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Comparison of microbial gene presence and activity in soil; developing methods to study gene expression using RNA extracted directly from soil

Reference	BBS/E/C/00031764
Principal Investigator / Supervisor	Professor Penny Hirsch
Co-Investigators / Co-Supervisors
Institution	Rothamsted Research
Department	Rothamsted Research Department
Funding type	Research
Value (£)	75,078
Status	Completed
Type	Institute Project
Start date	01/04/1997
End date	31/03/1999
Duration	24 months

Abstract

It is difficult to correlate the presence of any particular group of bacteria known to have important functions in soil, with their activity in soil under different conditions. Most soils contain at least 100 million bacteria per g, and it has been estimated that an almost infinite number of different species are present. However, only a minority of these can be cultured and many are fastidious and slow-growing. Novel approaches are needed to study both the activity and genetic diversity of soil bacterial populations which might be present at relatively low levels. Methods to detect genes in soil bacteria without relying on laboratory culture were developed in a previous project, 031471, concerned with monitoring GM bacteria after field release, using DNA extracted directly from soil, specific primers and PCR. DNA is present in all live bacteria whether or not they are active, but ribosomal (r) RNA and messenger (m) RNA are present in significantly increased quantities in active cells and rRNA sequences provide taxonomic identification of organisms. Methods to detect rRNA and mRNA in soil, in conjunction with existing methods for DNA, would provide a much more comprehensive understanding of soil microbial ecology than has been possible to date. Problems that such methods might address include demonstrating whether or not GM strains surviving in soil are dormant or active. Another question concerns the ecology of methane and ammonium oxidizers in agricultural soils: functional activity can be measured but the bacteria are very difficult to culture and little is known of the size and diversity of dormant and active populations under different conditions. Objectives 1996 New project in 1997. 1997 Develop RNA extraction from soil and verify it using GM bacteria. Investigate the relationship between the presence of rRNA from methane oxidizers in soils with high and low rates of methane oxidation. Determine the sensitivity of detection of mRNA which denotes expression of a marker gene in a GM bacterial inoculant in soil. There is considerable interest in the activity of nitrifying bacteria in soil which are very difficult to study using conventional approaches. Two major subgroups of ammonium-oxidizers are known to exist, but their relative importance is uncertain in soil. Primers specific to the ammonium monooxygenase (AMO) and the 16s rRNA genes from each of the two groups will be designed and used to establish the relative numbers of bacterial cells of each type and activity based on the relative numbers of each type of 16s rRNA.

Summary

unavailable

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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