Generation and Elucidation of Complex Bacterial Phenotypes Using High-Throughput Genomic Techniques

Jacob Reece Borden

doi:https://doi.org/10.21985/N2KM7N

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Generation and Elucidation of Complex Bacterial Phenotypes Using High-Throughput Genomic Techniques

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Using a novel DNA-microarray hybridization protocol, the diffusion distance during static microarray hybridization was estimated for Cy3- and Cy5-labeled cDNA probes as 3.8 and 2.6 mm, respectively, despite having almost identical molecular masses. Continuous mixing during microarray hybridization resulted in a 15-20% increase in signal intensity over arrays hybridized statically. However, no change was observed in the Cy3/Cy5 signal intensity ratio between continuously mixed and static hybridizations, suggesting that the observed dye bias in diffusion-distance estimates results from differences in the detection limits of Cy3 and Cy5-labeled cDNA. A Clostridium acetobutylicum ATCC 824 genomic library was constructed to identify genetic elements conferring superior growth in the presence of n-butanol. An initial protocol based on a single butanol challenge in batch culture yielded a library insert containing the entire CAC0003 coding region. A second protocol based on serial transfers into progressively higher butanol concentrations yielded the CAC1869 gene, while DNA-microarray-based visualization of library populations allowed us to examine the dynamic process of library enrichment. While strain 824(pCAC0003) had moderately improved butanol tolerance, strain 824(pCAC1869) showed an 80-90% increase in butanol tolerance over the plasmid-control strain, and 824(pCAC1869) grew to higher cell densities in challenged and unchallenged cultures. A second C. acetobutylicum genomic library was constructed with a constitutive promoter for expression of library inserts. Serial-enrichment in the presence of butyric acid yielded genomic regions involved in the synthesis of ribosomal RNA and conferring improved butyrate tolerance. The 354 bp promoter region of the 16S ribosomal DNA was identified as the minimal, resistance-conferring DNA fragment (pRDNA7). DNA microarrays were used to identify reproducible transcriptional differences between unstressed and butyrate-stressed 824(pRDNA7) and 824(pSOS95del). Genes overexpressed in 824(pRDNA7) include spore-formation genes CAC120-CAC1293, membrane protein CAC1292, alkaline shock-induced protein (CAC1734), and permease CAC3285 with homology to GadC and AdiC. Underexpressed genes include the osmosensor KdpATPase complex (CAC3677 - CAC3682) and several membrane-associated and cell-wall biogenesis proteins. Finally, ribosomal RNA content in 824(pRDNA7) was different from that in 824(pSOS95del). A two-library system was developed to screen for interacting genomic fragments. E. coli w3110 genomic DNA was used to construct a fosmid library and a plasmid library containing 35 kb and 3 kb genomic inserts, respectively. Enrichment for interacting genomic fragments that confer ethanol resistance demonstrated stable library maintenance, and yielded a single plasmid insert containing a transcriptional regulator (yrbA) and organic solvent transporter (yrbC) and a single fosmid insert containing four transcriptional regulators as well as additional tolerance-related genes.

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