Adam C. Martiny

Patterns and Implications of Gene Gain and Loss in the Evolution of Prochlorococcus

Prochlorococcus is a marine cyanobacterium that numerically dominates the mid-latitude oceans and is the smallest known oxygenic phototroph. Numerous isolates from diverse areas of the worlds oceans have been studied and shown to be physiologically and genetically distinct. All isolates described thus far can be assigned to either a tightly clustered high-light (HL)-adapted clade, or a more divergent low-light (LL)-adapted group. The 16S rRNA sequences of the entire Prochlorococcus group differ by at most 3%, and the four initially published genomes revealed patterns of genetic differentiation that help explain physiological differences among the isolates. Here we describe the genomes of eight newly sequenced isolates and combine them with the first four genomes for a comprehensive analysis of the core (shared by all isolates) and flexible genes of the Prochlorococcus group, and the patterns of loss and gain of the flexible genes over the course of evolution. There are 1,273 genes that represent the core shared by all 12 genomes. They are apparently sufficient, according to metabolic reconstruction, to encode a functional cell. We describe a phylogeny for all 12 isolates by subjecting their complete proteomes to three different phylogenetic analyses. For each non-core gene, we used a maximum parsimony method to estimate which ancestor likely first acquired or lost each gene. Many of the genetic differences among isolates, especially for genes involved in outer membrane synthesis and nutrient transport, are found within the same clade. Nevertheless, we identified some genes defining HL and LL ecotypes, and clades within these broad ecotypes, helping to demonstrate the basis of HL and LL adaptations in Prochlorococcus. Furthermore, our estimates of gene gain events allow us to identify highly variable genomic islands that are not apparent through simple pairwise comparisons. These results emphasize the functional roles, especially those connected to outer membrane synthesis and transport that dominate the flexible genome and set it apart from the core. Besides identifying islands and demonstrating their role throughout the history of Prochlorococcus, reconstruction of past gene gains and losses shows that much of the variability exists at the “leaves of the tree,” between the most closely related strains. Finally, the identification of core and flexible genes from this 12-genome comparison is largely consistent with the relative frequency of Prochlorococcus genes found in global ocean metagenomic databases, further closing the gap between our understanding of these organisms in the lab and the wild.

Genomic islands and the ecology and evolution of Prochlorococcus

Prochlorococcus ecotypes are a useful system for exploring the origin and function of diversity among closely related microbes. The genetic variability between phenotypically distinct strains that differ by less that 1% in 16S ribosomal RNA sequences occurs mostly in genomic islands. Island genes appear to have been acquired in part by phage-mediated lateral gene transfer, and some are differentially expressed under light and nutrient stress. Furthermore, genome fragments directly recovered from ocean ecosystems indicate that these islands are variable among cooccurring Prochlorococcus cells. Genomic islands in this free-living photoautotroph share features with pathogenicity islands of parasitic bacteria, suggesting a general mechanism for niche differentiation in microbial species.

Present and future global distributions of the marine Cyanobacteria Prochlorococcus and Synechococcus

The Cyanobacteria Prochlorococcus and Synechococcus account for a substantial fraction of marine primary production. Here, we present quantitative niche models for these lineages that assess present and future global abundances and distributions. These niche models are the result of neural network, nonparametric, and parametric analyses, and they rely on >35,000 discrete observations from all major ocean regions. The models assess cell abundance based on temperature and photosynthetically active radiation, but the individual responses to these environmental variables differ for each lineage. The models estimate global biogeographic patterns and seasonal variability of cell abundance, with maxima in the warm oligotrophic gyres of the Indian and the western Pacific Oceans and minima at higher latitudes. The annual mean global abundances of Prochlorococcus and Synechococcus are 2.9 ± 0.1 × 1027 and 7.0 ± 0.3 × 1026 cells, respectively. Using projections of sea surface temperature as a result of increased concentration of greenhouse gases at the end of the 21st century, our niche models projected increases in cell numbers of 29% and 14% for Prochlorococcus and Synechococcus, respectively. The changes are geographically uneven but include an increase in area. Thus, our global niche models suggest that oceanic microbial communities will experience complex changes as a result of projected future climate conditions. Because of the high abundances and contributions to primary production of Prochlorococcus and Synechococcus, these changes may have large impacts on ocean ecosystems and biogeochemical cycles.

Sequencing genomes from single cells by polymerase cloning

Genome sequencing currently requires DNA from pools of numerous nearly identical cells (clones), leaving the genome sequences of many difficult-to-culture microorganisms unattainable. We report a sequencing strategy that eliminates culturing of microorganisms by using real-time isothermal amplification to form polymerase clones (plones) from the DNA of single cells. Two Escherichia coli plones, analyzed by Affymetrix chip hybridization, demonstrate that plonal amplification is specific and the bias is randomly distributed. Whole-genome shotgun sequencing of Prochlorococcus MIT9312 plones showed 62% coverage of the genome from one plone at a sequencing depth of 3.5×, and 66% coverage from a second plone at a depth of 4.7 ×. Genomic regions not revealed in the initial round of sequencing are recovered by sequencing PCR amplicons derived from plonal DNA. The mutation rate in single-cell amplification is <2 × 105, better than that of current genome sequencing standards. Polymerase cloning should provide a critical tool for systematic characterization of genome diversity in the biosphere.

Phylogenetic conservatism of functional traits in microorganisms

A central question in biology is how biodiversity influences ecosystem functioning. Underlying this is the relationship between organismal phylogeny and the presence of specific functional traits. The relationship is complicated by gene loss and convergent evolution, resulting in the polyphyletic distribution of many traits. In microorganisms, lateral gene transfer can further distort the linkage between phylogeny and the presence of specific functional traits. To identify the phylogenetic conservation of specific traits in microorganisms, we developed a new phylogenetic metric—consenTRAIT—to estimate the clade depth where organisms share a trait. We then analyzed the distribution of 89 functional traits across a broad range of Bacteria and Archaea using genotypic and phenotypic data. A total of 93% of the traits were significantly non-randomly distributed, which suggested that vertical inheritance was generally important for the phylogenetic dispersion of functional traits in microorganisms. Further, traits in microbes were associated with a continuum of trait depths (τD), ranging from a few deep to many shallow clades (average τD: 0.101–0.0011 rRNA sequence dissimilarity). Next, we demonstrated that the dispersion and the depth of clades that contain a trait is correlated with the trait’s complexity. Specifically, complex traits encoded by many genes like photosynthesis and methanogenesis were found in a few deep clusters, whereas the ability to use simple carbon substrates was highly phylogenetically dispersed. On the basis of these results, we propose a framework for predicting the phylogenetic conservatism of functional traits depending on the complexity of the trait. This framework enables predicting how variation in microbial composition may affect microbially-mediated ecosystem processes as well as linking phylogenetic and trait-based patterns of biogeography.

Phosphate acquisition genes in Prochlorococcus ecotypes: evidence for genome-wide adaptation.

The cyanobacterium Prochlorococcus is the numerically dominant phototroph in the oligotrophic oceans. This group consists of multiple ecotypes that are physiologically and phylogenetically distinct and occur in different abundances along environmental gradients. Here we examine adaptations to phosphate (P) limitation among ecotypes. First, we used DNA microarrays to identify genes involved in the P-starvation response in two strains belonging to different ecotypes, MED4 (high-light-adapted) and MIT9313 (low-light-adapted). Most of the up-regulated genes under P starvation were unique to one strain. In MIT9313, many ribosomal genes were down-regulated, suggesting a general stress response in this strain. We also observed major differences in regulation. The P-starvation-induced genes comprise two clusters on the chromosome, the first containing the P master regulator phoB and most known P-acquisition genes and the second, absent in MIT9313, containing genes of unknown function. We examined the organization of the phoB gene cluster in 11 Prochlorococcus strains belonging to diverse ecotypes and found high variability in gene content that was not congruent with rRNA phylogeny. We hypothesize that this genome variability is related to differences in P availability in the oceans from which the strains were isolated. Analysis of a metagenomic library from the Sargasso Sea supports this hypothesis; most Prochlorococcus cells in this low-P environment contain the P-acquisition genes seen in MED4, although a number of previously undescribed gene combinations were observed.

Long-Term Succession of Structure and Diversity of a Biofilm Formed in a Model Drinking Water Distribution System

ABSTRACT In this study, we examined the long-term development of the overall structural morphology and community composition of a biofilm formed in a model drinking water distribution system with biofilms from 1 day to 3 years old. Visualization and subsequent quantification showed how the biofilm developed from an initial attachment of single cells through the formation of independent microcolonies reaching 30 μm in thickness to a final looser structure with an average thickness of 14.1 μm and covering 76% of the surface. An analysis of the community composition by use of terminal restriction fragment length polymorphisms showed a correlation between the population profile and the age of the sample, separating the samples into young (1 to 94 days) and old (571 to 1,093 days) biofilms, whereas a limited spatial variation in the biofilm was observed. A more detailed analysis with cloning and sequencing of 16S rRNA fragments illustrated how a wide variety of cells recruited from the bulk water initially attached and resulted in a species richness comparable to that in the water phase. This step was followed by the growth of a bacterium which was related to Nitrospira, which constituted 78% of the community by day 256, and which resulted in a reduction in the overall richness. After 500 days, the biofilm entered a stable population state, which was characterized by a greater richness of bacteria, including Nitrospira, Planctomyces, Acidobacterium, and Pseudomonas. The combination of different techniques illustrated the successional formation of a biofilm during a 3-year period in this model drinking water distribution system.

Taxonomic resolution, ecotypes and the biogeography of Prochlorococcus

In order to expand our understanding of the diversity and biogeography of Prochlorococcus ribotypes, we PCR-amplified, cloned and sequenced the 16S/23S rRNA ITS region from sites in the Atlantic and Pacific oceans. Ninety-three per cent of the ITS sequences could be assigned to existing Prochlorococcus clades, although many novel subclades were detected. We assigned the sequences to operational taxonomic units using a graduated scale of sequence identity from 80% to 99.5% and correlated Prochlorococcus diversity with respect to environmental variables and dispersal time between the sites. Dispersal time was estimated using a global ocean circulation model. The significance of specific environmental variables was dependent on the degree of sequence identity used to define a taxon: light correlates with broad-scale diversity (90% cut-off), temperature with intermediate scale (95%) whereas no correlation with phosphate was observed. Community structure was correlated with dispersal time between sample sites only when taxa were defined using the finest sequence similarity cut-off. Surprisingly, the concentration of nitrate, which cannot be used as N source by the Prochlorococcus strains in culture, explains some variation in community structure for some definitions of taxa. This study suggests that the spatial distribution of Prochlorococcus ecotypes is shaped by a hierarchy of environmental factors as well dispersal limitation.

Microbiomes in light of traits: A phylogenetic perspective.

Function in the tree of life How does the composition of microbial communities integrate functionally with the wider environment? Martiny et al. review how patterns of microbial species abundances in different environments and disease states can have strong evolutionary signals. Some environmental changes select the survival of organisms with conserved metabolisms requiring complex configurations of proteins and cofactors that have long evolutionary histories, such as methane producers. In contrast, surviving antibiotic exposure may only require a single gene that can be traded promiscuously among many unrelated organisms. So, depending on the key ingredient (whether it is temperature, light, nutrient, or a dose of antibiotic) and the evolutionary history of its complementary metabolism, shifting environmental conditions will have predictable effects at different levels within the microbial tree of life. Science, this issue p. 10.1126/science.aac9323 BACKGROUND Microbial communities—microbiomes—are intricately linked to human health and critical ecosystem services. New technologies allow the rapid characterization of hundreds of samples at a time and provide a sweeping perspective on microbiome patterns. However, a systematic understanding of what determines microbiome diversity and composition and its implications for system functioning is still lacking. A focus on the phenotypic characteristics of microorganisms—their traits—offers a path for interpreting the growing amount of microbiome data. Indeed, a variety of trait-based approaches have been proposed for plants and animal communities, and this approach has helped to clarify the mechanisms underlying community assembly, diversity-process relationships, and ecosystem responses to environmental change. Although there is a growing emphasis on microbial traits, the concept has not been fully appreciated in microbiology. However, a trait focus for microorganisms may present an even larger research opportunity than for macro-organisms. Not only do microorganisms play a central role in nutrient and energy cycling in most systems, but the techniques used to characterize microbiomes usually provide extensive molecular and phylogenetic information. ADVANCES One major difference between macro- and microorganisms is the potential for horizontal gene transfer (HGT) in microbes. Higher rates of HGT mean that many microbial traits might be unrelated to the history of the vertically descended parts of the genome. If true, then the taxonomic composition of a microbiome might reveal little about the health or functioning of a system. We first review key aspects of microbial traits and then recent studies that document the distribution of microbial traits onto the tree of life. A synthesis of these studies reveals that, despite the promiscuity of HGT, microbial traits appear to be phylogenetically conserved, or not distributed randomly across the tree of life. Further, microbial traits appear to be conserved in a hierarchical fashion, possibly linked to their biochemical and genetic complexity. For instance, traits such as pH and salinity preference are relatively deeply conserved, such that taxa within deep clades tend to share the trait. In contrast, other traits like the ability to use simple carbon substrates or to take up organic phosphorus are shallowly conserved, and taxa share these traits only within small, shallow clades. OUTLOOK The phylogenetic, trait-based framework that emerges offers a path to interpret microbiome variation and its connection to the health and functioning of environmental, engineered, and human systems. In particular, the taxonomic resolution of biogeographic patterns provides information about the traits under selection, even across entirely different systems. Parallels observed among human and free-living communities support this idea. For instance, microbial traits related to growth on different substrates (e.g., proteins, fats, and carbohydrates) in the human gut appear to be conserved at approximately the genus level, a resolution associated with the level of conservation of glycoside hydrolase genes in bacteria generally. A focus on two particular types of traits—response and effect traits—may also aid in microbiome management, whether that means maintaining human health or mitigating climate change impacts. Future work on microbial traits must consider three challenges: the influence of different trait measurements on cross-study comparisons; correlations between traits within and among microorganisms; and interactions among microbial traits, the environment, and other organisms. Our conclusions also have implications for the growing field of community phylogenetics beyond applications to microorganisms. Measuring and mapping the phylogenetic distribution of microbial traits. Microbial traits encompass a range of phenotypic characteristics that vary in complexity, including (clockwise from top) virus resistance, cellulose degradation, salinity preference, nitrogen fixation, biofilm formation, and the production of alkaline phosphatase. Each trait can be measured in innumerable ways. For instance, it can be described by discrete or continuous metrics (e.g., the presence of a gene versus the number of gene copies) of potential or realized phenotypes (e.g., those assayed by functional metagenomics versus in situ activity). [Credits: C. Wiehe; M. Maltz; J. Martiny; L. Riemann; J. Haagensen; K. Frischkorn] A focus on the phenotypic characteristics of microorganisms—their traits—offers a path for interpreting the growing amount of microbiome data. We review key aspects of microbial traits, as well as approaches used to assay their phylogenetic distribution. Recent studies reveal that microbial traits are differentially conserved across the tree of life and appear to be conserved in a hierarchical fashion, possibly linked to their biochemical complexity. These results suggest a predictive framework whereby the genetic (or taxonomic) resolution of microbiome variation among samples provides information about the traits under selection. The organizational parallels seen among human and free-living microbiomes seem to support this idea. Developments in this framework may offer predictions not only for how microbial composition responds to changing environmental conditions, but also for how these changes may alter the health or functioning in human, engineered, and environmental systems.

Microbial abundance and composition influence litter decomposition response to environmental change

Rates of ecosystem processes such as decomposition are likely to change as a result of human impacts on the environment. In southern California, climate change and nitrogen (N) deposition in particular may alter biological communities and ecosystem processes. These drivers may affect decomposition directly, through changes in abiotic conditions, and indirectly through changes in plant and decomposer communities. To assess indirect effects on litter decomposition, we reciprocally transplanted microbial communities and plant litter among control and treatment plots (either drought or N addition) in a grassland ecosystem. We hypothesized that drought would reduce decomposition rates through moisture limitation of decomposers and reductions in plant litter quality before and during decomposition. In contrast, we predicted that N deposition would stimulate decomposition by relieving N limitation of decomposers and improving plant litter quality. We also hypothesized that adaptive mechanisms would allow microbes to decompose litter more effectively in their native plot and litter environments. Consistent with our first hypothesis, we found that drought treatment reduced litter mass loss from 20.9% to 15.3% after six months. There was a similar decline in mass loss of litter inoculated with microbes transplanted from the drought treatment, suggesting a legacy effect of drought driven by declines in microbial abundance and possible changes in microbial community composition. Bacterial cell densities were up to 86% lower in drought plots and at least 50% lower on litter derived from the drought treatment, whereas fungal hyphal lengths increased by 13-14% in the drought treatment. Nitrogen effects on decomposition rates and microbial abundances were weaker than drought effects, although N addition significantly altered initial plant litter chemistry and litter chemistry during decomposition. However, we did find support for microbial adaptation to N addition with N-derived microbes facilitating greater mass loss in N plots than in control plots. Our results show that environmental changes can affect rates of ecosystem processes directly through abiotic changes and indirectly through microbial abundances and communities. Therefore models of ecosystem response to global change may need to represent microbial biomass and community composition to make accurate predictions.