Open Access

Genome sequence of the mud-dwelling archaeon Methanoplanus limicola type strain (DSM 2279T), reclassification of Methanoplanus petrolearius as Methanolacinia petrolearia and emended descriptions of the genera Methanoplanus and Methanolacinia

  • Markus Göker
  • , Megan Lu,
  • , Anne Fiebig
  • , Matt Nolan
  • , Alla Lapidus,
  • , Hope Tice
  • , Tijana Glavina Del Rio
  • , Jan-Fang Cheng
  • , Cliff Han,
  • , Roxanne Tapia,
  • , Lynne A. Goodwin,
  • , Sam Pitluck
  • , Konstantinos Liolios
  • , Konstantinos Mavromatis
  • , Ioanna Pagani
  • , Natalia Ivanova
  • , Natalia Mikhailova
  • , Amrita Pati
  • , Amy Chen
  • , Krishna Palaniappan
  • , Miriam Land,
  • , Shanmugam Mayilraj
  • , Manfred Rohde
  • , John C. Detter,
  • , Boyke Bunk
  • , Stefan Spring
  • , Reinhard Wirth
  • , Tanja Woyke
  • , James Bristow
  • , Jonathan A. Eisen,
  • , Victor Markowitz
  • , Philip Hugenholtz,
  • , Nikos C. Kyrpides,
  • and Hans-Peter Klenk
Corresponding author

DOI: 10.4056/sigs.5138968

Received: 15 March 2014

Accepted: 15 March 2014

Published: 15 June 2014


Methanoplanus limicola Wildgruber et al. 1984 is a mesophilic methanogen that was isolated from a swamp composed of drilling waste near Naples, Italy, shortly after the Archaea were recognized as a separate domain of life. Methanoplanus is the type genus in the family Methanoplanaceae, a taxon that felt into disuse since modern 16S rRNA gene sequences-based taxonomy was established. Methanoplanus is now placed within the Methanomicrobiaceae, a family that is so far poorly characterized at the genome level. The only other type strain of the genus with a sequenced genome, Methanoplanus petrolearius SEBR 4847T, turned out to be misclassified and required reclassification to Methanolacinia. Both, Methanoplanus and Methanolacinia, needed taxonomic emendations due to a significant deviation of the G+C content of their genomes from previously published (pre-genome-sequence era) values. Until now genome sequences were published for only four of the 33 species with validly published names in the Methanomicrobiaceae. Here we describe the features of M. limicola, together with the improved-high-quality draft genome sequence and annotation of the type strain, M3T. The 3,200,946 bp long chromosome (permanent draft sequence) with its 3,064 protein-coding and 65 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.


anaerobicmotilemesophilicmethanogenswampimproved-high-quality draftMethanomicrobiaceaeGEBA


Strain M3T (= DSM 2279 = ATCC 35062 = OCM 101) is the type strain of the species Methanoplanus limicola [1,2], one out of currently three species in the genus Methanoplanus [1,2]. Strain M3T was originally isolated from the mud of a drilling swamp near Baia, Naples Area, Italy [1]. The genus name was derived from the Neo-Latin therm “methanum”, pertaining to methane, and the Latin adjective “planus”, meaning a flat plate, which refers to its flat cell morphology [1]. The species epithet was derived from the Latin word limicola, a dweller in the mud, inhabitant of a swamp [1]. When Wildgruber et al. described the type strain of the novel species in 1982 [1] they not only realized the striking similarity to the square-shaped flat bacterium that was reported two years earlier by Walsby [3], but also classified it as the type strain of the type species in the type genus of Methanomicrobiales Family III, ‘Methanoplanaceae’ [1]. However, when years later 16S rRNA sequences became available for phylogenetic analyses it became clear that the strains which represent the species Methanoplanus are closely related to Methanomicrobiaceae (including the genera Methanomicrobium, Methanogenium, and Methanoculleus). Since that time, the genus Methanoplanus is generally placed within the Methanomicrobiaceae, and Methanoplanaceae Wildgruber et al. 1984 has fallen into disuse [4], although the genus Methanoplanus was never formally reclassified. In the 31 years since strain M3T was first characterized, only two follow-up projects have reported the use of M. limicola in comparative analyses; Ivanov and Stabnikova [5] used M. limicola for a study on the molecular phylogeny of methanogenic archaea based on the G+C content, and Liu et al. used the species in a study on air tolerance and water stress [6].

Here we present a summary classification and a set of features for M. limicola M3T, together with the description of the genomic sequencing and annotation.

Classification and features

The single genomic 16S rRNA sequence of M. limicola M3T was compared with the Greengenes database for determining the weighted relative frequencies of taxa and (truncated) keywords as previously described [7]. The most frequently occurring genera were Methanoculleus (51.9%), Methanoplanus (18.5%), Methanogenium (16.8%), Methanosphaerula (5.3%) and Methanomicrobium (3.7%) (52 hits in total). Regarding the two hits to sequences from members of the species, the average identity within HSPs was 99.9%, whereas the average coverage by HSPs was 92.8%. Regarding the five hits to sequences from other members of the genus, the average identity within HSPs was 96.6%, whereas the average coverage by HSPs was 95.0%. Among all other species, the one yielding the highest score was M. endosymbiosus (FR733674), which corresponded to an identity of 99.5% and an HSP coverage of 99.7%. (Note that the Greengenes database uses the INSDC (= EMBL/NCBI/DDBJ) annotation, which is not an authoritative source for nomenclature or classification.) The highest-scoring environmental sequence was EU420694 ('Archaeal and Kao-Mei Wetland clone KM07-Da-3'), which showed an identity of 95.7% and an HSP coverage of 98.0%. The most frequently occurring keywords within the labels of all environmental samples which yielded hits were 'temperatur' (4.7%), 'bioreactor' (4.4%), 'anaerob' (4.0%), 'methanogen' (3.3%) and 'archaeal' (2.9%) (198 hits in total) fit to the features known from the habitat of strain M3T. Environmental samples which yielded hits of a higher score than the highest scoring species were not found.

Figure 1 shows the phylogenetic neighborhood of M. limicola in a 16S rRNA based tree. The sequence of the single 16S rRNA gene copy in the genome does not differ from the previously published 16S rRNA sequence (M59143), which contains 23 ambiguous base calls.

Figure 1

Phylogenetic tree highlighting the position of M. limicola relative to the type strains of the other species within the family Methanomicrobiaceae. The tree was inferred from 1,271 aligned characters of the 16S rRNA gene sequence under the maximum likelihood (ML) criterion and rooted as previously described [7]. The branches are scaled in terms of the expected number of substitutions per site. Numbers adjacent to the branches are support values from 250 ML bootstrap replicates [8] (left) and from 1,000 maximum-parsimony bootstrap replicates [9] (right) if larger than 60%. Lineages with type-strain genome sequencing projects registered in GOLD [10] are labeled with one asterisk, those also listed as 'Complete and Published' with two asterisks [11-14] (for Methanoregula boonei and Methanosphaerula palustris see CP000780 and CP001338, respectively).

The tree depicted in Figure 1 reveals discrepancies between the current classification of the group and 16S rRNA phylogenetic analysis, as the genus Methanoplanus appeared polyphyletic, with M. petrolearius appearing as sister group of Methanolacinia payntneri with maximum support. We conducted a constraint analysis as previously described [15], enforcing the monophyly of all genera (which only affects Methanoplanus in this dataset, see Figure 1). The best-known ML tree had a log likelihood of -7,097.90, whereas the best tree found under the constraint had a log likelihood of -7,144.12. The constrained tree was significantly worse than the globally best one in the Shimodaira-Hasegawa test as implemented in RAxML [8] (α = 0.01). The best-known MP trees had a score of 1,090, whereas the best constrained trees found had a score of 1,115 and were significantly worse in the Kishino-Hasegawa test as implemented in PAUP* [9] (α = 0.01).

M. limicola M3T cells stain Gram negative [1] and are plate-shaped with sharp crystal-like edges 1−3 µm long and 1−2 µm wide (Figure 2 and [1]). Weak motility was observed and motility genes were identified in the genome (see below). Polar tufts of flagella were also reported [1], but not visible in Figure 2. Granules with putative reserve material were observed in thin section EM images, as were curious ‘bone-shaped’ cells [1]. Cell envelopes consist of an S-layer glycoprotein with a hexagonal surface pattern [1]. Cultures grow with H2 or formate as sole substrates supplemented with ≥ 0.1% acetate essentially required [1]. Growth temperatures span from 17−41°C (optimum 40°C) in the presence of 0.4−5.4% NaCl (optimum 1%) [1]. A summary of the classification and features is presented in Table 1.

Figure 2

Scanning electron micrograph of M. limicola M3T

Table 1

Classification and general features of M. limicola M3T according to the MIGS recommendations [16] published by the Genomic Standards Consortium [17].




       Evidence code

      Current classification

       Domain Bacteria

       TAS [18]

       Phylum Euryarchaeota

       TAS [19]

       Class Methanomicrobia

       TAS [20]

       Order Methanomicrobiales

       TAS [21-25]

       Family Methanomicrobiaceae

       TAS [21,22]

       Genus Methanoplanus

       TAS [1,2]

       Species Methanoplanus limicola

       TAS [1,2]

       Type strain M3

       TAS [1]

      Gram stain


       TAS [1]

      Cell shape


       TAS [1]


       weakly motile

       TAS [1]


       not reported

      Temperature range

       mesophile, 17-41°C

       TAS [1]

      Optimum temperature


       TAS [1]


       0.4 - 5.4% NaCl (w/v), optimum 1.0%

       TAS [5]


      Oxygen requirement


       TAS [1]

      Carbon source

       CO2, formate

       TAS [1]

      Energy metabolism

       methanogen, chemoorganotrophic

       TAS [1]



       swamps of fresh water and seawater

       TAS [1]


      Biotic relationship

       free living

       TAS [1]





      Biosafety level


       TAS [26]



       mud of drilling swamp

       TAS [1]


      Geographic location

       near Baia, Naples Area, Italy

       TAS [1]


      Sample collection time

       1981 or earlier












       not reported



       not reported

Evidence codes - 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). Evidence codes are from of the Gene Ontology project [27].


No chemotaxonomical results were reported for strain M3T, except for an estimation of 47.5% for the G+C content of the genome determined by a melting point in 0.1 × SSC [1].

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing on the basis of its phylogenetic position [28], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [29]. The genome project is deposited in the Genomes On Line Database [10] and the complete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI) using state of the art sequencing technology [30]. A summary of the project information is shown in Table 2.

Table 2

Genome sequencing project information





      Finishing quality



      Libraries used

       Three genomic libraries: one 454 pyrosequence standard library, one 454 PE library (5 kb insert size), one Illumina library


      Sequencing platforms

       Illumina GAii, 454 GS FLX Titanium


      Sequencing coverage

       834.5 × Illumina; 33.4 × pyrosequence



       Newbler version 2.3, Velvet 1.0.13, phrap version SPS - 4.24


      Gene calling method


      INSDC ID

       CM001436, AHKP00000000

      GenBank Date of Release

       January 24, 2012

      GOLD ID


      NCBI project ID


      Database: IMG



      Source material identifier

       DSM 2279

      Project relevance

       Tree of Life, GEBA

Growth conditions and DNA isolation

M. limicola strain M3T, DSM 2279, was grown anaerobically under H2/CO2 gas phase in DSMZ medium 141 (Methanogenium medium; MMG medium + 0.1% acetate; substrate: H2 or formate; stimulated by YE or peptone, + vitamins) [31] at 35−40°C. DNA was isolated from 0.5-1 g of cell paste using MasterPure Gram-positive DNA purification kit (Epicentre MGP04100) following the standard protocol as recommended by the manufacturer with modification st/LALM for cell lysis as described in Wu et al. 2009 [29]. DNA is available through the DNA Bank Network [32].

Genome sequencing and assembly

The genome was sequenced using a combination of Illumina and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website [33]. Pyrosequencing reads were assembled using the Newbler assembler (Roche). The initial Newbler assembly consisting of 760 contigs in ten scaffolds was converted into a phrap [34] assembly by making fake reads from the consensus, to collect the read pairs in the 454 paired end library. Illumina GAii sequencing data (3,470.2 Mb) was assembled with Velvet [35] and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. The 454 draft assembly was based on 332.3 Mb 454 draft data and all of the 454 paired end data. Newbler parameters are -consed -a 50 -l 350 -g -m -ml 20. The Phred/Phrap/Consed software package [34] was used for sequence assembly and quality assessment in the subsequent finishing process. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with gapResolution [33], Dupfinisher [36], or sequencing cloned bridging PCR fragments with subcloning. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F. Chang, unpublished). A total of 159 additional reactions were necessary to close some gaps and to raise the quality of the final sequence. Illumina reads were also used to correct potential base errors and increase consensus quality using a software Polisher developed at JGI [37]. The error rate of the final genome sequence is less than 1 in 100,000. Together, the combination of the Illumina and 454 sequencing platforms provided 867.9 x coverage of the genome. The final assembly contained 421,665 pyrosequence and 44,481,858 Illumina reads.

Genome annotation

Genes were identified using Prodigal [38] as part of the DOE-JGI [39] genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [40]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) non-redundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. These data sources were combined to assert a product description for each predicted protein. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes - Expert Review (IMG-ER) platform [41].

Genome properties

The genome consists of one scaffold (circularity not experimentally proven) of 3,200,946 bp length with a 42.2% G+C content (Table 3 and Figure 3). Of the 3,128 genes predicted, 3,064 were protein-coding genes, and 65 RNAs; 122 pseudogenes were also identified. The majority of the protein-coding genes (60.8%) were assigned a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.

Table 3

Genome Statistics



      % of Total

Genome size (bp)



DNA coding region (bp)



DNA G+C content (bp)



Number of replicons


Extrachromosomal elements


Total genes



RNA genes



rRNA operons


tRNA genes



Protein-coding genes



Pseudo genes



Genes with function prediction (proteins)



Genes in paralog clusters



Genes assigned to COGs



Genes assigned Pfam domains



Genes with signal peptides



Genes with transmembrane helices



CRISPR repeats


*but five genes for 5S rRNA

Figure 3

Graphical map of the chromosome. From bottom to the top: 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 (black), GC skew (purple/olive).

Table 4

Number of genes associated with the general COG functional categories








       Translation, ribosomal structure and biogenesis




       RNA processing and modification








       Replication, recombination and repair




       Chromatin structure and dynamics




       Cell cycle control, cell division, chromosome partitioning




       Nuclear structure




       Defense mechanisms




       Signal transduction mechanisms




       Cell wall/membrane biogenesis




       Cell motility








       Extracellular structures




       Intracellular trafficking and secretion, and vesicular transport




       Posttranslational modification, protein turnover, chaperones




       Energy production and conversion




       Carbohydrate transport and metabolism




       Amino acid transport and metabolism




       Nucleotide transport and metabolism




       Coenzyme transport and metabolism




       Lipid transport and metabolism




       Inorganic ion transport and metabolism




       Secondary metabolites biosynthesis, transport and catabolism




       General function prediction only




       Function unknown




       Not in COGs

Insights into the genome sequence

The G+C content in the genus Methanoplanus

When calculated from the genome sequences, the G+C content of M. limicola DSM 2279 amounts to 42.2%, whereas the previously published value, determined using traditional (“wet-lab”) techniques, is 47.5% [1]. Similarly, the G+C content of M. petrolearius was given as 50% [42], whereas the analysis of the genome sequence of the type strain SEBR 4837T (DSM 11571) yielded 47.4% [11]. It was frequently stated in the literature that “organisms that differ by more than 10 mol% do not belong to the same genus and that 5 mol% is the common range found within a species” [43]. A recent study [44] has shown that when calculated from genome sequences the G+C content varies at most 1% within species and that larger variances are caused by the limitations of the traditional techniques for analyses. It has thus been recommended to conduct emendations of species descriptions in the case of discrepancies larger than 1%, and to also conduct emendations of genus descriptions if the species emendations yield values that do not fit into the range of the G+C content given in the literature for the respective genus [44].

Considerations about the polyphyletic genus Methanoplanus

The phylogenetic tree presented in Figure 1 shows Methanoplanus as a polyphyletic taxon with the members of Methanomicrobium and Methanolacinia interspersed between the members of Methanoplanus. Given the high bootstrap support for the branches in that section of the phylogenetic tree, this situation calls for some attention, mainly due to the location of M. petrolearius [42]. The conflict between 16S rRNA gene data and the classification is significant, as revealed by the bootstrap values and the paired-site tests described above.

The problematic local structure of the phylogenetic tree might be caused by the fact that most of the five species located in the respective part of the tree were already decribed in the early days of Archaea research when only a limited number of reference sequences were available: M. limicola dates from 1982 [1], M. endosymbiosus from 1986 [45], M. petrolearius from 1997 [42], M. paynteri from 1983 [46] (renamed in 1989 [47]), and Methanomicrobium mobilis even from 1968 [48]. State-of-the-art techniques for the initial taxonomic characterization of the then novel bacteria were much less advanced than today, e.g. Sanger sequencing had just been invented (in 1977) when M. limicola was characterized with DNA-RNA hybridizations as decisive technique [49], and still not yet generally used for taxonomic work when M. endosymbiosus was characterized four years later. When the latest of the three Methanoplanus species with a validly published name, M. petrolearius, was added in 1997 16S rRNA sequences were used, but the ones from M. paynteri (closest neighbor in the phylogenetic tree in Figure 1) and M. mobilis were not yet available or at least not used for comparative analyses [42].

The completion of the Sequencing Orphan Species (SOS) initiative early last year [50], closed the last gaps in the availability of high-quality 16S rRNA reference sequences for phylogenetic trees. However, a decade after the first genome-based investigations into the history of the domain Archaea [51] and the systematic overview of their evolution, physiology, and molecular biology [52], a significant fraction of draft genome sequences as such generated in the genomic Encyclopedia of Bacteria and Archaea [29] are still very much needed to cover all of the diversity of the Archaea, especially from difficult-to-grow organisms and from type strains of remote clades such as the Methanomicrobiaceae.

With all these limitations, a closer inspection of the positions of the members of Methanoplanus in Figure 1 might still be worthwhile. M. petrolearius appears to be clearly separated from the other two members of the genus, M. limicola and M. endosymbiosus, but closely linked to M. paynteri with a 99.8% 16S rRNA gene sequence identity. Table 5 shows a summary of the features of all members of the genera Methanoplanus and Methanolacinia, indicating that based on the higher optimal growth temperature, the lack of observed flagella and observed motility (although the flagellin genes are encoded in the genome), the usage of CO2+2-propanol as a substrate, and the higher G+C content of the genome [42], M. petrolearius clusters rather with M. paynteri than with the other two members of Methanoplanus.

Table 5

Features of the type strains within the genera Methanoplanus and Methanolacinia.

      M. limicola [1] DSM 2279

      M. endosymbiosus [45] DSM 3599

      M. petrolearius [42] DSM 11571

      M. paynteri [46,47] DSM 2545



      marine ciliate

      oil well

      marine sediment

Temperature range (Topt) °C

      17−41 (32)

      16−36 (32)

      28−43 (37)

      unknown (40)


      motile, flagella

      flagella reported



pH range (pHopt)

      ND (6.5−7.5)

      6.1−8.0 (6.8−7.3)

      5.3−8.2 (7.0)


NaCl conc. % (opt.)

      0.4−5.4 (1)

      0−4.5 (1.5)

      0−5 (1−3)

Substrates used

      H2+CO2, formate

      H2+CO2, formate

      H2+CO2, formate, CO2+2-propanol

      H2+CO2, CO2+2-propanol, CO2+2-butanol, CO2+2-cyclopentanol

G+C content

      42.2% genome      (was 47.5% pre-genome)

      38.7% melting curve

      47.4% genome      (was 50% pre-genome)

      44.8% buoyant density

Although the genome sequence of M. petrolearius SEBR 4847T (DSM 11571) was recently published [11], the one for Ml. paynteri was still lacking, as well as information about a wet lab DNA-DNA hybridization (DDH) between the type strains of the two species. Given the high degree of 16S rRNA sequence identity between the two strains (99.8%), established thresholds of species delimitations, 97% [53], even under recently published relaxed recommendations, 98.2−99% [54], definitely demands such an analysis for the purpose of species discrimination. Whereas the rather large difference of 2.6% in the G+C content of the two genomes (Table 5, based on currently available mixed data from genome sequence and buoyant density measurement) predicts a rather low DDH value as the outcome of such an experiment, the recently observed significant deviations between previously published G+C values and G+C values inferred from genome sequences [44] do not, however, allow for definitive conclusions from the difference in G+C values.

For this reason, we have obtained a draft genome sequence for M. paynteri DSM 2546T using Illumina-MiSeq as a sequencing platform in order to obtain paired-end reads of 250 bp and Velvet [35] for the assembly. The draft genome comprised 54 contigs and is available from NCBI under the accession number AXDV00000000 and from IMG under the object ID pending. Digital DDH similarities between Ml. paynteri DSM 2546T (AXDV00000000) and M. petrolearius SEBR 4847T (DSM 11571, CP002117) were calculated with the GGDC web server [55,56] version 2.0 [57] under the recommended settings. The inter-genomic distance (formula 2) was 0.0753, corresponding to a DDH estimate of 48.50% ± 2.61%. The probability of a DDH value > 70% was accordingly only 0.1514.

In conclusion, from the topology of 16S rRNA gene sequence-based phylogenetic tree supported by the distribution of the characteristic features listed in Table 4 we can conclude that strain SEBR 4847T should rather be classified as a member of the genus Methanolacinia than as M. petrolearius, whereas the digital DDH results clearly indicate that Ml. paynteri (represented by the type strain G-2000, DSM 2545) and M. petrolearius (represented by the type strain SEBR 4847, DSM 11571) are distinct species. Thus, we propose Methanolacinia petrolearia comb. nov. to accommodate M. petrolearius, with SEBR 4847 being the type strain.

The situation between M. limicola (type species of Methanoplanus) and M. endosymbiosus is only slightly better than the relationship between M. paynteri and M. petrolearius discussed above. Based on the above reported Greengenes analysis the 16S rRNA gene sequences of the two type strains show 99.5% sequence identity and an HSP coverage of 99.7%. Again, by all accepted standards of species discrimination [53,54] such a close similarity would call for a DDH experiment to resolve the close relationship, but such data are not available. Also a digital DDH cannot be performed because only the genome sequence of M. limicola presented here is available, but not that of DSM 3599, the type strain of M. endosymbiosus. Table 5 indicates that the two strains share almost all of the listed features (except habitat), except for a 3.5% difference in the G+C content, which, in case it would be confirmed and not biased by a technical artifact in the melting curve measurement done for M. endosymbiosus, indicated a sufficiently low level of DDH to distinguish the two species [44]. Nevertheless, the probability that the digital DDH value between the two type strains might surpass the 70% species discrimination (once the genome sequence of M. endosymbiosus is resolved) threshold cannot be neglected. It might be too early to draft the obituary for M. endosymbiosus, but it is better to be prepared in case the once trispecific polyphyletic genus Methanoplanus becomes monospecific, an event that may occur once the drafts of all needed type strain genomes (the core objective of GEBA) are deciphered. Depending on the availability of enough cell material, M. endosymbiosus should now be scheduled as a sequencing target for the upcoming phases the GEBA, e.g. the Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes (KMG-I) projects [58], to resolve the question about the exact relationship between M. limicola and M. endosymbiosus.

Taxonomic consequences

As explained in detail above, the differences in the reported G+C contents of M. limicola and M. petrolearius to the ones calculated from their genome sequences justifies an emendation of the species descriptions. Moreover, M. petrolearius should be placed within the genus Methanolacinia. The descriptions of the two genera should be emended accordingly.

Emended description of the species Methanoplanus limicola Wildgruber et al. 1982

The description of the species Methanoplanus limicola is the one given by Wildgruber et al. 1982 [1], with the following modification.

The G+C content is 42%.

Emended description of the species Methanoplanus petrolearius Ollivier et al. 1997

The description of the species Methanoplanus petrolearius is the one given by Ollivier et al. 1997 [42], with the following modification.

The G+C content is 47%.

Description of Methanolacinia petrolearia, comb. nov.

Basonym: Methanoplanus petrolearius Ollivier et al. 1997

The description of the species is the same as given for Methanoplanus petrolearius Ollivier et al. 1997 with the emendation given above.

Emended description of the genus Methanoplanus

The description is the one given by Wildgruber et al. [1] with the following modifications:

The G+C content is 39-42%.

Emended description of the genus Methanolacinia

The description is the one given by Zellner et al. [47] with the following modifications:

The G+C content is 45-47%.



We would like to gratefully acknowledge the help of Evelyne-Marie Brambilla (DSMZ) for DNA extractions and quality control. This work was performed under the auspices of the US Department of Energy 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, UT-Battelle and Oak Ridge National Laboratory under contract DE-AC05-00OR22725, as well as German Research Foundation (DFG) INST 599/1-2. AL was in part supported by the Russian Ministry of Science Mega-grant no.11.G34.31.0068; SJ O'Brien Principal Investigator. The Council of Scientific and Industrial Research (CSIR, India) and DAAD, Germany, provided a Fellowship to Shanmugam Mayilraj.

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.


  1. Wildgruber G, Thomm M, König H, Ober K, Rucchiuto T and Stetter KO. Methanoplanus limicola, a plate-shaped methanogen representing a novel family, the Methanoplanaceae. Arch Microbiol. 1982; 132:31-36 View Article
  2. . Validation List no. 14. Validation of the publication of new names and new combinations previously effectively published outside the IJSB. Int J Syst Bacteriol. 1984; 34:270-271 View Article
  3. Walsby EA. A square bacterium. Nature. 1980; 283:69-71 View Article
  4. Huber H, Huber G, Stetter KO. Genus VI. Methanoplanus Wildgruber, Thomm and Stetter. 1984, 270VP (Effective publication: Wildgruber, Thomm and Stetter in Wildgruber, Thomm, König, Ober, Ricchiuto and Stetter 1982, 36). In: Boone DR, Castenholz RW, Garrity (eds): Bergey's Manual of Systematic Bacteriology, second edition, vol. 1 (The Archaea and the deeply branching and phototrophic Bacteria), Springer-Verlag, New York, 2001, pp. 259-261..
  5. Ivanov VN and Stabnikova EV. Use of the level of G+C n DNA for studying the molecular phylogeny of methanogenic archaebacteria. [in Russian] Mikrobiologiia. 1999; 68:710-715PubMed
  6. Liu CT, Miyaki T, Aono T and Oyaizu H. Evaluation of methanogenic strains and their ability to endure aeration and water stress. Curr Microbiol. 2008; 56:214-218 View ArticlePubMed
  7. Göker M, Lapidus A, Spring S, Yasawong M, Lucas S, Glavina del Rio T, Chen F, Tice H, Cheng JF and Goodwin LA. Complete genome sequence of Ignisphaera aggregans type strain (AQ1.S1T). Stand Genomic Sci. 2010; 3:66-75 View ArticlePubMed
  8. Stamatakis A, Hoover P and Rougemont J. A rapid bootstrap algorithm for the RAxML web servers. Syst Biol. 2008; 57:758-771 View ArticlePubMed
  9. Swofford DL. PAUP*: Phylogenetic Analysis Using Parsimony (*and Other Methods), Version 4.0 b10. Sinauer Associates, Sunderland, 2002.
  10. Pagani I, Liolios K, Jansson J, Chen IM, Smirnova T, Nosrat B, Markowitz VM and Kyrpides NC. The Genomes OnLine Database (GOLD) v.4: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res. 2012; 40:D571-D579 View ArticlePubMed
  11. Brambilla E, Ngatchou-Djao OD, Daligault H, Lapidus A, Lucas S, Hammon N, Nolan M, Tice H, Cheng JF and Han C. Complete genome sequence of Methanoplanus petrolearius type strain (SEBR 4847T). Stand Genome Sci. 2010; 2:203-211 View Article
  12. Anderson IJ, Sieprawska-Lupa M, Lapidus A, Nolan M, Copeland A, Glavina del Rio T, Tice H, Dalin E, Barry K and Saunders E. Complete genome sequence of Methanoculleus marisnigri Romesser et al 1981 type strain JR1. Stand Genomic Sci. 2009; 1:189-196 View ArticlePubMed
  13. Anderson IJ, Sieprawska-Lupa M, Goltsman E, Lapidus A, Copeland A, Glavina del Rio T, Tice H, Dalin E, Barry K and Pitluck S. Complete genome sequence of Methanocorpusculum labreanum type strain Z. Stand Genomic Sci. 2009; 1:197-203 View ArticlePubMed
  14. Anderson I, Ulrich LE, Lupa B, Susanti D, Porat I, Hooper SD, Lykidid 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
  15. Abt B, Han C, Scheuner C, Lu M, Lapidus A, Nolan M, Lucas S, Hammon N, Deshpande S and Cheng JF. Complete genome sequence of the termite hindgut bacterium Spirochaeta coccoides type strain (SPN1T), reclassification in the genus Sphaerochaeta as Sphaerochaeta coccoides comb. nov. and emendations of the family Spirochaetaceae and the genus Sphaerochaeta. Stand Genomic Sci. 2012; 6:194-209 View ArticlePubMed
  16. 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
  17. Field D, Amaral-Zettler L, Cochrane G, Cole JR, Dawyndt P, Garrity GM, Gilbert J, Glöckner FO, Hirschman L and Karsch-Mzrachi I. The Genomic Standards Consortiu.m PLoS Biol. 2011; 9:e1001088 View ArticlePubMed
  18. Woese CR, Kandler O and Wheelis ML. Towards a natural system of organisms. Proposal for the domains Archaea and Bacteria. Proc Natl Acad Sci USA. 1990; 87:4576-4579 View ArticlePubMed
  19. Garrity GM, Holt JG. Phylum AII. Euryarchaeota phy. nov. In: Garrity GM, Boone DR, Castenholz RW (eds), Bergey's Manual of Systematic Bacteriology, Second Edition, Volume 1, Springer, New York, 2001, p. 211-355.
  20. Garrity GM, Bell JA, Lilburn T. Taxonomic outline of the Procaryotes. In: Garrity GM, Bell JA, Lilburn TG (eds), Taxonomic Outline of the Procaryotes, Bergey's Manual of Systematic Bacteriology, Second Edition. Release 4.0, Fourth Edition, Springer-Verlag, New York, 2003, p. 1-39.
  21. Balch WE, Fox GE, Magrum LJ, Woese CR and Wolfe RS. Methanogens: reevaluation of a unique biological group. Microbiol Rev. 1979; 43:260-296PubMed
  22. . Validation List no. 6. Validation of the publication of new names and new combinations previously effectively published outside the IJSB. Int J Syst Bacteriol. 1981; 31:215-218 View Article
  23. Garrity GM, Labeda DP and Oren A. Judicial Commission of the International Committeeon Systematics of Prokaryotes; XIIth International (IUMS) Congress of Bacteriology and Applied Microbiology. Minutes of the meetings, 3, 4 and 6 August 2008, Istanbul, Turkey. Int J Syst Evol Microbiol. 2011; 61:2775-2780 .View Article
  24. . The nomenclatural types of the orders Acholeplasmatales, Halanaerobiales, Halobacteriales, Methanobacteriales, Methanococcales, Methanomicrobiales, Planctomycetales, Prochlorales, Sulfolobales, Thermococcales, Thermoproteales and Verrucomicrobiales are the genera Acholeplasma, Halanaerobium, Halobacterium, Methanobacterium, Methanococcus, Methanomicrobium, Planctomyces, Prochloron, Sulfolobus, Thermococcus, Thermoproteus and Verrucomicrobium, respectively. Opinion 79. Int J Syst Evol Microbiol. 2005; 55:517-518 .View ArticlePubMed
  25. Euzéby JP and Tindall BJ. Nomenclatural type of orders: corrections necessary according to Rules 15 and 21a of the Bacteriological Code (1990 Revision), and designation of appropriate nomenclatural types of classes and subclasses. Request for an opinion. Int J Syst Evol Microbiol. 2001; 51:725-727PubMed
  26. BAuA. 2010, Classification of bacteria and archaea in risk groups. TRBA 466, p. 131.Web Site
  27. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS and Eppig JT. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000; 25:25-29 View ArticlePubMed
  28. Klenk HP and Göker M. En route to a genome-based classification of Archaea and Bacteria? Syst Appl Microbiol. 2010; 33:175-182 View ArticlePubMed
  29. Wu D, Hugenholtz P, Mavromatis K, Pukall R, Dalin E, Ivanova NN, Kunin V, Goodwin L, Wu M and Tindall BJ. A phylogeny-driven Genomic Encyclopaedia of Bacteria and Archaea. Nature. 2009; 462:1056-1060 View ArticlePubMed
  30. Mavromatis K, Land ML, Brettin TS, Quest DJ, Copeland A, Clum A, Goodwin L, Woyke T, Lapidus A and Klenk HP. The fast changing landscape of sequencing technologies and their impact on microbial genome assemblies and annotation. PLoS ONE. 2012; 7:e48837 View ArticlePubMed
  31. List of growth media used at DSMZ: Web Site
  32. Gemeinholzer B, Dröge G, Zetzsche H, Haszprunar G, Klenk HP, Güntsch A, Berendsohn WG and Wägele JW. The DNA Bank Network: the start from a German initiative. Biopreserv Biobank. 2011; 9:51-55 View ArticlePubMed
  33. . Web Site
  34. Phrap and Phred for Windows. MacOS, Linux, and Unix. Web Site
  35. Zerbino DR and Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008; 18:821-829 View ArticlePubMed
  36. Han C, Chain P. Finishing repeat regions automatically with Dupfinisher. In: Proceedings of the 2006 international conference on bioinformatics & computational biology. Arabnia HR, Valafar H (eds), CSREA Press. June 26-29, 2006: 141-146.
  37. Lapidus A, LaButti K, Foster B, Lowry S, Trong S, Goltsman E. POLISHER: An effective tool for using ultra short reads in microbial genome assembly and finishing. AGBT, Marco Island, FL, 2008.
  38. Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW and Hauser LJ. Prodigal Prokaryotic Dynamic Programming Genefinding Algorithm. BMC Bioinformatics. 2010; 11:119 View ArticlePubMed
  39. Mavromatis K, Ivanova NN, Chen IM, Szeto E, Markowitz VM and Kyrpides NC. The DOE-JGI Standard operating procedure for the annotations of microbial genomes. Stand Genomic Sci. 2009; 1:63-67 View ArticlePubMed
  40. Pati A, Ivanova N, Mikhailova N, Ovchinikova G, Hooper SD, Lykidis A and Kyrpides NC. GenePRIMP: A Gene Prediction Improvement Pipeline for microbial genomes. Nat Methods. 2010; 7:455-457 View ArticlePubMed
  41. Markowitz VM, Ivanova NN, Chen IMA, Chu K and Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics. 2009; 25:2271-2278 View ArticlePubMed
  42. Ollivier B, Cayol JL, Patel BKC, Magot M, Fardeau ML and Garcia JL. Methanoplanus petrolearius sp. nov., a novel methanogenic bacterium from an oil-producing well. FEMS Microbiol Lett. 1997; 147:51-56 View ArticlePubMed
  43. Rossello-Mora R and Amann R. The species concept for prokaryotes. FEMS Microbiol Rev. 2001; 25:39-67 View ArticlePubMed
  44. Meier-Kolthoff JP, Klenk HP and Göker M. Taxonomic use of the G+C content and DNA:DNA hybridization in the genomic age. Int J Syst Evol Microbiol. 2014; 64:352-256.. View ArticlePubMed
  45. van Bruggen JJA, Zwart KB, Hermans JGF, van Hove EM, Stumm CK and Vogels GD. Isolation and characterization of Methanoplanus endodymbiosus sp. nov., an endosymbiont of the marine sapropelic ciliate Metopus contortus Quennerstedt. Arch Microbiol. 1986; 144:367-374 View Article
  46. Rivard CJ, Henseon JM, Thomas MV and Smith PH. Isolation and characterization of Methanobacterium paynteri sp. nov., a mesophilic methanogen isolated from marine sediments. Appl Environ Microbiol. 1983; 46:484-490PubMed
  47. Zellner G, Messner P, Kneifel H, Tindall BJ, Winter J and Stackebrandt E. Methanolacinia gen nov., incorporating Methanomicrobium paynteri as Methanolacinia paynteri comb. nov. J Gen Appl Microbiol. 1989; 35:185-202 View Article
  48. Paynter MJB and Hungate RE. Characterization of Methanobacterium mobilis, sp. n., isolated the bovine rumen. J Bacteriol. 1968; 95:1943-1951PubMed
  49. Tu JK, Prangishvili D, Huber H, Wildgruber G, Zillig W and Stetter KO. Taxonomic relations between archaebacteria including 6 novel genera examined by cross hybridizations of DNA and 16s rRNA. J Mol Evol. 1982; 18:109-114 View ArticlePubMed
  50. Yarza P, Spröer C, Swiderski J, Mrotzek N, Spring S, Tindall BJ, Gronow S, Pukall R, Klenk HP and Lang E. Sequencing orphan species initiative (SOS): Filling the gaps in the 16S rRNA gene sequence database for all species with validly published names. Syst Appl Microbiol. 2013; 36:69-73 View ArticlePubMed
  51. Forterre P, Gribaldo S, Brochier-Armanet C. Natural history of the archaeal domain. In: RA Garrett & HP Klenk (eds) Archaea: evolution, physiology, and molecular biology. Wiley-Blackwell 2007, pp 17-28.
  52. Garrett RA. HP Klenk HP (eds) Archaea: evolution, physiology, and molecular biology. Wiley-Blackwell 2007.
  53. Stackebrandt E and Goebel BM. Taxonomic note: a place for DNA-DNA reassiciations and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol. 1994; 44:846-849 View Article
  54. Meier-Kolthoff JP, Göker M, Spröer C and Klenk HP. When should a DDH experiment be mandatory in microbial taxonomy? Arch Microbiol. 2013; 195:413-418 View ArticlePubMed
  55. Auch AF, Klenk HP and Göker M. Standard operating procedure for calculating genome-to-genome distances based on high-scoring segment pairs. Stand Genomic Sci. 2010; 2:142-148 View ArticlePubMed
  56. Auch AF, Von Jan M, Klenk HP and Göker M. Digital DNA-DNA hybridization for microbial species delineation by means of genome-to-genome sequence comparison. Stand Genomic Sci. 2010; 2:117-134 View ArticlePubMed
  57. Meier-Kolthoff JP, Auch AF, Klenk HP and Göker M. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics. 2013; 14:60 View ArticlePubMed
  58. Kyrpides NC, Wojke T, Eisen JA, Garrity GM, Whitman WB, Hugenholtz P and Klenk HP. Genomic encyclobedia of type strains, phase I: the one thousand microbial genomes (KMG-I) project. Stand Genomic Sci. . 2013; 9:628-634