MacGregor Lab Research

Barbara MacGregor is a microbial ecologist, whose research interests may be described as “Who is there, and what are they doing?” – simple questions, but difficult to answer for the complex mixture of species often found in natural microbial communities. Molecular biological methods based on detection, sequencing, and phylogenetic identification of ribosomal RNA (rRNA) and the genes encoding it (rDNA) now allow description of microbial populations without isolation and culturing. rRNA is found in all species, it shows little evidence of cross-species transfer, and its production is growth-rate regulated, making it a good indicator for the presence of active microbial populations. Specific RNA or DNA sequences are amplified from environmental samples by the polymerase chain reaction (PCR), and compared with known sequences to infer their line of descent. There are a variety of methods for estimating the concentrations of different rRNAs or rDNAs in a sample, giving an idea of the community composition. However, because “functional” (enzyme-encoding) genes can be exchanged among even distantly-related species, rRNA phylogeny is not a reliable guide to physiology: for example, it cannot be assumed that a new rDNA sequence comes from a photosynthetic species, even if its closest known evolutionary relatives are photosynthesizers.

• New methods to directly link species identity with carbon source utilization
This linkage is being established via carbon isotopic characterization of rRNA, either by taking advantage of natural-abundance differences in 13C/12C ratios among substrates, or by addition of 13C- or 14C-labeled substrates. In collaboration with Dr. Nicole Dubilier (Max Planck Institute for Marine Microbiology; Bremen, Germany), we are measuring the carbon isotopic composition of host and symbiont rRNAs to determine the contribution of methane-oxidizing symbiotic bacteria to the growth of mussels living near oceanic hydrocarbon seeps and vents. Because methane is naturally depleted in 13C, methane-derived compounds tend to be isotopically “light” compared to those produced from other carbon sources.

Together with Dr. Dieter Juchelka (Thermo Electron GmbH, Bremen, Germany), we have recently developed a technique to measure the isotopic composition of individual nucleic acids in an RNA sample, using an HPLC/isotope-ratio mass spec approach. This approach will greatly reduce concerns about sample purity, since a single compound is measured at a time. We hope this technique will allow us to work with smaller samples that will be easier to obtain from natural settings. A joint proposal (MacGregor, Martens, Teske & Arnosti) for instrument acquisition has been submitted to NSF in order to purchase an IsoLink interface and to establish the technology at the MASC Department.

• Direct profiling of microbial communities without PCR
We are also developing methods for rRNA-based profiling of microbial communities without a PCR step, to directly identify the species using different carbon substrates. We have found that rRNAs of similar size but different sequences can be separated on non-denaturing polyacrylamide gels. Adam Friedman, a UNC undergraduate, is working with Dr. Stuart Strand (University of Washington – Seattle) to identify bacteria taking up radiolabeled acetate in an activated sludge wastewater treatment plant. A better understanding of microbial processes in treatment plants should help make them more efficient and reliable.

• Direct detection of microbial enzymes in environmental samples
As part of a collaboration (MacGregor/Arnosti/Teske labs), we are exploring the possibility of directly identifying the microbial enzymes involved in degrading high molecular weight carbon compounds. From the amino acid sequences of these enzymes, we hope to be able to work backwards, using RNA- and DNA-targeted probes, to find and identify the species that produced them.