nai

Project Progress

In most ecosystems, consortia of microorganisms orchestrate key processes in geochemical cycling, but little is known about ecological and evolutionary responses of these communities to cyclic and transient environmental shifts. The goal of this project is to employ advanced molecular techniques that will allow us to efficiently describe microbial population structures and to build deoxyribonucleic acid (DNA) microarrays for monitoring gene expression patterns both in laboratory cultures and in naturally occurring microbial mats.

We have developed high throughput technology for analyzing complex microbial populations and have applied it to studies of microbial communities in Guaymas Basin and the Rio Tinto. Serial Analysis of Gene Tags (SAGT) ligates together the polymerase chain reaction (PCR) products from orthologous hypervariable regions in ribosomal ribonucleic acid (rRNA) genes to form large concatemers. A single DNA sequencing reaction of a cloned concatemer can include as many as 20-30 orthologous hypervariable regions represented in a population of nucleic acid molecules. In this way, samples loaded onto a 96-channel capillary sequencing machine can provide information about thousands of microorganisms in an analyzed sample. The first step in the SAGT technique involves PCR amplification of small subunit rRNA genes from genomic DNA extracted from an environmental sample using conserved primers that contain a type II restriction site and flank a hypervariable region (20-100 bp) of the rRNA gene. The primer sequences are removed from the amplicons by digestion with this type II enzyme, leaving a short base pair extension. Digested fragments are ligated to form concatemers that are then cloned and sequenced. The primer pairs can be designed to amplify any of the rapidly evolving regions in rRNA genes. Alternatively, we can design primers to amplify regions that are not rapidly evolving and hence restrict the granularity of the analysis, but at the same time identify population members that are more distantly related. We have a manuscript ready for submission that shows the comparison of the SAGT technique with more expensive full length rRNA analyses from microbial populations in sediments of Guaymas Basin. We are now applying this technique to analysis of microbial populations in the Rio Tinto and have been able to fully describe microbial populations at several sites in this river system.

The complementary component of our ecogenomics project is the use of DNA microarrays to characterize gene expression patterns throughout the diel cycle for organisms isolated from microbial mats. We have constructed a genomic library of the mat-forming cyanobacterium Microcoleus chthonoplastes using DNA extracted from a culture of a strain collected from the Sippewissett salt marsh, in West Falmouth, Massachusetts. We have sequenced 10,241 clones with insert sizes of 650 – 1150 bp, and used this information to select the sequences to array. Selection criteria for including a gene was based on a BLASTX hit to a known cyanobacterial sequence with an e value <= 1x10^-4 and a Pfam score >= 25. This filter ensured that clones on the array were from Microcoleus and that they contained a gene coding region. After removing redundant clones, we selected 1090 sequences to array. These sequences were PCR amplified from the vector, and the DNAs printed onto glass slides for expression profiling studies.

Preliminary experiments have used the array to examine differences in gene expression during the day and night. We took laboratory cultures of Microcoleus, placed them into a 14 hr light / 10 hr dark cycle for 3 days, and then collected cells after two hours of light exposure and after two hours of darkness. As expected, a number of genes directly involved in photosynthesis were expressed at higher levels in the day samples as compared to the night samples. A large number of other genes involved in growth, replication, and many other processes were also upregulated during the day. At night, we found “light repressed proteins” showing increased expression, as well as a number of regulatory proteins. These data have also shown us a number of unexpected putative results, such as an increase in expression of an iron transporter at night.

To help better understand and verify these results, we are currently in the process of assembling a time course of expression data across an entire diel cycle. This information should provide us with valuable information about the precise timing and daily expression patterns of many of these genes. Once we have collected these data on diel variations, we plan to take environmental samples directly from the Sippewissett marsh and see how closely our laboratory manipulations can match what is actually happening in the environment.