Chapter Three - Coupling FACS and Genomic Methods for the Characterization of Uncultivated Symbionts

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Abstract

Symbioses between microbes are likely widespread and functionally relevant in diverse biological systems; however, they are difficult to discover. Most microbes remain uncultivated, symbioses can be relatively rare or dynamic, and intercellular connections can be delicate. Thus, traditional methods such as microscopy are inadequate for efficient discovery and precise characterization of novel interactions, their metabolic basis, and the species involved. High-throughput metagenomic sequencing of entire microbial communities has revolutionized the field of microbial ecology; however, genomic signals from symbionts can get buried in sequences from abundant organisms and evidence for direct links between microbial species cannot be gained from bulk samples. Thus, a specialized approach to the characterization of symbioses between naturally occurring microbes is required. This chapter presents methods for combining fluorescence-activated cell sorting to isolate and separate uncultivated symbionts with molecular biology techniques for DNA amplification in order to characterize uncultivated symbionts through genomic and metagenomic techniques.

Introduction

Symbioses occur between microorganisms in diverse biological systems. These associations range from delicate connections at cell surfaces to integration of one symbiont into the cell of another, similar to an organelle. The metabolic basis for the symbioses, the types of cellular structures that link cells, their dynamics, and the relative autonomy of the symbiotic partners, are likely as diverse and ecologically relevant as microbes themselves.

The majority of microbes remain uncultivated, so study of complex natural microbial communities is required to discover and characterize novel symbioses. Coupling of microscopy to fluorescence in situ hybridization (FISH) has been a successful approach to studying uncultivated symbioses (Orphan, House, Hinrichs, McKeegan, & DeLong, 2001). However, these traditional methods are limited for several reasons. Morphological features of the symbioses can be damaged during sample handling. This is relevant to epiphytic symbioses or symbioses between delicate cells. Use of untreated samples could reduce artifacts and preserve intercellular connections, but garnering enough precise measurements of the symbionts’ cellular parameters for statistical power is tedious. To characterize metabolisms of symbionts, studies have applied stable isotope analysis along with FISH (Orphan et al., 2001). However, assessing the full metabolisms and evolutionary histories of symbionts is limited without genomic or metagenomic analysis and it would be difficult to recover intact cells from FISH preparations for whole-genome sequencing. An ideal approach to characterizing uncultivated symbionts will combine high-throughput methods that yield data on cellular parameters, phylogeny, and metabolism.

Fluorescence-activated cell sorting (FACS) is a widely chosen method for separating and concentrating cells from complex samples prior to genomic analysis. Other methods such as serial dilution (Zhang et al., 2006), micromanipulation (Hongoh et al., 2008, Kvist et al., 2007), laser-capture microdissection (Navin et al., 2011), Raman tweezers (Brehm-Stecher and Johnson, 2004, Huang et al., 2009), and microfluidics (Blainey et al., 2011, Marcy et al., 2007) have also been used effectively to separate cells prior to genetic or genomic analysis. However, for characterizing uncultivated symbionts, these methods fall short of FACS in a number of ways.

FACS machines deposit single events (individual cells or multiple cells in association) into a variety of vessels quickly and accurately (Ibrahim and van den Engh, 2003, Ibrahim and van den Engh, 2007). Precise assessment of pigment, cell size, membrane features, and genetic composition (cell-cycle analysis) can be obtained in minutes for thousands of events (Ibrahim & van den Engh, 2007). Contaminating DNA is limited because only a very small droplet of liquid from the source sample and sheath fluid accompanies each sorted event (Stepanauskas & Sieracki, 2007). Finally, FACS is a relatively gentle. The laminar flow fluidics of FACS prevents disruption of cells during sorting and this is critical in the study of potentially delicate symbioses (Ibrahim & van den Engh, 2007).

Genomic analysis can dramatically increase understanding of the basis for cellular interactions and the ecosystem function of symbionts beyond measuring cellular parameters (Tripp et al., 2010). However, current next-generation sequencing applications require more nucleic acids than can be obtained from thousands of sorted microbes. Thus, coupling FACS to nucleic acid amplification methods is essential.

MDA is a major advance over using PCR for whole-genome amplification (WGA) (Cheung and Nelson, 1996, Dean et al., 2001, Telenius et al., 1992, Zhang et al., 1992). MDA uses the Phi29 DNA polymerase to generate high quantities of double-stranded DNA that can be longer than 10 kb, making it an excellent starting material for sequencing applications and genomic analyses (Fig. 3.1).

Besides MDA, other nucleic acid amplification techniques can also be useful in characterizing microbial symbioses (Fig. 3.1). Especially in delicate associations, one symbiotic partner may remain unknown. In these cases, FACS can be coupled to nested PCR, which is applied to sorted single events (host–symbiont complexes), to amplify phylogenetically relevant genes for sequencing. This is a relatively low-cost method of identifying symbiotic partners before further sequencing efforts (Thompson et al., 2012) (Fig. 3.1).

Together FACS, MDA, and nested PCR can provide researchers with information on the species specificity of symbiotic associations, metabolic potential, ecological function, and evolutionary history of uncultivated microbial symbionts. This chapter focuses on how FACS can be coupled with genomic analysis to identify and characterize uncultivated symbionts (Fig. 3.1).

Section snippets

Flow cytometer features

While several high-throughput flow sorters are available, the BD Biosciences Influx™ Cell Sorter has been the FACS of choice for several recent studies on uncultivated symbionts (Cuvelier et al., 2010, Tripp et al., 2010, Vaulot et al., 2012, Zehr et al., 2008). The Influx™ can be equipped with 10 laser paths supporting collection of 24 data parameters, with the choice of lasers dependent on target cell types. A 488 nm laser (Sapphire Coherent) is commonly used to study phytoplankton as it

Metagenomic Sequencing of Uncultured Symbiont Populations

Genomic analysis can be instrumental in determining the metabolism of uncultivated symbionts (Tripp et al., 2010). However, genome sizes of symbiotic partners can be vastly different. Especially in symbioses between bacteria (smaller genomes) and microbial eukaryotes, whose genomes can be much larger or present in multiple copy numbers, it is advantageous to isolate the one partner in an enriched sample to ensure adequate genome coverage following sequencing. WGA via MDA is effective at

Determining Host Identity and Metabolism

Analysis of individual symbiotic associations is critical to unequivocally link the identity of an uncultivated symbiont to its host or symbiotic partner. Furthermore, relatively high-throughput analysis of single associations is essential to explore the species specificity of the association and this can be accomplished with nested PCR using universal primers for phylogenetically informative genes.

The physical connection between cells engaged in symbiosis can vary in its tolerance to sample

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