Experiments

The isolation of metabolically and physiologically diverse microbes provides an in-depth, hands-on experience with the metabolic properties and (eco)physiological traits of important microbes. The isolations focus on key members of the community of the microbial mats that we collect from the nearby Elkhorn Slough and maintain in the laboratory. In particular, we isolate purple sulfur bacteria, purple non-sulfur bacteria, green bacteria, colorless sulfur bacteria, luminescent bacteria, lactic acid bacteria, propionic acid bacteria, Clostridia, sulfate-reducing bacteria, Fe(III)-reducing bacteria, Mn(II)-oxidizing bacteria. These isolations form a valuable complement to the concepts on microbial physiology and metabolism that we develop around the ecophysiology of these microorganisms, as well as the causes and consequences of their niche construction.

To complement the ‘culture-based’ physiological and genetic experiments described above and below, HMC students also carry out cultivation-independent PCR-based analyses of various ecosystems. While many students arrive with some knowledge of standard 16S rRNA-based analysis of microbial communities, very few have prior experience in characterizing the diversity of ecologically relevant functional genes. Thus, we have implemented a ‘functional gene analysis’ experiment in which students utilize the ammonia monooxygenase (amoA) gene as a molecular marker to examine the distribution and diversity of uncultivated ammonia-oxidizing archaea (AOA). In previous years we have extracted DNA from a wide variety of local ecosystems chosen by the students, including: Elkhorn Slough estuary sediments, multiple depths in Monterey Bay, Hopkins intertidal rocks and beach sand, as well as material from the biological ‘sand filter’ from the neighboring Monterey Bay Aquarium. Students learn how to generate, sequence, and analyze archaeal amoA clone libraries using a variety of cutting-edge phylogenetic and statistical approaches. At the end of the course, the students’ extensive archaeal amoA dataset was deposited in GenBank, providing a useful resource to the scientific community. Coupled with numerous lectures highlighting how key functional genes (e.g. amoA, nifH, nirK, nirS, dsrA, mcrA, etc.) can be used to study the diversity of specific groups of biogeochemically important microbes in the natural environment, this amoA-based experiment provides students with valuable hands-on research experience in molecular microbial ecology.

Other cultivation-independent methods with which students gain experience include fluorescence in-situ hybridization (FISH) using phylogenetic markers and single-cell whole genome amplification using a microfluidics chip to sort single cells from environmental samples.

Population genetics provides a means of understanding diversity in terms of the evolutionary processes that generate it.  Marine vibrios are isolated from sea anemones located at two geographically distinct sites.  The nucleotide sequence of three housekeeping genes and one ecologically significant gene, plus 16S is obtained from a total of ~100 strains and the resulting data subjected to a range of analyses to determine the contribution of mutation, recombination, migration, selection and drift to patterns of vibrio diversity.  The project introduces students to the very latest approaches in multi-locus sequence analysis, including hands-on experience with methods for assessing recombination, selection, drift, and the partitioning of diversity across space and time.

Rapid generation time and large population size means that evolution in microbial populations cannot be ignored.  The evolution of antibiotic resistance in marine vibrios is examined in real time.  Fitness costs associated with the evolution of resistance to quinolone antibiotics are determined by competition assays.  The molecular changes in candidate genes are determined by DNA seqence analysis.  The ability of selection to ameliorate maladaptive pleiotropic effects are examined through selection experiments.  The project introduces students to hands-on experience in experimental evolution and shows how genotype (including molecular details) can be connected to phenotype and fitness through evolutionary time. 

Student Presentations