Research

Temporal/Spatial Patterns of Diversity

Since 2000, in conjunction with the Caron Lab , the Fuhrman Lab has collected and analyzed water at multiple depths from San Pedro Ocean Time Series (SPOT) as part of the USC Microbial Observatory. Using a combination of molecular tools including gene-specific sequencing (16S rRNA, 18S rRNA, myoviral g23), metagenomics, and transcriptomics, we continue to assess the microbial populations (viral, bacterial, archaeal, eukaryotes) in conjunction with environmental parameters and biological metrics (microscopic and flow cytometric bacteria and virus counts, secondary production rates from thymidine and leucine incorporation, etc.).

The monthly time series enables us to assess long-term patterns and networks of seasonality, co-occurrence, and diversity for all players in the microbial food web. These patterns allow us to use real-world variations of natural complex communities to address fundamental questions about the factors controlling microbial biomass, diversity, activities, and biogeochemical roles.

We are also assessing patterns at different temporal (weekly, daily, sub-daily) and spatial scales (regional to global). Daily time series have shown unexpectedly sudden shifts in microbial communities that were previously missed by more routine monthly or weekly sampling. Our recent work showed that the dominant phytoplankton during the spring bloom can change on a near-daily basis, much more rapidly than first thought, and can include multiple harmful algal bloom species in rapid succession. We also found that Euryarchaea can peak to ~40% of the prokaryote community, in other words these poorly-known microbes can briefly bloom to make up the dominant organism in the near-surface ocean. We also analyze our data in the context of regional and global diversity patterns via our own sampling of three regional sites, which include SPOT plus a site in the Port of Los Angeles as well as a matched site near relatively pristine Catalina Island. In addition, we compare our data to those from major global sampling networks, such as the Earth Microbiome Project, and Ocean Sampling Day, both of which Dr. Fuhrman serves as a scientific advisor.

Selected Publications: Chow et al. 2012, Cram et al. 2015, Needham et al. 2013, Needham and Fuhrman 2016

Mock Communities to Assess Amplicon Sequencing Pipelines. Our Recommended 3-Domain PCR Primers and Pipline(s)

Although not widely recognized, high quality microbial community composition analysis requires periodic calibration and checking, just like any chemical assay. Small differences in PCR protocols and analytical pipelines can introduce major changes in results. Using a mock community of clones that were generated (by David Needham in our lab) to represent microbes common in mesotrophic and oligotrophic marine environments, we assessed the accuracy and precision of our 16S rRNA gene sequencing pipeline. We have selected a primer pair and analytical pipeline particularly well suited to marine and other microbiome projects. Our 515-926 primer pair not only amplifies the ssu rRNA (16S or 18S) of the vast majority of known organisms in all three domains, but testing with our prokaryotic mock communities shows that the results are remarkably quantitative. When we compared the observed vs expected abundances from our 27-member mock community, a log-log plot shows the r2 value is 0.95 (see figure to the left). This compares to an  r2 ~0.5 for the popular 515-806 primers used by many labs.

Based on our results, we strongly encourage the use of a mock community for anyone using rRNA “tag sequencing,” and it is important to include the mock communities blindly within sets of samples (not run alone). Not only did we find that popular 515-806 primers poorly quantified ssu rRNA gene abundance, but it also poorly amplified members of the SAR11 cluster, the most abundant bacteria in the global surface ocean; we also found that our downstream analyses, especially clustering, were greatly informed by tracking expected vs. observed classifications and operational taxonomic unit (OTU)-generation of the mock community. Several “standard” aspects of popular analytical pipelines caused incorrect merging or splitting of mock community OTUs, but we found ways to avoid that.

 

Chloroplasts. Of significant note, the 16S sequences of chloroplasts in eukaryotic phytoplankton (except dinoflagellates, which have aberrant chloroplast genomes) provide a valuable assessment of their community composition, far less affected by copy number variations than more classic 18S rRNA gene sequence analysis.

For a set of recommendations on how to use the Fuhrman Lab’s mock communities and PCR primers , please see our Methods and Publications.

Select Publications: Parada et al. 2015, Needham and Fuhrman 2016, Walters et al. 2016

Viral and protistian control of bacterial and archaeal populations

“Predatory or top-down regulation refers to the limitation of bacteria below levels supportable by resources alone” –Pace and Cole 1994, Microbial Ecology

Bacterial and archaeal populations are shaped by resource availability as well as viral lysis and protist grazing. Bottom-up and top-down controls are ultimately equally important.  We use dilution experiments, time-series data, network analysis, and statistical tools to simultaneously assess top-down and bottom-up regulation on community-wide abundance and diversity.

Select Publications: Chow et al. 2014, Cram et al. 2016

Human Impacts on Microbial Communities

To study how anthropogenic inputs affect the microbial community composition and function, we analyze samples from the Port of Los Angeles, with from the SPOT site  and also a nearshore site near Catalina Island as minimally impacted controls. Using a variety of tools, we are assessing the resistance of the naturally occurring community to pollutants like heavy metals and various aromatic hydrocarbons.

We also apply a cutting edge combination of Stable Isotope Probing (SIP) and high throughput sequencing to reveal which of the community members can metabolize pollutants and incorporate them into their biomass, directly linking diversity and function.

Viral/host patterns

Viral lysis helps to control microbial populations, and viruses impact community composition and microbial evolution.  We are addressing several aspects of the interactions between viruses and cellular microorganisms. In recent years we transitioned from fingerprinting-based analysis of myoviruses via PCR of the g23 gene, to sequencing of that gene in high-throughput manner, to the most recent work on viral metagenomics to assess the entire viral community. We have used time series (daily, monthly) to statistically link viruses with hosts and to assess the fundamental nature of virus-host networks as the contrast with bacteria-grazer networks. We are using a combination of bioinformatics, statistical networks, cultures, time series data, and single amplified genomes (SAGs), to address several questions, including how host ranges differ between viruses and the extents that viruses impact different members of food webs.

 

We also work with Computational Biology colleagues (labs of Dr. Fengzhu Sun and Dr. Ting Chen) to use bioinformatics and word patterns to identify viral sequences, distinguish viral sequences from those in cellular organisms, and to match viruses with hosts.

Select Publications: Chow et al. 2012, Chow et al. 2014, Needham et al. 2013

What phylogenetic resolution is needed in microbial ecological research? Ecological Species and "Microdiversity"

A major issue in microbial ecology research is how to best cluster similarly-functioning related organisms together and how to split ones that are related but ecologically different. This is essentially trying to define ecological species. While microbiologists frequently use marker genes and apply standard cutoffs of similarity, e.g. 99% similar 16S rRNA (or, 97%, which we consider too coarse), it is not clear what levels are truly most appropriate for ecological research, and how it may differ depending on the environment and scientific questions at hand.
By taking advantage of the most recent sequencing capabilities, and combining time-series data and information from the literature and major databases, we are able to address questions relating to suitable genes for study and the level of resolution needed to relate diversity to ecological processes without excessive lumping or splitting. These are not expected to be the same for all questions and for all organisms. For example, we expect that resource utilization may be studied at a coarser sequence resolution than viral susceptibility.  This work uses both marker genes and metagenomes.

Metagenomics and Metatranscriptomics

Our time-series samples offer unique opportunities to “go back in time” and re-analyze archived material using the most modern techniques. Using metagenomics and bioinformatics tools such as Anvi’o, we are addressing questions such as “How do genomic variants change over time?” in ways that can resolve these changes at the gene or allele level.


In concert with metagenomics, we are also mapping parallel metatranscriptomes to observe changing patterns of gene expression between adjacent sites and also over seasons. better understand what functions particular organisms are performing under different conditions.

Cultured Marine Archaea

We maintain several mixed enrichment cultures containing Marine Group I archaeal strains (Thaumarchaeota) that were enriched from SPOT, originally based on cultures and protocols generously shared by Professor Alyson Santoro. Isolates are instrumental in understanding fundamental processes, and we are currently using ours to assess genomic features, global distributions, and virus infectivity, among other things.