Ecophysiological characterization of Crenothrix polyspora

Methane-oxidizing bacteria (MOB) represent a functional guild of ubiquitous microorganisms that are able to use the greenhouse gas methane as electron donor and carbon source. MOB play a major role in global carbon cycling and represent nature`s largest biological sink for atmospheric methane, which is the most abundant organic gas in the atmosphere. Except of Methylocella silvestris, which represents a facultative methanotroph, all known MOB are obligate methanotrophs and are not able to grow on carbon sources containing C-C bonds. Using cultivation-independent techniques, we recently identified and functionally characterized a very unusual methane oxidizer: Crenothrix polyspora (Proc. Natl. Acad. Sci. USA 7: 2363-2367 (Track II)). This, yet not cultivable, filamentous microorganism has been described already in 1870 by Ferdinand Cohn. C. polyspora has a complex life cycle and is infamous for occasional mass development in drinking water treatment facilities. However, despite the technical importance of C. polyspora, the identity and ecophysiology of this microorganism remained unresolved for more than a century. The main reason for this long-lasting gap of knowledge about C. polyspora is that until now, all attempts to culture this microorganism failed, so that it cannot be studied by the traditional cultivation-dependent approaches of microbiology. Without depending on cultivation based approaches, we were able to identify C. polyspora as a novel Gammaproteobacterium closely related to previously known type I methanotrophs. Consistent with this finding we could demonstrate that C. polyspora is capable of oxidizing methane and using methane as well as methanol as carbon source. Furthermore, C. polyspora seems to be able to exploit other carbon sources such as acetate and glucose, a very uncommon feature amongst methane-oxidizing bacteria. Thus, C. polyspora represents the first recognized filamentous methane oxidizer, has the most complex morphological life cycle of all known members of this functional guild, and even seems to be a facultative methanotroph. The aim of the proposed project is learn more about the ecophysiology of this filamentous methane oxidizer and to gain initial insights into the basics and function of the complex life cycle of C. polyspora. To achieve this aim we will follow a two-pronged strategy. Firstly, we will use the information retrieved by metagenomic analyses of C. polyspora to decipher the molecular basics of the proposed metabolic versatility of this microorganism. Secondly, we will incubate physically enriched C. polyspora biomass under various different environmental conditions (e.g. different substrates, substrate concentrations, temperature, pH) to define the substrate spectrum and the environmental parameters permissive for the growth and metabolic activity of C. polyspora. The latter approach will reveal initial information about the function of the so called Micro- and Macrogonidia formed by C. polyspora during its life cycle, and furthermore, will disclose which environmental parameters trigger their formation, leading to a better understanding of this uncommon microbial feature. Among other cultivation-independent tools, confocal Raman-microspectroscopy in combination with Fluorescence in situ hybridization (FISH; Raman-FISH Huang and Stoecker Environ. Microbiol. 9: 1878-1889) will be an essential tool for the ecophysiological analysis of C. polyspora in the scope of the proposed project. This novel tool for microbial ecology allows not only to monitor the incorporation of stable isotope-labelled substrates into microbial cells on a single cell level, but furthermore will enable us to quantify the amount of assimilated substrate and to determine into which cellular compounds the respective substrates are incorporated. In addition, confocal Raman-microspectroscopy allows insights into the chemical composition of an analysed microbial cell and thus represents an ideal tool for the direct analysis and comparison of the gonidia and the vegetative cells of C. polyspora on a single cell level. Also Raman-FISH has already proven its potential to become a powerful extension of the existing toolbox for in situ structure function analyses, this novel method will require further optimization to meet all demands of the proposed project. Hence, an integral part of the project will deal with the further development of this novel and promising tool. We expect that this advancement of the Raman-FISH method will further strengthen the toolbox for in situ studies and hence will be beneficial for microbial ecology.

Methane-oxidizing bacteria (MOB) represent a functional guild of ubiquitous microorganisms that are able to use the greenhouse gas methane as electron donor and carbon source. MOB play a major role in global carbon cycling and represent nature`s largest biological sink for atmospheric methane, which is the most abundant organic gas in the atmosphere. Except of Methylocella silvestris, which represents a facultative methanotroph, all known MOB are obligate methanotrophs and are not able to grow on carbon sources containing C-C bonds. Using cultivation-independent techniques, we recently identified and functionally characterized a very unusual methane oxidizer: Crenothrix polyspora (Proc. Natl. Acad. Sci. USA 7: 2363-2367 (Track II)). This, yet not cultivable, filamentous microorganism has been described already in 1870 by Ferdinand Cohn. C. polyspora has a complex life cycle and is infamous for occasional mass development in drinking water treatment facilities. However, despite the technical importance of C. polyspora, the identity and ecophysiology of this microorganism remained unresolved for more than a century. The main reason for this long-lasting gap of knowledge about C. polyspora is that until now, all attempts to culture this microorganism failed, so that it cannot be studied by the traditional cultivation-dependent approaches of microbiology. Without depending on cultivation based approaches, we were able to identify C. polyspora as a novel Gammaproteobacterium closely related to previously known type I methanotrophs. Consistent with this finding we could demonstrate that C. polyspora is capable of oxidizing methane and using methane as well as methanol as carbon source. Furthermore, C. polyspora seems to be able to exploit other carbon sources such as acetate and glucose, a very uncommon feature amongst methane-oxidizing bacteria. Thus, C. polyspora represents the first recognized filamentous methane oxidizer, has the most complex morphological life cycle of all known members of this functional guild, and even seems to be a facultative methanotroph. The aim of the proposed project is learn more about the ecophysiology of this filamentous methane oxidizer and to gain initial insights into the basics and function of the complex life cycle of C. polyspora. To achieve this aim we will follow a two-pronged strategy. Firstly, we will use the information retrieved by metagenomic analyses of C. polyspora to decipher the molecular basics of the proposed metabolic versatility of this microorganism. Secondly, we will incubate physically enriched C. polyspora biomass under various different environmental conditions (e.g. different substrates, substrate concentrations, temperature, pH) to define the substrate spectrum and the environmental parameters permissive for the growth and metabolic activity of C. polyspora. The latter approach will reveal initial information about the function of the so called Micro- and Macrogonidia formed by C. polyspora during its life cycle, and furthermore, will disclose which environmental parameters trigger their formation, leading to a better understanding of this uncommon microbial feature. Among other cultivation-independent tools, confocal Raman-microspectroscopy in combination with Fluorescence in situ hybridization (FISH; Raman-FISH Huang and Stoecker Environ. Microbiol. 9: 1878-1889) will be an essential tool for the ecophysiological analysis of C. polyspora in the scope of the proposed project. This novel tool for microbial ecology allows not only to monitor the incorporation of stable isotope-labelled substrates into microbial cells on a single cell level, but furthermore will enable us to quantify the amount of assimilated substrate and to determine into which cellular compounds the respective substrates are incorporated. In addition, confocal Raman-microspectroscopy allows insights into the chemical composition of an analysed microbial cell and thus represents an ideal tool for the direct analysis and comparison of the gonidia and the vegetative cells of C. polyspora on a single cell level. Also Raman-FISH has already proven its potential to become a powerful extension of the existing toolbox for in situ structure function analyses, this novel method will require further optimization to meet all demands of the proposed project. Hence, an integral part of the project will deal with the further development of this novel and promising tool. We expect that this advancement of the Raman-FISH method will further strengthen the toolbox for in situ studies and hence will be beneficial for microbial ecology.