Molecular ecology of facultative methanotrophs
Rahman, M. Tanvir (2010) Molecular ecology of facultative methanotrophs. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b2485038~S15
Methylocella spp. are facultative methanotrophs, able to grow not only on one
carbon molecules e.g., methane or methanol, but also on multi-carbon compounds
e.g., acetate, succinate and malate.
PCR-based molecular techniques were developed and validated to target the 16S
rRNA genes of all the species of the genus Methylocella present in environmental
samples. DNA extracted from a variety of environmental samples screened by PCR
to determine the environmental distribution of Methylocella spp. Methylocella were
found to be widely distributed, not only in acidic environments, but also in neutral
and alkaline environments. New primers targeting the mmoX gene of Methylocella
spp. that encoded the α-subunit of methane monooxygenases were designed. A
SYBR® green-based real-time quantitative PCR assay was developed and validated
using these Methylocella mmoX gene-targeting primers. The abundance of
Methylocella spp. present in selected environmental samples was quantified using
the newly developed real-time quantitative PCR assay
A series of 13CH4 DNA-SIP experiments were carried either in the presence or
absence of 12C acetate in microcosms containing Moor House peat soil to investigate
the effect of acetate (0.5 mM) on the ability of Methylocella silvestris to oxidize
methane. Methane oxidation data indicated that acetate repressed the ability of
Methylocella silvestris to oxidize methane in peat soil microcosms. 13CH4 DNA-SIP
experiments revealed that in presence of acetate, Methylocella silvestris did not
utilize methane as a carbon source, suggesting that acetate might be the alternative
source of carbon. However, when 13C-labelled acetate DNA-SIP experiments were
carried out, Methylocella spp. were not found to be dominant acetate utilizers in the
peat soil microcosms. Methylocella spp. seem to have been outcompeted by more
efficient acetate utilizers such as Brevundimonas and Burkholderia.
To identify genes that might be involved in the utilization of methane or acetate,
Methylocella silvestris whole genome transcriptomics experiments were carried out.
All the genes of the sMMO gene cluster were found to be highly upregulated during
growth on methane. In addition to the sMMO gene cluster, a gene encoding Fur was
also found to be highly upregulated during growth on methane. During growth on
acetate, a gene cluster encoding glycine dehydrogenase was found to be highly
upregulated. Microarray experiments carried out here provided potential candidate
genes for further characterization by gene knockout based studies. Further work is
also required to validate the microarray findings.
A study was carried out on forest soils derived from Swedish islands that were at
different successional stages. All islands were found to be positive for the presence
of Methylocella spp., and identity of composition of other methanotrophs were
determined using a pmoA-diagnostic microarray. All the islands were dominated by
Methylococcus, Methylosinus and methanotrophs of the uncultivated RA14 clade.
The diversity of methanotrophs was higher in late successional islands compared to
mid and early successional islands. In addition the diversity of methanotrophs
decreased as the soil depth increased.
|Item Type:||Thesis or Dissertation (PhD)|
|Subjects:||Q Science > QR Microbiology|
|Library of Congress Subject Headings (LCSH):||Methanotrophs -- Genetics|
|Official Date:||July 2010|
|Institution:||University of Warwick|
|Theses Department:||Department of Biological Sciences|
|Supervisor(s)/Advisor:||Murrell, J. C. (J. Colin)|
|Sponsors:||University of Warwick ; University of Warwick. Dept. of Biological Sciences|
|Extent:||xxi, 306 leaves : ill., charts, maps|
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