Functional genomics of Methylococcus capsulatus

Johan R. Lillehaug

UoB

Science
    Faculty

MBI

Research
- Fjose
- Fladmark
- Goksøyr
- Helland
- Jensen
- Lillehaug
- Male
- Raae
- Seo
- Ziegler
- Walther
- Aasland

1. Functional genomics of the methane-oxidising bacterium Methylococcus capsulatus (Bath): – protein expression and functions.
[Joint project with prof. Harald B. Jensen]

The project is a continuation of the M. capsulatus genome sequencing collaboration (Project 140785/420 “Gas and biotechnology (GABI)”), supported by the Norwegian Research Council and the University of Bergen. At the University of Bergen a combined expertise in bioinformatics, microbiology, bacterial metabolism, gene expression, protein structure/function and enzymology, and general molecular biology, now addresses the functional genomics of M. capsulatus, and we collaborate with leading international groups.

Methanotrophs are a group of gram-negative bacteria with the ability to use methane as a sole source of carbon and energy. The simple growth requirements of methanotrophs make them attractive as sources for single-cell protein and bulk chemicals. They also have a important potential in bioremediation and heterologous protein expression. 

Methylococcus capsulatus (Bath) is the main constituent of Bioprotein, a single cell protein product based on natural gas. Bioprotein is produced in a novel fermentor constructed by Norferm DA (http://www.norferm.com) at Tjeldbergodden in Møre og Romsdal, supplied with gas from the Heidrun gasfield in the Åsgård – Heidrun structure, and represents the first product to be commercialized from a methanotroph.

The metabolism of methane gas to biomass by Methylococcus capsulatus (Bath) is based on the expression of the enzyme methane monooxygenase (MMO), which oxidises methane to methanol. There are two different MMOs expressed in M. capsulatus, one soluble enzyme (sMMO) and one membrane-bound enzyme (pMMO). The expression of these two enzymes appears to be controlled by the concentration of copper ions in the growth medium. Important in relation to the carbon and nitrogen metabolism is the uptake of nutrients, including important metal ions, into the cell.  Proteins involved in this process are located/associated to the surface-side of the outer membrane, in the outer membrane, in the periplasmic space, in the inner membrane and in the cytoplasm. The copper-regulation of the methane monooxygenases occurs both at the DNA and protein level (reviewed by Murrell et al. 2000), and copper ions also regulates the production of the extensive intracellular membranes of the bacterium (Prior and Dalton 1985). The bacterium appears to have multiple pathways involved in the metabolism of methane and duplications of several of the important enzyme encoding genes in the methane oxidation pathways, so-called genomic redundancy (Ward et al. 2004). The expression of several of these genes appears also to be depending on the availability of copper ions (4, 9, and 12).

Our long term aim is to analyse the metal ion regulation of enzyme activities and protein expression, in particular the copper ion homeostasis. At present, we are particularly engaged in understanding how outer membrane proteins and surface associated proteins are involved in the uptake of copper ions and how the copper level affects regulation of gene expression at the transcription level.

 It is an integrated part of our proteomic research to study transport proteins and the protein translocation machinery responsible for the transport of the involved proteins to the various compartments of the cell. Regulator proteins will also be included. Knowledge of such translocation systems and regulator proteins would appear to be of great importance for biotechnological utilisation of the organism. At present we have revealed that at least 50 proteins are being influenced by a change in copper concentration in the medium above and below a critical concentration of about 0.8 μM Cu++.

Until quite recently (Fjellbirkeland et al. 1997, 2000, 2001) nothing was known about the proteins of the outer membrane of M. capsulatus. 2D-PAGE has revealed some 100 different polypeptide-spots in the outer membrane, and we have now analysed the most abundant by mass spectrometry (8). We have also established 2D-PAGE for the soluble proteins of M. capsulatus, and analysed many of these, in particular those that are regulated by the copper level of the growth medium (9).

References (M. capsulatus project):

1. Fjellbirkeland A, Kleivdal H, Joergensen C, Thestrup H, Jensen HB.1997. Outer membrane proteins of Methylococcus capsulatus (Bath). Arch Microbiol 168:128-135
2. Fjellbirkeland A, V. Bemanian, I. R. McDonald, J. Colin Murrell, H. B. Jensen. 2000. Molecular analysis of an outer membrane protein, MopB, of Methylococcus capsulatus (Bath) and structural comparisons with proteins of the OmpA family. Archives of Microbiology.173:346-351.
3. Fjellbirkeland A, Kruger PG, Bemanian V, Høgh BT, Murrell JC, Jensen HB. 2001. The C-terminal part of the surface-associated protein MopE of the methanotroph Methylococcus capsulatus (Bath) is secreted into the growth medium. Archives of Microbiology.176:197-203.
4. Odd Andre Karlsen, Frode Berven,  Graham Stafford, Øivind Larsen, J. Colin Murrell, Harald B. Jensen, Anne Fjellbirkeland, 2003. The surface-associated and secreted MopE protein of the Methyloccus capsulatus (Bath) responds to changes in the concentration of copper in the growth medium. Applied and Environmental  Microbiology. 69(4):2386-2388.
5. Berven FS, Karlsen OA, Murrell JC, Jensen HB (2003) Multiple polypeptide forms observed in two-dimensional gels of Methylococcus capsulatus (Bath) polypeptides are generated during the separation procedure. Electrophoresis  24, 757-761
6. Berven FS, Flikka K, Jensen HB, Eidhammer I (2004) BOMP: A program to predict integral β-barrell outer membrane proteins encoded within genomes of gram-negative bacteria. Nucleic acid research 32 Web Server issue.
7. Naomi Ward, Larsen Ø, Sakwa J., Bruseth L., Khouri H., Durkin S., Dimitrov G., Jiang L:, Scanlan D., Kang K., Lewis M., Nelson K., Methe B., Wu M., Heidelberg J.F., Paulsen I.T., Fouts D., Ravel J., Trettelin H., Ren Q., Read T., DeBoy R., Seshadri R., Salzberg SL., Jrnsen H.B., Birkeland N.K., Nelson W., Dodson R.J., Grindhaug S.H., Holt I., Eidhammer I., Jonassen I., Vanaken S., Utter back T., Feldbluyim T.M., Fraser C.M., Lillehaug J.R., Eisen J.A. 2004. Genomic Insights into Methanotrophy: The Complete Genome Sequence of Methylococcus (Bath). PLoS Biology http://www.plosbiology.org/ Oct 2004 vol 2 issue 10.
8. Murrell JC, McDonald IR, Gilbert B (2000) Regulation of expression of methane monooxygenase by copper ions. Trends Microbiol 8: 221-225.
9. Karlsen OA, Ramsevik L, Bruseth L, Larsen Ø, Jensen HB, Lillehaug JR. 2005 Characterization of a prokaryotic hemerythrin from the methanotrophic bacterium Methylococcus capsulatus (Bath). FEBS Journal 272, 1-13.
10. Berven FS, Karlsen OA, Straume AH, Flikka K, Murrell JC, Fjellbirkeland A, Lillehaug JR, Eidhammer I, Jensen HB, (2004) Analysing the outer membrane subproteome of Methylococcus capsulatus using proteomics and novel biocomputing tools (PhD thesis Frode Berven, to be submitted).
11. Bruseth L et al. (2005). Exploring the essential copper-switch in Methylococcus capsulatus (Bath). Regulation of genes by copper depletion. In Preparation.
12. Bruseth L et al. (2005). A novel surface associated protein complex in Methylococcus capsulatus (Bath) senses environmental changes in metal ion compositions. In Preparation.
13. Bruseth L et al. (2005). Copper homeostasis in Methylococcus capsulatus (Bath). Leave them or love them: An intrinsic duality in response to copper ions elucidates the issue of copper homeostasis and toxicity. In Preparation.