Methylomonas: Classification, Detection and Impact in our Environment

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Methylomonas: Classification, Detection and Impact in Our EnvironmentAcielle Angeli C. Garcera

Institute of Biology, College of Science, University of the Philippines Diliman

Methanotrophs to which Methylomonas belong, are obligately methylotrophic bacteria

which utilizes methane as their primary carbon and energy source2. They are a subset of a larger

group of bacteria – the methylotrophs which encompass all microbes who can utilize one-carbon

compounds more reduced than CO23,5

. They can be divided into three groups – the group I

methanotrophs belonging to either the - or -Proteobacteria, the group II methanotrophs

belonging to -Proteobacteria and group X belonging to -Proteobacteria5. Initial classifications

of Whittenbury et al. (1970) delegated methanotrophs into either group I or group X based on

their morphology, fine structure and intracytoplasmic membranes15. Knowledge of the carbon

assimilation pathways of these groups of bacteria further strengthened the distinction between

groups I and II methanotrophs and added another group – group X3.

The genus, Methylomonas, belong to the group I methanotrophs specifically the subgroup

A which employs the RuMP pathway for formaldehyde formation5. They are Gram-negative

bacteria whose difficulty to be isolated into pure cultures and whose lack of obvious phenotypic

differences with other methanotrophic bacteria makes them hard to classify. Methylomonas are

further divided into two clusters: those that contain carotenoid pigments and those that are

mesophilic with a G+C content quite similar to a sub-group of Methylococcus (48-55 mol %)2.

However, extensive studies of Bowman, et al. where they employed extensive phenotypic

surveys, DNA hybridization techniques and phospholipid fatty acid (PFLA) analysis has shown

that of the two clusters of Methylomonas only the ones that contained carotenoids can be truly

considered as belonging to the genus Methylomonas. The other cluster is more closely related to



Methylococcus. These carotenoid-containing Methylomonas are: M. methanica, M. aurantiaca

and M. fodinarum2.

Methylomonas are described as rodlike to coccobacillary in shape with dimensions 0.5-

1.0 µm in width and 1.0 – 2.0 µm in length. They are motile with a single flagellum and possess

pink and orange carotenoids. They are capsulated mesophiles growing at an optimum

temperature of 25-35°C, optimum pH of 6.5-7 and less than 1.5% NaCl2.

In the oxidation of methane, CO2 must first be converted to methanol by methane

monooxygenase (MMO) which has two forms: a particulate membrane bound form (pMMO)

found to be present in all methanotrophs except for one genus and a soluble cytoplasmic form

(sMMO) present mostly in group II and X methanotrophs and very few group I strains one of

which is M. methanica strain 68-1, the first group I methanotroph discovered to have this

enzyme8,10,11. The structural differences of the two enzymes have implications on their ecological

roles in soil methane oxidation where sMMOs oxygen requirements which are much lower than

those of pMMOs (17 micro M for sMMO; 0.1 miro M for pMMOs) imply that methanotrophs

with sMMOs can compete for methane better in deep soils where there is low oxygen tension10

.

The possible occurrence of Methylomonas in deeper soil levels may account for its rarity

in being isolated from root-associated soils but this is not conclusive since group II

methanotrophs are found to be the frequent isolates from these areas4,5,7

. However, investigations

of the root microflora of freshwater macrophytes Pontederia cordata, Sparganium eurycarpum

and Sagittaria latifolia revealed Methylomonas strains from the three well-established

Methylomonas species ( M. methanica, M. aurantiaca and M. fodinarum)4. Also, Methylomonas



methanica was isolated from submerged rice roots6. Thus the distribution of Methylomonas –

whether more abundantly in deeper or shallower soils merits more attention.

In the detection of these bacteria, several assays have been developed. Among them is the

indirect fluorescent antibody staining technique which permits the autecological study of

Methylomonas methanica revealing its distribution to be highly concentrated in the deeper parts

of the aquatic sediments. Although highly effective in soil samples, the tecnhique resulted in low

recovery rates for M. methanica from water samples. Despite these dismal rates, it is still

considered accurate in providing a relative if not actual concentrations of M. methanica due to

the consistency of the results obtained 13. More common approaches involved the development of

gene probes and molecular markers such as the general gene probe mxaF (encodes the subunit

of methanol dehydrogenase) which can discriminate between methanotrophs and other gram-

negative methylotrophs but whose highly conservative nature amongst the methanotrophs makes

it infeasible for the formation of group-specific or genus-specific probes12

. A more accurate

probe would be the use of the pmoA which encodes for the subunit of pMMOs and is thus

found only in methanotrophs and absent from other methylotrophs. The combination of this gene

probe with PCR techniques as well as other sub-group assays allow for the detection of the

diversity of methanotrophs in soil samples. Methylomonas was found to be positive to only one

of the specific sub-groups assays coupled with real-time PCR analysis9. The phylogenetic results

of the pmoA gene probe is highly similar to those produced by the 16s rRNA analysis further

implicating its validity. To observe these organisms in situ, stable isotope probing (SIP) may be

employed. It was through this method that type I methanotrophs were found to be actively

assimilating methane in high pH environment (around pH 10)11.



The detection of methanotrophs in the environment is important for the determination of

their roles in bioremediation and global warming. The increasing abundance of methane in the

atmosphere may be an important factor in global warming since it absorbs more infrared

radiation than CO23,5. The role of methanotrophs in the carbon cycle should then be extensively

studied since knowledge of such may lead to preventive measures for extreme global warming.

Methanotrophs’ coversion of methane into CO2 can be of two paths: assimilatory in which the

CO2 is released into the environment entirely and dissimilatory in which CO2 is absorbed by the

body as part of its biomass10

. The former contributes greatly to global warming and although the

path they utilize is mostly dissimilatory thus reducing the greenhouse gases it would still be

beneficial if studies to ascertain the entirety of such would be made and if possible how the

methanotrophs following the assimilatory path can be converted to the other 10

. Methanotrophic

bacteria whose main habitat is methane’s primary sink, the soil, can assimilate only as much as

10 Tg/year of the 520 Tg/per total atmospheric methane of which a significant but rarely

measured amount comes from gas emissions of human activities5. It would then be prudent,

faced with the current conditions of the environment, that apart from studying how the activities

of methanotrophic bacteria can be enhanced, more efforts be placed into acquiring the necessary

numbers of methane production and increasing awareness of the general public to the said issues.

Apart from global warming, methanotrophs can also be useful in bioremediation. Eversince the

discovery of sMMOs in Methylomonas methanica, its potential use in the in-situ degradation of

TCE attracted attention1,8,14

. Its abiliy to rapidly degrade TCE, one of the most common

contaminants of groundwater, even with limited amounts of copper required for the optimum

functioning of sMMOs has led scientists to characterize it and explore its extensive use in

bioremediation especially in situ ones8,14

.



As new ways of determining phylogenetic characteristics of microorganisms are

developed, the classification of Methylomonas may be shifted yet again. The universality of its

energy source implies that although its detection can be tricky, it is ubiquitous enough – found

on both terrestrial and aquatic environments. Its importance in critical environmental processes

cannot be denied and thus studies specializing in this as well as efforts in isolating and further

characterizing this methanotroph should further be intensified as this could determine the future

outcome of the world we live in.

References:

1.

Auman, A.J., C.C. Speake, and M.E. Lidstrom. 2001. nifH sequences and nitrogen fixation in type I antype II methanotrophs. Appl. Environ. Microbiol. 67(9):4009-4016.

2. Bowman, J.P., L.I. Sly, P.D. Nichols, and A.C. Hayward. 1993. Revised taxonomy of the

methanotrophs: description of Methylobacter gen. nov., emendation of Methylococcus, validation of

Methylosinus and Methylocystis species, and a proposal that the Family Methylococcaceae includes only

group I methanotrophs. Int. J. Syst. Bacteriol. 43(4):735-753.

3. Brusseau, G.A., E.S. Bulygina, and R.S. Hanson. 1994. Phylogenetic analysis and development of

probes for differentiating methylotrophic bacteria. Appl. Environ. Microbiol. 60(2):626-6364. Calhoun, A. and G.M. King. 1998. Characterization of root-associated methanotrophs from three

freshwater macrophytes: Pontederia cordata, Sparganium eurycarpum and Sagittaria latifolia. Appl.

Environ. Microbiol. 64(3):1099-1105.5. Hanson, R.S. and T.E. Hanson. 1996. Methanotrophic Bacteria. Microbiol. Rev. 6(2):439-471

6. Horz, H.P., M.T. Yinga and W. Liesack. 2001. Detection of methanotroph diversity on roots of

submerged rice plants by molecular retrieval of pmoA-based terminal restriction fragment length

polymorphism profiling. Appl. Environ. Microbiol. 67:4177-4185.

7. King, G.M. 1994. Association of methanotrophs with roots and rhizomes of aquatic vegetation Appl.

Environ. Microbiol. 60:3220-3227.

8. Koh, S-C, J.P. Bowman, and G.S. Sayler. 1993. Soluble methane monooxygenase production and

trichloroethylene degradation by a type I methanotroph, Methylomonas methanica 68-1. Appl. Environ.

Microbiol. 59(4):960-9679. Kolb, S., C. Knief, S. Stubner, and R. Conrad. 2003. Quantitative detection of methanotrophs in soil by

novel pmoA-targeted real-time PCR assays. Appl. Environ. Microbiol. 69(5):2423-2429.

10. Mancinelli, R. L. 1995. The regulation of methane oxidation in soil. Annual Review of Microbiology11. McDonald, I.R., L. Bodrossy, Y. Chen, and J.C. Murrell. 2008. Molecular ecology techniques for the

study of aerobic methanotrophs. 74(5):1305-1315.

12. McDonald, I.R. and J.C. Murrell. 1997. The methanol dehydrogenase structural gene mxaF and its use as

functional gene probe for methanotrophs and methylotrophs. Appl. Environ. Microbiol. 63(8):3218-3224.

13. Reed, W.M. and P.R. Rugan. 1978. Distribution of Methylomonas methanica and Methylosinustrichosporium in Cleveland Harbor as determined by an indirect fluorescent antibody-membrane filter

technique. Appl. Environ. Microbiol. 35(2):422-430.

14. Shigematsu T., S. Hanada, M. Eguchi, Y. Kamagata, T. Kanagawa, and R. Kurane. 1999. Solublemethane monooxygenase gene clusters from trichloroethylene-degrading Methylomonas sp. strains and

detection of methanotrophs during in situ bioremediation. Appl. Environ. Microbiol. 65(12):5198-5206.

15. Whittenbury, R., K.C. Phillips, and J.F. Wilkinson. 1970. Enrichment, isolation and some properties of

methane utilizing bacteria. J. Gen. Microbiol. 61:205-218

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Methylomonas: Classification, Detection and Impact in our Environment