Project description:Comparison of probe-target dissociations of probe Eub338 and Gam42a with native RNA of P. putida, in vitro transcribed 16s rRNA of P. putida, in vitro transcribed 16S rRNA of a 2,4,6-trinitrotoluene contaminated soil and an uncontaminated soil sample. Functional ANOVA revealed no significant differences in the dissociation curves of probe Eub338 when hybridised to the different samples. On the opposite, the dissociation curve of probe Gam42a with native RNA of P. putida was significantly different than the dissociation curves obtained with in vitro transcribed 16S rRNA samples. Keywords: Microbial diversity, thermal dissociation analysis, CodeLink microarray
Project description:The survival, pollutant degradation activity and transcriptome response was monitored in Sphingomonas sp. LH128 inoculated into soil. Cultivable cell numbers were determined by plating, while phenanthrene degradation was monitored by HPLC. The genetic base for the adaptive strategy of LH128 in soil was investigated by using microarray consisting 7,200 gene-coding ORFs. During 4 hours of incubation, 510 genes were differentially expressed (317 increased and 193 reduced expression) while 610 genes were differentially expressed (318 increased and 292 reduced) after 10 days of incubation. Genes with increased expression comprised of gene encoding PAH catabolic enzymes, stress resistance, oxidative stress tolerance, outer membrane proteins/porins and efflux pump proteins while the downregulated genes comprised of genes encoding flagellar biosynthesis, ribosomal proteins and ATPase. Transcriptomic response of phenanthrene degrading Sphingomonas sp. LH128 inoculated into phenanthrene contaminated soil after 4h and after 10 days of incubation was studied using genome-wide gene expression analysis. For this purpose, the strain was pregrown in minimal medium and inoculated at appropriated celld densitites. RNA was extracted both from soil and and from initial inoculum and cDNA was synthesized and labeled with Cy3. Transcriptomic response in soil of three replicates per conditions after both incubation duration were analyzed and compared with the initial inoculum
Project description:The experiment was designed to test the interactions of Spartina alterniflora, its microbiome, and the interaction of the plant-microbe relationship with oil from the Deepwater Horizon oil spill (DWH). Total RNA was extracted from leaf and root microbiome of S. alterniflora in soils that were oiled in DWH oil spill with or without added oil, as well as those grown in unoiled soil with or without added oil. The work in its entirety characterizes the transport, fate and catabolic activities of bacterial communities in petroleum-polluted soils and within plant tissues.
Project description:Microbial community analysis with DNA oligonucleotide microarrays targeting ribosomal RNA (rRNA) provides a highly parallel interrogation of nucleic acids isolated from environmental samples. High fidelity readout is essential for accurate interpretation of hybridisations. We describe the hybridisation of in vitro transcribed 16S rRNA from an uncontaminated and 2,4,6-trinitrotoluene contaminated soil to an oligonucleotide microarray containing group- and species-specific perfect match (PM) probes and their 2 corresponding mismatch (MM) probes. Thermal dissociation analysis was used to determine the specificity of each PM-MM probe set. Functional ANOVA often discriminated PM-MM probe sets when Td values (temperature at 50% probe-target dissociation) could not. Maximum discrimination for many PM and MM probes often occurred at temperatures greater than the Td. Comparison of signal intensities measured prior to dissociation analysis from hybridisations of the two soil samples revealed significant differences in domain-, group- and species-specific probes. Functional ANOVA showed significantly different dissociation curves for 11 PM probes when hybridisations from the two soil samples were compared, even though initial signal intensities for 3 of the 11 did not vary. This approach provides a highly parallel, multi-level analysis that incorporates MM probes and dissociation curves into high fidelity microarray analysis of complex environmental nucleic acid profiles. Keywords: Microbial diversity, thermal dissociation analysis
