Open Access

Complete genome sequence of Methanocorpusculum labreanum type strain Z

  • Iain J. Anderson
  • , Magdalena Sieprawska-Lupa
  • , Eugene Goltsman
  • , Alla Lapidus
  • , Alex Copeland
  • , Tijana Glavina Del Rio
  • , Hope Tice
  • , Eileen Dalin
  • , Kerrie Barry
  • , Sam Pitluck
  • , Loren Hauser,
  • , Miriam Land,
  • , Susan Lucas
  • , Paul Richardson
  • , William B. Whitman
  • and Nikos C. Kyrpides
Corresponding author

DOI: 10.4056/sigs.35575

Received: 24 September 2009

Published: 29 September 2009

Abstract

Methanocorpusculum labreanum is a methanogen belonging to the order Methanomicrobiales within the archaeal kingdom Euryarchaeota. The type strain Z was isolated from surface sediments of Tar Pit Lake in the La Brea Tar Pits in Los Angeles, California. M. labreanum is of phylogenetic interest because at the time the sequencing project began only one genome had previously been sequenced from the order Methanomicrobiales. We report here the complete genome sequence of M. labreanum type strain Z and its annotation. This is part of a 2006 Joint Genome Institute Community Sequencing Program project to sequence genomes of diverse Archaea.

Keywords:

archaeamethanogenMethanomicrobiales

Introduction

Methanocorpusculum labreanum is a methanogen belonging to the order Methanomicrobiales within the archaeal kingdom Euryarchaeota. Strain Z is the type strain of this species. It was isolated from surface sediments of Tar Pit Lake at the La Brea Tar Pits in Los Angeles [1]. Most of the other described members of this family have been isolated from anaerobic digesters or waste water [2]. The genus covers organisms with a wide temperature range. One psychrotolerant strain was isolated from a Russian pond polluted with paper mill waste water [3], while other strains were found in heated sediment at a hydrothermal vent site [4]. Methanocorpusculum species may be common in subsurface environments as they were the most prominent genus found in a coal bed in Indiana [5] and in shale in northern Michigan [6].

Methanogens have been divided into two groups known as Class I and Class II based on phylogeny [7]. Class I includes the orders Methanococcales, Methanobacteriales, and Methanopyrales, which use H2/CO2 or formate as substrates for methanogenesis, although some can also use alcohols as electron donors. Class II includes the orders Methanosarcinales and Methanomicrobiales. Some of the Methanosarcinales are capable of using various methyl compounds as substrates for methanogenesis including acetate, methylamines, and methanol, but Methanomicrobiales are restricted to the same substrates as the Class I methanogens [2]. Therefore, Methanomicrobiales are phylogenetically closer to Methanosarcinales but physiologically more similar to Class I methanogens, making them an interesting target for genome sequencing. In a 2006 Community Sequencing Program (CSP) project, we proposed sequencing two members of the order Methanomicrobiales: M. labreanum and Methanoculleus marisnigri. Previously only one genome was available from this order, that of Methanospirillum hungatei. Methanocorpusculum labreanum and Methanoculleus marisnigri are phylogenetically distant from each other and from Methanospirillum hungatei (Figure 1), and they represent the three families within the order Methanomicrobiales. We report here the sequence and annotation of M. labreanum type strain Z.

Figure 1

Phylogenetic tree of 16S rRNA of selected Methanomicrobiales showing the distance between the three organisms for which complete genomes are available – Methanospirillum hungatei, Methanocorpusculum labreanum, and Methanoculleus marisnigri. The tree uses sequences aligned within the Ribosomal Database Project (RDP), and the tree was constructed with the RDP Tree Builder [8]. Methanosarcina barkeri was used as the outgroup. The numbers indicate bootstrap values based on 100 replicates.

Organism information

Methanocorpusculum labreanum Z was isolated from surface sediments at the La Brea Tar Pits [1]. A polypropylene bottle was filled with half surface sediment and half lake water. In an anaerobic chamber the contents of the bottle were mixed to suspend the sediment, and 0.5 ml of the slurry was added to 5 ml enrichment medium. The enrichment medium contained sodium formate, trypticase peptone, and salts. The gas phase was H2-CO2 at a ratio of 4:1 and a pressure of 152 kPa. The physiological characteristics of M. labreanum were described as follows [1]. The cells were coccoid with a diameter of 0.4-2.0 μm. They were irregular in shape under some growth conditions, such as higher salt or with added acetate. Motility was not observed and no flagella were observed. Growth was observed on H2/CO2 or formate, but not with acetate, propionate, methanol, trimethylamine, or ethanol. Growth was observed in a narrow window of pH, from 6.5 to 7.5, with pH 7.0 as the optimal value. Growth was observed between 25 and 40°C, with an optimum at 37°C. M. labreanum can tolerate a wide range of salt concentration, from 0 to 30 g/L NaCl. Acetate was stimulatory at lower salt concentrations. Either trypticase peptone, yeast extract, or cysteine was required for growth. The features of M. labreanum Z are presented in Table 1.

Table 1

Classification and general features of Methanocorpusculum labreanum Z in accordance with the Minimum Information about a Genome Sequence (MIGS) recommendations [9].

MIGS ID

Property

Term

Evidence Code

Current classification

Domain Archaea

Phylum Euryarchaeota

Class Methanomicrobia

Order Methanomicrobiales

Family Methanocorpusculaceae

Genus Methanocorpusculum

TAS [10]

Species Methanocorpusculum labreanum

TAS [1]

Gram stain

negative

TAS [1]

Cell shape

irregular coccus

TAS [1]

Motility

nonmotile

TAS [1]

Sporulation

nonsporulating

Temperature range

25-40°C

TAS [1]

Optimum temperature

37°C

TAS [1]

MIGS-6.3

Salinity

0-30 g/L NaCl

TAS [1]

MIGS-22

Oxygen requirement

anaerobe

TAS [1]

Carbon source

CO2, acetate

TAS [1]

Energy source

H2/CO2, formate

TAS [1]

MIGS-6

Habitat

sediment

TAS [1]

MIGS-15

Biotic relationship

free-living

TAS [1]

MIGS-14

Pathogenicity

none

Biosafety level

1

Isolation

sediment

TAS [1]

MIGS-4

Geographic location

Tar Pit Lake, La Brea Tar Pits

TAS [1]

MIGS-5

Isolation time

1989

TAS [1]

MIGS-4.1 MIGS-4.2

Latitude-longitude

34.107811/-118.599658

MIGS-4.3

Depth

0-5 cm

TAS [1]

MIGS-4.4

Altitude

not applicable

Evidence codes - IDA: Inferred from Direct Assay (first time in publication); TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [10]. If the evidence code is IDA, then the property should have been directly observed, for the purpose of this specific publication, for a live isolate by one of the authors, or an expert or reputable institution mentioned in the acknowledgements.

Genome sequencing information

Genome project history

M. labreanum was selected for sequencing based upon its phylogenetic position relative to other methanogens of the order Methanomicrobiales. It is part of a 2006 Joint Genome Institute Community Sequencing Program project that included six diverse archaeal genomes. A summary of the project information is shown in Table 2. The complete genome sequence was finished in January, 2007. The GenBank accession number for the project is CP000559. The genome project is listed in the Genomes OnLine Database (GOLD) [11] as project Gc00506. Sequencing was carried out at the Joint Genome Institute (JGI) Production Genomics Facility (PGF). Quality assurance was done by JGI-Stanford. Finishing was done at JGI-PGF. Annotation was done by JGI-Oak Ridge National Laboratory (ORNL) and by JGI-PGF.

Table 2

Genome sequencing project information

MIGS ID

Characteristic

Details

MIGS-28

Libraries used

3kb, 6kb and 40kb (fosmid)

MIGS-29

Sequencing platform

ABI3730, 454

MIGS-31.2

Sequencing coverage

34x

MIGS-31

Finishing quality

Finished

Sequencing quality

less than one error per 50kb

MIGS-30

Assembler

Newbler, Paracel

MIGS-32

Gene calling method

CRITICA, Glimmer

GenBank ID

CP000559

GenBank date of release

February 2, 2007

GOLD ID

Gc00506

NCBI project ID

18109

IMG Taxon ID

640069317

MIGS-13

Source material identifier

DSM 4855

Project relevance

Tree of Life

DNA isolation, genome sequencing and assembly

The methods for DNA isolation, genome sequencing and assembly for this genome have previously been published [12].

Genome annotation

Protein-coding genes were identified using a combination of CRITICA [13] and Glimmer [14] followed by a round of manual curation using the JGI GenePRIMP pipeline [15]. GenePRIMP points out cases where gene start sites may be incorrect based on alignment with homologous proteins. It also highlights genes that appear to be broken into two or more pieces, due to a premature stop codon or frameshift, and genes that are disrupted by transposable elements. All of these types of broken and interrupted genes are labeled as pseudogenes. Genes that may have been missed by the gene calling programs are also identified in intergenic regions. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Signal peptides were identified with SignalP [16], and transmembrane helices were determined with TMHMM [17]. CRISPR elements were identified with the CRISPR Recognition Tool (CRT) [18]. Paralogs are hits of a protein against another protein within the same genome with an e-value of 10-2 or lower. The tRNAScanSE tool [19] was used to find tRNA genes. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes Expert Review (IMG-ER) platform [20].

Genome properties

The genome of M. labreanum Z consists of a single circular chromosome (Figure 2). The genome size of 1.80 Mbp is similar to those of Class I methanogens, but smaller than the genomes of Methanosarcina species and the other Methanomicrobiales, which range between 2.5 and 5.8 Mbp. The G+C percentage is 50.0%, higher than that of most other sequenced methanogens. There are 1,830 genes, of which 1,765 are protein-coding genes and the remaining 65 are RNA genes. There were only 26 pseudogenes identified, constituting 1.4% of the total genes. The properties and statistics of the genome are summarized in Table 3, and genes belonging to COG functional categories are listed in Table 4.

Figure 2

Graphical circular map of the chromosome of Methanocorpusculum labreanum Z. From outside to the center: Genes on forward strand (colored by COG categories), Genes on reverse strand (colored by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew.

Table 3

Genome statistics

Attribute

Value

% of total

Genome size (bp)

1,804,962

100.00%

DNA coding region (bp)

1,600,673

88.68%

DNA G+C content (bp)

902,600

50.01%

Number of replicons

1

Extrachromosomal elements

0

Total genes

1830

100.00%

RNA genes

65

3.55%

rRNA operons

3

Protein-coding genes

1765

96.45%

Pseudogenes

26

1.42%

Genes in paralog clusters

745

42.21%

Genes assigned to COGs

1358

76.94%

Genes assigned Pfam domains

1335

75.64%

Genes with signal peptides

406

23.00%

Genes with transmembrane helices

368

20.85%

CRISPR repeats

2

Table 4

Numbers of genes associated with the 25 general COG functional categories.

Code

value

% of total

COG category

E

130

7.4

Amino acid transport and metabolism

G

54

3.1

Carbohydrate transport and metabolism

D

10

0.6

Cell cycle control, cell division, chromosome partitioning

N

5

0.3

Cell motility

M

35

2.0

Cell wall/membrane/envelope biogenesis

B

2

0.1

Chromatin structure and dynamics

H

129

7.3

Coenzyme transport and metabolism

Z

0

0.0

Cytoskeleton

V

13

0.7

Defense mechanisms

C

134

7.6

Energy production and conversion

W

0

0.0

Extracellular structures

S

172

9.7

Function unknown

R

219

12.4

General function prediction only

P

95

5.4

Inorganic ion transport and metabolism

U

17

1.0

Intracellular trafficking, secretion, and vesicular transport

I

24

1.4

Lipid transport and metabolism

Y

0

0.0

Nuclear structure

F

49

2.8

Nucleotide transport and metabolism

O

57

3.2

Posttranslational modification, protein turnover, chaperones

A

0

0.0

RNA processing and modification

L

65

3.7

Replication, recombination and repair

Q

8

0.5

Secondary metabolites biosynthesis, transport and catabolism

T

30

1.7

Signal transduction mechanisms

K

77

4.4

Transcription

J

147

8.3

Translation, ribosomal structure and biogenesis

-

293

16.6

Not in COGs

Insights from the genome sequence

The genome sequence of M. labreanum Z shows some similarities to Class I methanogens and some to Methanosarcinales but also has some unique features. In common with Class I methanogens, M. labreanum uses a partial reductive TCA cycle to synthesize 2-oxoglutarate, and it has the Eha membrane-bound hydrogenase. Similar to Methanosarcinales, M. labreanum has the Ech membrane-bound hydrogenase. A unique feature of M. labreanum and the other Methanomicrobiales is the presence of anti- and anti-anti-sigma factors, which is surprising as Archaea do not use sigma factors. Phylogenetic analysis of methanogenesis and cofactor biosynthesis enzymes suggest that Methanomicrobiales form a group distinct from other methanogens, and therefore methanogens can be split in to three classes [12]. Surprisingly M. labreanum lacks the F420-nonreducing hydrogenase, which has been proposed to couple Coenzyme M-Coenzyme B heterodisulfide reduction and ferredoxin reduction for the first step of methanogenesis in the cytoplasm of Methanomicrobiales [21]. In place of this hydrogenase, M. labreanum may use the membrane-bound hydrogenase Mbh or energy-converting hydrogenase Ech to couple heterodisulfide reduction to a transmembrane ion gradient [12].

Declarations

Acknowledgments

This work was performed under the auspices of the US Department of Energy’s Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under Contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, and Los Alamos National Laboratory under Contract No. DE-AC02-06NA25396. L. H. and M. L. were supported by the Department of Energy under contract DE-AC05-000R22725. M. S.-L., and W. B. W. were supported by DOE contract number DE-FG02-97ER20269.


This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

References

  1. Zhao Y, Boone DR, Mah RA, Boone JE and Xun L. Isolation and characterization of Methanocorpusculum labreanum sp. nov. from the LaBrea Tar Pits. Int J Syst Bacteriol. 1989; 39:10-13
  2. Garcia JL, Ollivier B and Whitman WB. The order Methanomicrobiales. Prokaryotes. 2006; 3:208-230 View Article
  3. Simankova MV, Kotsyurbenko OR, Lueders T, Nozhevnikova AN, Wagner B, Conrad R and Friedrich MW. Isolation and characterization of new strains of methanogens from cold terrestrial habitats. Syst Appl Microbiol. 2003; 26:312-318 View ArticlePubMed
  4. Dhillon A, Lever M, Lloyd KG, Albert DB, Sogin ML and Teske A. Methanogen diversity evidenced by molecular characterization of methyl coenzyme M reductase A (mcrA) genes in hydrothermal sediments of the Guaymas Basin. Appl Environ Microbiol. 2005; 71:4592-4601 View ArticlePubMed
  5. Strąpoć D, Picardal FW, Turich C, Schaperdoth I, Macalady JL, Lipp JS, Lin YS, Ertefai TF, Schubotz F and Hinrichs KU. Methane-producing microbial community in a coal bed of the Illinois basin. Appl Environ Microbiol. 2008; 74:2424-2432 View ArticlePubMed
  6. Waldron PJ, Petsch ST, Martini AM and Nüsslein K. Salinity constraints on subsurface archaeal diversity and methanogenesis in sedimentary rock rich in organic matter. Appl Environ Microbiol. 2007; 73:4171-4179 View ArticlePubMed
  7. Bapteste É, Brochier C and Boucher Y. Higher-level classification of the Archaea: evolution of methanogenesis and methanogens. Archaea. 2005; 1:353-363 View ArticlePubMed
  8. Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed-Mohideen AS, McGarrell DM, Marsh T and Garrity GM. The ribosomal database project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res. 2009; 37:D141-D145 View ArticlePubMed
  9. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ and Angiuoli SV. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008; 26:541-547 View ArticlePubMed
  10. Xun L, Boone DR and Mah RA. Deoxyribonucleic acid hybridization study of Methanogenium and Methanocorpusculum species, emendation of the genus Methanocorpusculum, and transfer of Methanogenium aggregans to the genus Methanocorpusculum as Methanocorpusculum aggregans comb. nov. Int J Syst Bacteriol. 1989; 39:109-111
  11. Liolios K, Mavromatis K, Tavernarakis N and Kyrpides NC. The Genomes OnLine Database (GOLD) in 2007: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res. 2008; 36:D475-D479 View ArticlePubMed
  12. Anderson I, Ulrich LE, Lupa B, Susanti D, Porat I, Hooper SD, Lykidis A, Sieprawska-Lupa M, Dharmarajan L and Goltsman E. Genomic characterization of Methanomicrobiales reveals three classes of methanogens. PLoS ONE. 2009; 4:e5797 View ArticlePubMed
  13. Badger JH and Olsen GJ. CRITICA: coding region identification tool invoking comparative analysis. Mol Biol Evol. 1999; 16:512-524PubMed
  14. Delcher AL, Harmon D, Kasif S, White O and Salzberg SL. Improved microbial gene identification with GLIMMER. Nucleic Acids Res. 1999; 27:4636-4641 View ArticlePubMed
  15. Pati A. GenePRIMP: A Gene Prediction Improvement Pipeline for microbial genomes. (Submitted).
  16. Emanuelsson O, Brunak S, von Heijne G and Nielsen H. Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc. 2007; 2:953-971 View ArticlePubMed
  17. Krogh A, Larsson B, von Heijne G and Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol. 2001; 305:567-580 View ArticlePubMed
  18. Bland C, Ramsey TL, Sabree F, Lowe M, Brown K, Kyrpides NC and Hugenholtz P. CRISPR recognition tool (CRT): a tool for automatic detection of clustered regularly interspaced palindromic repeats. BMC Bioinformatics. 2007; 8:209 View ArticlePubMed
  19. Lowe TM and Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997; 25:955-964 View ArticlePubMed
  20. Markowitz VM, Mavromatis K, Ivanova NN, Chen IMA, Chu K and Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics. 2009; 25:2271-2278PubMed
  21. Thauer RK, Kaster AK, Seedorf H, Buckel W and Hedderich R. Methanogenic archaea: ecologically relevant differences in energy conservation. Nat Rev Microbiol. 2008; 6:579-591 View ArticlePubMed