu, 6%), and the proportion of the CAT and CAC (His,
40% and 60%, respectively) codons used, relates to the
Methanococcus/Archaeoglobus cooS genes. The relatively
high G+C content in the C. hydrogenoformans cooS sequence (50.4%) is similar to that in the A. fulgidus cooS
sequence (51.5%).
4. Discussion
In this study, we report the sequence of the genes encoding the CODH system, which is an essential part of the
energy and biosynthesis pathways of methanogens [1], but
appears to be only a supplemental metabolic pathway in
most CO-metabolizing bacteria (e.g. R. rubrum and M.
thermoacetica) [2]. CO represents an essential substrate
for C. hydrogenoformans. Based on sequence and physiological information, we propose that the cooF and cooS
genes might have originated within the archaeal domain
and were then transferred horizontally to the bacterial
domain [11^13]. The newly introduced genes would represent additional metabolic capabilities and would allow the
recipient anaerobic bacteria to thrive in new habitats and
exploit additional nutrient and energy sources (i.e. CO).
CODHs have been divided into two groups, the Mo^
Fe^£avin enzymes from aerobes and the Ni^Fe enzymes
from anaerobes [1]. Aerobic bacteria appear to use an O2 resistant CODH that shows no similarity to anaerobic
CODH. In anaerobic bacteria, the catalytic unit of the
CODH system is the CooS subunit which is phylogeneti-
FEMSLE 9619 27-9-00
246
J.M. Gonzälez, F.T. Robb / FEMS Microbiology Letters 191 (2000) 243^247
cally related to the CooS of Methanococcus and Archaeoglobus. cooS genes from other methanogenic archaea,
such as M. thermoautotrophicum and M. soehngenii, cluster together and are distinct from the cooS genes from
anaerobic bacteria and Methanococcus/Archaeoglobus. A
functionally essential cysteine-rich motif, GX2 CX2 CX2 GPCRIX4ÿ6 PX1ÿ3 GX1 CGX0ÿ2 A, is conserved in both anaerobic bacteria and the Methanococcus/Archaeoglobus
group. C. hydrogenoformans cooS shows high similarity
to other anaerobic bacteria and to the Methanococcus/Archaeoglobus cooS genes.
Codon usage analysis, and speci¢cally the di¡erential
use of Arg codons in the cooF and cooS genes, points to
the possibility of di¡erent origins of these two functionally
related genes. We consider it signi¢cant that the cooS gene
contains no AGA/AGG Arg codons, whereas cooF has
84% of the Arg codons in this group, which is characteristic of thermophilic archaea. This suggests two possible
scenarios for the origin of cooF and cooS genes: (i) the
cooF and cooS were transferred either at di¡erent time
points after the bacteria/archaea split and/or they originated from di¡erent donor species ; or (ii) these genes
have shown a di¡erential unselected mutation rate during
their evolution in the bacterium C. hydrogenoformans. The
last possibility seems unlikely due to the high G+C content of these genes with respect to the relatively low C.
hydrogenoformans G+C genome content of 39% [8]. As
well, we consider it unlikely that the C. hydrogenoformans
cooF gene is an archaeal cooF analog that originated from
non-cooF bacterial Fe/S proteins due to di¡erences in codon usage and G+C content. An iron^sulfur cluster-binding protein annotated from the bacterium T. maritima [13]
is an exception in the bacteria since it shows high preference for the AGG and AGA Arg-encoding codons; the
genome sequence of T. maritima provides very strong evidence supporting wide spread lateral gene transfer [13]
from archaea into bacteria sharing these sulfur-rich geothermal habitats.
Directional transference of genetic information from archaea to bacteria therefore appears to us to be the most
likely origin of C. hydrogenoformans CODH genes. Horizontal gene transfer is an area of active speculation
[13,14]. For horizontal gene transfer to be feasible, we
presume that physical proximity among anaerobic bacteria
and the potential archaeal donor would favor DNA exchange. Supporting this assumption, associations among
anaerobic bacteria and methanogens have been reported
[15,16] as well as the existence of methanogens in hydrothermal vents [17]. C. hydrogenoformans is a strictly anaerobic bacterium isolated from hydrothermal springs that
uses CO, producing H2 and CO2 [8]. Interestingly, methanogens in hydrothermal vents are known to produce CO
under certain conditions, such as hydrogen de¢ciency [18].
Interspecies CO transfers in natural volcanic environments
may be examples of bacterial^archaeal interactions resulting in a mutualistic relationship. Physical proximity and
mutualistic metabolism could provide the appropriate conditions required for exchanging genetic information, resulting in a more e¤cient utilization of available resources.
Acknowledgements
This study was funded by grants from the Markey
Foundation and NSF Lexen program. Contribution no.
596 from the Center of Marine Biotechnology.
References
[1] Ferry, J.G. (1995) CO dehydrogenase. Annu. Rev. Microbiol. 49,
305^333.
[2] Meyer, O. and Schlegel, H.G. (1983) Biology of aerobic carbon oxidizing bacteria. Annu. Rev. Microbiol. 37, 277^310.
[3] Meyer, O. (1989) Aerobic carbon monoxide-oxidizing bacteria. In:
Autotrophic Bacteria (Schlegel, H.G. and Bowien, B., Eds.), pp. 331^
350. Science Tech Publishers, Madison, WI.
[4] Kerby, R.L., Hong, S.S., Ensign, S.A., Coppoc, L.J., Ludden, P.W.
and Roberts, G.P. (1992) Genetic and physiological characterization
of the Rhodospirillum rubrum carbon monoxide dehydrogenase system. J. Bacteriol. 174, 5284^5294.
[5] Morton, T.A., Runquist, J.A., Ragsdale, S.W., Shanmugasundaran,
T., Wood, H.G. and Ljungdahl, L.G. (1991) The primary structure of
the subunits of carbon monoxide dehydrogenase/acetyl-CoA synthase
from Clostridium thermoaceticum. J. Biol. Chem. 266, 23824^23828.
[6] Ferry, J.G. (1992) Biochemistry of methanogens. Crit. Rev. Biochem.
Mol. Biol. 27, 473^503.
[7] Moyer-Zinkhan, D. and Thauer, R.K. (1990) Anaerobic lactate oxidation to 3 CO2 by Archaeoglobus fulgidus via the carbon monoxide
dehydrogenase pathway ^ demonstration of the acetyl-CoA carbon^
carbon cleavage reaction in cell extracts. Arch. Microbiol. 153, 215^
218.
[8] Svetlichny, V.A., Sokolova, T.G., Gerhardt, M., Ringpfeil, M., Kostrikina, N.A. and Zavarzin, G.A. (1991) Carboxydothermus hydrogenoformans gen. nov., sp. nov., a CO-utilizing thermophilic anaerobic bacterium from hydrothermal environmenta of Kunashir Island.
Syst. Appl. Microbiol. 14, 254^260.
[9] Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J.
(1990) Basic local alignment search tool. J. Mol. Biol. 215, 403^410.
[10] Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTALW: Improving the sensitivity of progressive multiple sequence
alignment through sequence weighting, positions-speci¢c gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673^4680.
[11] Syvanen, M. (1994) Horizontal gene transfer: Evidence and possible
consequences. Annu. Rev. Genet. 28, 237^261.
[12] Cohan, F.M. (1996) The role of genetic exchange in bacterial evolution. ASM News 62, 631^636.
[13] Nelson, K.E., Clayton, R.A., Gill, S.R., Gwinn, M.L., Dodson, R.J.,
Haft, D.H., Hickey, E.K., Peterson, J.D., Nelson, W.C., Ketchum,
K.A., McDonald, L.C., Utterback, T.R., Malek, J.A., Linher, K.D.,
Garrett, M.M., Stewart, A.M., Cotton, M.D., Pratt, M.S., Phillips,
C.A., Richardson, D., Heidelberg, J., Sutton, G.G., Fleischmann,
R.D., Eisen, J.A., White, O., Salzberg, S.L., Smith, H.O., Venter,
J.G. and Fraser, C.M. (1999) Evidence for lateral gene transfer between Archaea and Bacteria from genome sequence of Thermotoga
maritima. Science 399, 323^329.
[14] Doolittle, W.F. (1999) Phylogenetic classi¢cation and the universal
tree. Science 284, 2124^2128.
[15] Pak, K.-R. and Bartha, R. (1998) Mercury methylation by interspecies hydrogen and acetate transfer between sul¢dogens and methanogens. Appl. Environ. Microbiol. 64, 1987^1990.
FEMSLE 9619 27-9-00
J.M. Gonzälez, F.T. Robb / FEMS Microbiology Letters 191 (2000) 243^247
[16] Schnurer, A., Schink, B. and Svensson, B.H. (1996) Clostridium ultunense sp. nov., a mesophilic bacterium oxidizing acetate in syntrophic
association with a hydrogenotrophic methanogenic bacterium. Int. J.
Syst. Bacteriol. 46, 1145^1152.
[17] Barns, S.M., Fundynga, R.E., Je¡ries, M.W. and Pace, N.R. (1994)
247
Remarkable Archaea diversity in a Yellowstone National Park hot
spring environment. Proc. Natl. Acad. Sci. USA 91, 1609^1613.
[18] Conrad, R. and Thauer, R.K. (1983) Carbon monoxide production
by Methanobacterium thermoautotrophicum. FEMS Microbiol. Lett.
20, 229^232.
FEMSLE 9619 27-9-00