Open Access

Complete genome sequence of Methanocorpusculum labreanum type strain Z

  • Iain J. Anderson1,
  • Magdalena Sieprawska-Lupa2,
  • Eugene Goltsman1,
  • Alla Lapidus1,
  • Alex Copeland1,
  • Tijana Glavina Del Rio1,
  • Hope Tice1,
  • Eileen Dalin1,
  • Kerrie Barry1,
  • Sam Pitluck1,
  • Loren Hauser1, 3,
  • Miriam Land1, 3,
  • Susan Lucas1,
  • Paul Richardson1,
  • William B. Whitman2 and
  • Nikos C. Kyrpides1
Standards in Genomic Sciences20091:1020197

DOI: 10.4056/sigs.35575

Published: 29 September 2009

Abstract

Methanocorpusculum labreanum is a methanogen belonging to the order Methanomicrobiales within the archaeal phylum 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

archaea methanogen Methanomicrobiales

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.

Classification and features

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 according to the MIGS recommendations [9]

MIGS ID

Property

Term

Evidence Code

 

Current classification

Domain Archaea

TAS [1012]

 

Phylum Euryarchaeota

TAS[13, 14]

 

Class “Methanomicrobia

TAS[15]

 

Order Methanomicrobiales

TAS[16]

 

Family Methanocorpusculaceae

TAS[17,18]

 

Genus Methanocorpusculum

TAS [19]

 

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

Latitude-longitude

34.107811/-118.599658

 

MIGS-4.2

   

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 [19]. If the evidence code is IDA, then the property was directly observed for a living isolate by one of the authors or another expert 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

34×

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

Growth conditions and DNA isolation

The methods for DNA isolation, genome sequencing and assembly for this genome have previously been published [21].

Genome annotation

Protein-coding genes were identified using a combination of CRITICA [22] and Glimmer [23] followed by a round of manual curation using the JGI GenePRIMP pipeline [24]. 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 [25], and transmembrane helices were determined with TMHMM [26]. CRISPR elements were identified with the CRISPR Recognition Tool (CRT) [27]. 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 [28] 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 [29].

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 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 [21]. 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 [30]. 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 [21].

Declarations

Acknowledgements

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.

Authors’ Affiliations

(1)
Joint Genome Institute
(2)
Microbiology Department, University of Georgia
(3)
Oak Ridge National Laboratory

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Copyright

© The Author(s) 2009