Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Genomics:GTL Program Projects
University of Massachusetts, Amherst
33
Continued Progress in the use of Microarray Technology to Predict Gene Regulation and Function in Geobacter sulfurreducens
Barbara Methé1*(bmethe@tigr.org), Jennifer Webster1, Kelly Nevin2, and Derek Lovley2
1The Institute for Genomic Research, Rockville, MD and 2University of Massachusetts, Amherst, MA
Geobacter species represent a rare example in environmental microbiology in which microorganisms closely related to those which predominate in the environment and carry out environmental processes of interest can be readily cultivated in the laboratory. Molecular analyses designed to avoid culture bias, have demonstrated that microorganisms in the family Geobacteraceae are the dominant dissimilatory metal-reducing microorganisms in subsurface environments in which organic contaminants are being degraded with the reduction of Fe(III) and in aquatic sediments where dissimilatory metal reduction is important. In addition to their importance in global carbon, nutrient and metal cycles interest in Geobacter spp. stems from their potential as agents of bioremediation and capacity to create electricity.
Since completion of the G. sulfurreducens genome sequence, global gene expression profiling has been undertaken through the application of microarray technology. Experiments for querying whole genome PCR-based arrays currently being pursued include the examination of wild type G. sulfurreducens gene expression profiles under relevant physiological conditions and the testing of mutant strains in which a selected gene has been knocked out versus their wild type counterpart. Various data mining techniques including cluster analyses and analysis of variance are being employed to examine results from individual experiments and collectively across multiple experiments. These efforts have provided new insights into Geobacter physiology and regulatory networks.
For example, cells grown with chelated Fe(III) as the electron acceptor had higher levels of transcripts for omcB (GSU2737), an outer-membrane c-type cytochrome that is essential for Fe(III) reduction. Several other c-type cytochrome genes also appeared to be up regulated including a putative c-type cytochrome (GSU1334) which based on current genome comparative analyses is unique to Geobacter lineages. A substantial proportion (30%) of the significantly expressed genes during Fe(III) reduction were genes of unknown function, or hypothetical proteins. These results suggest differences in the physiology of Fe(III) reduction among microorganisms which perform this metabolic process. An unexpected result was significantly higher levels of transcripts for genes which have a role in metal efflux, potentially suggesting the importance of maintaining metal homeostasis during release of soluble metals when reducing Fe(III). This includes at least six transporter genes that are members of the resistance-nodulation-division (RND) superfamily of efflux transporters such as representatives of the transmembrane spanning heavy metal efflux pump czcA family (GSU0830, GSU1332) and one gene encoding for the membrane fusion protein of the czcB family (GSU0829). In contrast, transcript levels for other members of the czcA and czcB families in the G. sulfurreducens genome (GSU2135, GSU2136, GSU3400) were comparable during growth on Fe(III) and fumarate. This suggests that these czc family members are paralogs with different physiological roles and regulation. Common themes appearing across multiple experiments include the importance of transporter expression and the expression of a group of genes related to protein folding which for example are down regulated under conditions such as growth of Geobacter as a biofilm and with Fe(III) as an electron acceptor, in contrast they are up regulated in a mutant strain in which the rpoE sigma factor has been knocked out.
The next phase of the microarray component is building upon these previous successes while extending the flexibility and power of this technique. One example is the adaptation of methods to effect linear amplification of total RNA. Development of this protocol is important to producing high quality hybridizations from samples where the quantity of RNA that can ultimately be obtained from that sample is limited. High quality microarray hybridizations typically require several micrograms of total RNA per replicate. In order to obtain sufficient statistical power for meaningful analyses of microarray data it is necessary to replicate both biological samples as well as within sample replication (technical replication). Cell growth under conditions such as on poorly crystalline iron oxide media (an environmentally relevant growth condition of G. sulfurreducens) produces less total RNA upon extraction which can potentially hamper microarray efforts. Linear amplification (as opposed to geometric amplification with traditional PCR) of the total RNA allows the production of sufficient RNA quantities representative of the original proportions of the mRNA transcript population to facilitate the necessary replication of hybridizations to ensure a meaningful outcome post data analysis. The protocol described briefly here is currently being successfully tested on the G. sulfurreducens microarray and is a modification of the work of the classic “Eberwine” T7-Amplification method.
Amplified sense RNA is produced using random hexamers in a standard manner for first strand cDNA synthesis to create antisense cDNA. This product (antisense cDNA) is now used as the template in a second strand synthesis along with random nonamers to which a viral T3 promoter is attached. This results in a double-stranded cDNA in which the T3 promoter has been incorporated into the second strand. This second strand is in the sense orientation. An in vitro transcription (IVT) reaction can then be used to transcribe copious quantities of sense RNA from the T3 promoter sites. The resulting IVT product now serves as the template for standard cDNA synthesis and indirect fluorescent (Cy-Dye) labeling. These resulting targets can be used for hybridization to both PCR and oligonucleotide-based arrays.
One example where linear amplification has been applied is to the examination of gene expression patterns of G. sulfurreducens when grown using (insoluble) iron oxide as a sole electron acceptor. An overall improvement in hybridization intensity was realized with targets prepared from linear amplified RNA in comparison to unamplified RNA targets. Further, quantitative RT-PCR of a subset of genes from each RNA population (amplified and unamplified) have revealed that the trend of each gene (either significant elevation, depression or no change in gene expression) was the same in both RNA populations. The use of this linear RNA amplification technique in future will include the examination of global gene expression patterns from RNA extracted directly from environments in which Geobacter spp. are dominant members of the microbial community.
* Presenting author
