Jason Raymond

The Effect of Oxygen on Biochemical Networks and the Evolution of Complex Life

The evolution of oxygenic photosynthesis and ensuing oxygenation of Earths atmosphere represent a major transition in the history of life. Although many organisms retreated to anoxic environments, others evolved to use oxygen as a high–potential redox couple while concomitantly mitigating its toxicity. To understand the changes in biochemistry and enzymology that accompanied adaptation to O2, we integrated network analysis with information on enzyme evolution to infer how oxygen availability changed the architecture of metabolic networks. Our analysis revealed the existence of four discrete groups of networks of increasing complexity, with transitions between groups being contingent on the presence of key metabolites, including molecular oxygen, which was required for transition into the largest networks.

Niche adaptation and genome expansion in the chlorophyll d-producing cyanobacterium Acaryochloris marina

Acaryochloris marina is a unique cyanobacterium that is able to produce chlorophyll d as its primary photosynthetic pigment and thus efficiently use far-red light for photosynthesis. Acaryochloris species have been isolated from marine environments in association with other oxygenic phototrophs, which may have driven the niche-filling introduction of chlorophyll d. To investigate these unique adaptations, we have sequenced the complete genome of A. marina. The DNA content of A. marina is composed of 8.3 million base pairs, which is among the largest bacterial genomes sequenced thus far. This large array of genomic data is distributed into nine single-copy plasmids that code for >25% of the putative ORFs. Heavy duplication of genes related to DNA repair and recombination (primarily recA) and transposable elements could account for genetic mobility and genome expansion. We discuss points of interest for the biosynthesis of the unusual pigments chlorophyll d and α-carotene and genes responsible for previously studied phycobilin aggregates. Our analysis also reveals that A. marina carries a unique complement of genes for these phycobiliproteins in relation to those coding for antenna proteins related to those in Prochlorococcus species. The global replacement of major photosynthetic pigments appears to have incurred only minimal specializations in reaction center proteins to accommodate these alternate pigments. These features clearly show that the genus Acaryochloris is a fitting candidate for understanding genome expansion, gene acquisition, ecological adaptation, and photosystem modification in the cyanobacteria.

A nanostructure-initiator mass spectrometry-based enzyme activity assay

We describe a Nanostructure-Initiator Mass Spectrometry (NIMS) enzymatic (Nimzyme) assay in which enzyme substrates are immobilized on the mass spectrometry surface by using fluorous-phase interactions. This “soft” immobilization allows efficient desorption/ionization while also enabling the use of surface-washing steps to reduce signal suppression from complex biological samples, which results from the preferential retention of the tagged products and reactants. The Nimzyme assay is sensitive to subpicogram levels of enzyme, detects both addition and cleavage reactions (sialyltransferase and galactosidase), is applicable over a wide range of pHs and temperatures, and can measure activity directly from crude cell lysates. The ability of the Nimzyme assay to analyze complex mixtures is illustrated by identifying and directly characterizing β-1,4-galactosidase activity from a thermophilic microbial community lysate. The optimal enzyme temperature and pH were found to be 65°C and 5.5, respectively, and the activity was inhibited by both phenylethyl-β-d-thiogalactopyranoside and deoxygalactonojirimycin. Metagenomic analysis of the community suggests that the activity is from an uncultured, unsequenced γ-proteobacterium. In general, this assay provides an efficient method for detection and characterization of enzymatic activities in complex biological mixtures prior to sequencing or cloning efforts. More generally, this approach may have important applications for screening both enzymatic and inhibitor libraries, constructing and screening glycan microarrays, and complementing fluorous-phase organic synthesis.

Complete Genome Sequence of the Aerobic CO-Oxidizing Thermophile Thermomicrobium roseum

In order to enrich the phylogenetic diversity represented in the available sequenced bacterial genomes and as part of an “Assembling the Tree of Life” project, we determined the genome sequence of Thermomicrobium roseum DSM 5159. T. roseum DSM 5159 is a red-pigmented, rod-shaped, Gram-negative extreme thermophile isolated from a hot spring that possesses both an atypical cell wall composition and an unusual cell membrane that is composed entirely of long-chain 1,2-diols. Its genome is composed of two circular DNA elements, one of 2,006,217 bp (referred to as the chromosome) and one of 919,596 bp (referred to as the megaplasmid). Strikingly, though few standard housekeeping genes are found on the megaplasmid, it does encode a complete system for chemotaxis including both chemosensory components and an entire flagellar apparatus. This is the first known example of a complete flagellar system being encoded on a plasmid and suggests a straightforward means for lateral transfer of flagellum-based motility. Phylogenomic analyses support the recent rRNA-based analyses that led to T. roseum being removed from the phylum Thermomicrobia and assigned to the phylum Chloroflexi. Because T. roseum is a deep-branching member of this phylum, analysis of its genome provides insights into the evolution of the Chloroflexi. In addition, even though this species is not photosynthetic, analysis of the genome provides some insight into the origins of photosynthesis in the Chloroflexi. Metabolic pathway reconstructions and experimental studies revealed new aspects of the biology of this species. For example, we present evidence that T. roseum oxidizes CO aerobically, making it the first thermophile known to do so. In addition, we propose that glycosylation of its carotenoids plays a crucial role in the adaptation of the cell membrane to this bacteriums thermophilic lifestyle. Analyses of published metagenomic sequences from two hot springs similar to the one from which this strain was isolated, show that close relatives of T. roseum DSM 5159 are present but have some key differences from the strain sequenced.

Integrating Markov Clustering and Molecular Phylogenetics to Reconstruct the Cyanobacterial Species Tree from Conserved Protein Families

Attempts to classify living organisms by their physical characteristics are as old as biology itself. The advent of protein and DNA sequencing--most notably the use of 16S ribosomal RNA--defined a new level of classification that now forms our basic understanding of the history of life on earth. High-throughput sequencing currently provides DNA sequences at an unprecedented rate, not only providing a wealth of information but also posing considerable analytical challenges. Here we present comparative genomics-based methods useful for automating evolutionary analysis between any number of species. As a practical example, we applied our method to the well-studied cyanobacterial lineage. The 24 cyanobacterial genomes compared here occupy a wide variety of environmental niches and play major roles in global carbon and nitrogen cycles. By integrating phylogenetic data inferred for upward of 1,000 protein-coding genes common to all or most cyanobacteria, we have reconstructed an evolutionary history of the phylum, establishing a framework for resolving key issues regarding the evolution of their metabolic and phenotypic diversity. Greater resolution on individual branches can be attained by telescoping inward to the larger set of conserved proteins between fewer taxa. The construction of all individual protein phylogenies allows for quantitative tree scoring, providing insight into the evolutionary history of each protein family as well as probing the limits of phylogenetic resolution. The tools incorporated here are fast, computationally tractable, and easily extendable to other phyla and provide a scaleable framework for contrasting and integrating the information present in thousands of protein-coding genes within related genomes.

The genome of Heliobacterium modesticaldum, a phototrophic representative of the Firmicutes containing the simplest photosynthetic apparatus.

Despite the fact that heliobacteria are the only phototrophic representatives of the bacterial phylum Firmicutes, genomic analyses of these organisms have yet to be reported. Here we describe the complete sequence and analysis of the genome of Heliobacterium modesticaldum, a thermophilic species belonging to this unique group of phototrophs. The genome is a single 3.1-Mb circular chromosome containing 3,138 open reading frames. As suspected from physiological studies of heliobacteria that have failed to show photoautotrophic growth, genes encoding enzymes for known autotrophic pathways in other phototrophic organisms, including ribulose bisphosphate carboxylase (Calvin cycle), citrate lyase (reverse citric acid cycle), and malyl coenzyme A lyase (3-hydroxypropionate pathway), are not present in the H. modesticaldum genome. Thus, heliobacteria appear to be the only known anaerobic anoxygenic phototrophs that are not capable of autotrophy. Although for some cellular activities, such as nitrogen fixation, there is a full complement of genes in H. modesticaldum, other processes, including carbon metabolism and endosporulation, are more genetically streamlined than they are in most other low-G+C gram-positive bacteria. Moreover, several genes encoding photosynthetic functions in phototrophic purple bacteria are not present in the heliobacteria. In contrast to the nutritional flexibility of many anoxygenic phototrophs, the complete genome sequence of H. modesticaldum reveals an organism with a notable degree of metabolic specialization and genomic reduction.

Hydrothermal ecotones and streamer biofilm communities in the Lower Geyser Basin, Yellowstone National Park.

In Yellowstone National Park, a small percentage of thermal features support streamer biofilm communities (SBCs), but their growth criteria are poorly understood. This study investigates biofilms in two SBC hosting, and two non-SBC springs. Sequencing of 16S rRNA clones indicates changing community structure as a function of downstream geochemistry, with many novel representatives particularly among the Crenarchaeota. While some taxonomic groups show little genetic variation, others show specialization by sample location. The transition fringe environment between the hotter chemosynthetic and cooler photosynthetic zones hosts a larger diversity of organisms in SBC bearing springs. This transition is proposed to represent an ecotone; this is the first description of an ecotone in a hydrothermal environment. The Aquificales are ubiquitous and dominate among the Bacteria in the hottest environments. However, there is no difference in species of Aquificales from SBC and non-SBC locations, suggesting they are not responsible for the formation of SBCs, or that their role in SBC formation is competitively suppressed in non-SBC sites. In addition, only SBC locations support Thermotogales-like organisms, highlighting the potential importance these organisms may have in SBC formation. Here, we present a novel view of SBC formation and variability in hydrothermal ecosystems.

Coordinating environmental genomics and geochemistry reveals metabolic transitions in a hot spring ecosystem

We have constructed a conceptual model of biogeochemical cycles and metabolic and microbial community shifts within a hot spring ecosystem via coordinated analysis of the “Bison Pool” (BP) Environmental Genome and a complementary contextual geochemical dataset of ∼75 geochemical parameters. 2,321 16S rRNA clones and 470 megabases of environmental sequence data were produced from biofilms at five sites along the outflow of BP, an alkaline hot spring in Sentinel Meadow (Lower Geyser Basin) of Yellowstone National Park. This channel acts as a >22 m gradient of decreasing temperature, increasing dissolved oxygen, and changing availability of biologically important chemical species, such as those containing nitrogen and sulfur. Microbial life at BP transitions from a 92°C chemotrophic streamer biofilm community in the BP source pool to a 56°C phototrophic mat community. We improved automated annotation of the BP environmental genomes using BLAST-based Markov clustering. We have also assigned environmental genome sequences to individual microbial community members by complementing traditional homology-based assignment with nucleotide word-usage algorithms, allowing more than 70% of all reads to be assigned to source organisms. This assignment yields high genome coverage in dominant community members, facilitating reconstruction of nearly complete metabolic profiles and in-depth analysis of the relation between geochemical and metabolic changes along the outflow. We show that changes in environmental conditions and energy availability are associated with dramatic shifts in microbial communities and metabolic function. We have also identified an organism constituting a novel phylum in a metabolic “transition” community, located physically between the chemotroph- and phototroph-dominated sites. The complementary analysis of biogeochemical and environmental genomic data from BP has allowed us to build ecosystem-based conceptual models for this hot spring, reconstructing whole metabolic networks in order to illuminate community roles in shaping and responding to geochemical variability.

The Evolutionary Transition from Anoxygenic to Oxygenic Photosynthesis

Publisher Summary This chapter reviews briefly the early geologic evidence for photosynthesis and the structural and functional differences between anoxygenic and oxygenic photosynthesis, and then goes on to discuss the evolutionary transitions that have led us to the current situation in which oxygenic photosynthetic organisms dominate the biosphere, and anoxygenic organisms have largely retreated to specific environmental niches. Understanding the historical milestone provides unique insight not only into the evolutionary process, but also into how organism-environment interactions effect global-scale changes, ranging from carbon cycling and sequestration to composition of the atmosphere and oceans. The sequence identity decreases and the structural similarity diminish with increase in evolutionary distance between proteins. The chapter also focuses on the Type II reaction centers (RCs) that are found in two classes of anoxygenic phototrophs: purple photosynthetic bacteria and green filamentous photosynthetic bacteria (RC II) as well as photosystem II of oxygenic photosynthetic organisms (PS II). A number of lines of evidence agree that early RC complexes were found in anoxygenic phototrophs and were probably protein homodimers. The transition from these primitive anoxygenic complexes with a simple protein complement and bacteriochlorophyll pigments to the oxygenic PS II with chlorophyll pigments, an Mn-containing an oxygen evolving complex (OEC), and a much more complex protein complement was a remarkably dramatic evolutionary development that is still very poorly understood.

Visualization of the phylogenetic content of five genomes using dekapentagonal maps

The methods presented here summarize phylogenetic relationships of genomes in visually appealing and informative figures. Dekapentagonal maps depict phylogenetic information for orthologous genes present in five genomes, and provide a pre-screen for putatively horizontally transferred genes. If the majority of individual gene phylogenies are unresolved, bipartition histograms provide a means of uncovering and analyzing the plurality consensus. Analyses of genomes representing five photosynthetic bacterial phyla and of the prokaryotic contributions to the eukaryotic cell illustrate the utility of the methods.