Global metabolome analysis of Dunaliella tertiolecta, Phaeobacter italicus R11 Co-cultures using thermal desorption - Comprehensive two-dimensional gas chromatography - Time-of-flight mass spectrometry (TD-GC×GC-TOFMS).

Dunaliella tertiolecta is a marine microalgae that has been studied extensively as a potential carbon-neutral biofuel source (Tang et al., 2011). Microalgae oil contains high quantities of energy-rich fatty acids and lipids, but is not yet commercially viable as an alternative fuel. Carefully optimised growth conditions, and more recently, algal-bacterial co-cultures have been explored as a way of improving the yield of D. tertiolecta microalgae oils. The relationship between the host microalgae and bacterial co-cultures is currently poorly understood. Here, a complete workflow is proposed to analyse the global metabolomic profile of co-cultured D. tertiolectra and Phaeobacter italicus R11, which will enable researchers to explore the chemical nature of this relationship in more detail. To the best of the authors' knowledge this study is one of the first of its kind, in which a pipeline for an entirely untargeted analysis of the algal metabolome is proposed using a practical sample preparation, introduction, and data analysis routine.

Dynamic Subspecies Population Structure of Vibrio cholerae in Dhaka, Bangladesh.

Cholera has been endemic to the Ganges Delta for centuries. Although the causative agent, Vibrio cholerae, is autochthonous to coastal and brackish water, cholera occurs continually in Dhaka, the inland capital city of Bangladesh which is surrounded by fresh water. Despite the persistence of this problem, little is known about the environmental abundance and distribution of lineages of V. cholerae, the most important being the pandemic generating (PG) lineage consisting mostly of serogroup O1 strains. To understand spatial and temporal dynamics of PG lineage and other lineages belonging to the V. cholerae species in surface water in and around Dhaka City, we used qPCR and high-throughput amplicon sequencing. Seven different freshwater sites across Dhaka were investigated for six consecutive months, and physiochemical parameters were measured in situ. Total abundance of V. cholerae was found to be relatively stable throughout the 6-month sampling period, with 2 × 105 to 4 × 105 genome copies/L at six sites and around 5 × 105 genome copies/L at the site located in the most densely populated part of Dhaka City. PG O1 V. cholerae was present in high abundance during the entire sampling period and composed between 24 and 92% of the total V. cholerae population, only showing occasional but sudden reductions in abundance. In instances where PG O1 lost its dominance, other lineages underwent a rapid expansion while the size of the total V. cholerae population remained almost unchanged. Intraspecies richness of V. cholerae was positively correlated with salinity, conductivity, and total dissolved solids (TDS), while it was negatively correlated with dissolved oxygen (DO) concentration in water. Interestingly, negative correlation was observed specifically between PG O1 and salinity, even though the changes in this variable were minor (0-0.8 ppt). Observations in this study suggest that at the subspecies level, population composition of naturally occurring V. cholerae can be influenced by fluctuations in environmental factors, which can lead to altered competition dynamics among the lineages.

Roseobacters in a Sea of Poly- and Paraphyly: Whole Genome-Based Taxonomy of the Family Rhodobacteraceae and the Proposal for the Split of the "Roseobacter Clade" Into a Novel Family, Roseobacteraceae fam. nov.

The family Rhodobacteraceae consists of alphaproteobacteria that are metabolically, phenotypically, and ecologically diverse. It includes the roseobacter clade, an informal designation, representing one of the most abundant groups of marine bacteria. The rapid pace of discovery of novel roseobacters in the last three decades meant that the best practice for taxonomic classification, a polyphasic approach utilizing phenotypic, genotypic, and phylogenetic characteristics, was not always followed. Early efforts for classification relied heavily on 16S rRNA gene sequence similarity and resulted in numerous taxonomic inconsistencies, with several poly- and paraphyletic genera within this family. Next-generation sequencing technologies have allowed whole-genome sequences to be obtained for most type strains, making a revision of their taxonomy possible. In this study, we performed whole-genome phylogenetic and genotypic analyses combined with a meta-analysis of phenotypic data to review taxonomic classifications of 331 type strains (under 119 genera) within the Rhodobacteraceae family. Representatives of the roseobacter clade not only have different environmental adaptions from other Rhodobacteraceae isolates but were also found to be distinct based on genomic, phylogenetic, and in silico-predicted phenotypic data. As such, we propose to move this group of bacteria into a new family, Roseobacteraceae fam. nov. In total, reclassifications resulted to 327 species and 128 genera, suggesting that misidentification is more problematic at the genus than species level. By resolving taxonomic inconsistencies of type strains within this family, we have established a set of coherent criteria based on whole-genome-based analyses that will help guide future taxonomic efforts and prevent the propagation of errors.

Modular Molecular Weaponry Plays a Key Role in Competition Within an Environmental Vibrio cholerae Population.

The type VI secretion system (T6SS) operons of Vibrio cholerae contain extraordinarily diverse arrays of toxic effector and cognate immunity genes, which are thought to play an important role in the environmental lifestyle and adaptation of this human pathogen. Through the T6SS, proteinaceous "spears" tipped with antibacterial effectors are injected into adjacent cells, killing those not possessing immunity proteins to these effectors. Here, we investigate the T6SS-mediated dynamics of bacterial competition within a single environmental population of V. cholerae. We show that numerous members of a North American V. cholerae population possess strain-specific repertoires of cytotoxic T6SS effector and immunity genes. Using pairwise competition assays, we demonstrate that the vast majority of T6SS-mediated duels end in stalemates between strains with different T6SS repertoires. However, horizontally acquired effector and immunity genes can significantly alter the outcome of these competitions. Frequently observed horizontal gene transfer events can both increase or reduce competition between distantly related strains by homogenizing or diversifying the T6SS repertoire. Our results also suggest temperature-dependent outcomes in T6SS competition, with environmental isolates faring better against a pathogenic strain under native conditions than under those resembling a host-associated environment. Taken altogether, these interactions produce density-dependent fitness effects and a constant T6SS-mediated arms race in individual V. cholerae populations, which could ultimately preserve intraspecies diversity. Since T6SSs are widespread, we expect within-population diversity in T6SS repertoires and the resulting competitive dynamics to be a common theme in bacterial species harboring this machinery.

Simultaneous Quantification of Vibrio metoecus and Vibrio cholerae with Its O1 Serogroup and Toxigenic Subpopulations in Environmental Reservoirs.

Vibrio metoecus is a recently described aquatic bacterium and opportunistic pathogen, closely related to and often coexisting with Vibrio cholerae. To study the relative abundance and population dynamics of both species in aquatic environments of cholera-endemic and cholera-free regions, we developed a multiplex qPCR assay allowing simultaneous quantification of total V. metoecus and V. cholerae (including toxigenic and O1 serogroup) cells. The presence of V. metoecus was restricted to samples from regions that are not endemic for cholera, where it was found at 20% of the abundance of V. cholerae. In this environment, non-toxigenic O1 serogroup V. cholerae represents almost one-fifth of the total V. cholerae population. In contrast, toxigenic O1 serogroup V. cholerae was also present in low abundance on the coast of cholera-endemic regions, but sustained in relatively high proportions throughout the year in inland waters. The majority of cells from both Vibrio species were recovered from particles rather than free-living, indicating a potential preference for attached versus planktonic lifestyles. This research further elucidates the population dynamics underpinning V. cholerae and its closest relative in cholera-endemic and non-endemic regions through culture-independent quantification from environmental samples.

Culture-independent tracking of Vibrio cholerae lineages reveals complex spatiotemporal dynamics in a natural population.

Populations of the bacterium Vibrio cholerae consist of dozens of distinct lineages, with primarily (but not exclusively) members of the pandemic generating lineage capable of causing the diarrhoeal disease cholera. Assessing the composition and temporal dynamics of such populations requires extensive isolation efforts and thus only rarely covers large geographic areas or timeframes exhaustively. We developed a culture-independent amplicon sequencing strategy based on the protein-coding gene viuB (vibriobactin utilization) to study the structure of a V. cholerae population over the course of a summer. We show that the 26 co-occurring V. cholerae lineages continuously compete for limited space on nutrient-rich particles where only a few of them can grow to large numbers. Differential abundance of lineages between locations and size-fractions associated with a particle-attached or free-swimming lifestyle could reflect adaptation to various environmental niches. In particular, a major V. cholerae lineage occasionally grows to large numbers on particles but remain undetectable using isolation-based methods, indicating selective culturability for some members of the species. We thus demonstrate that isolation-based studies may not accurately reflect the structure and complex dynamics of V. cholerae populations and provide a scalable high-throughput method for both epidemiological and ecological approaches to studying this species.

Phaeobacter inhibens induces apoptosis-like programmed cell death in calcifying Emiliania huxleyi.

The model coccolithophore, Emiliania huxleyi, forms expansive blooms dominated by the calcifying cell type, which produce calcite scales called coccoliths. Blooms last several weeks, after which the calcified algal cells rapidly die, descending into the deep ocean. E. huxleyi bloom collapse is attributed to E. huxleyi viruses (EhVs) that infect and kill calcifying cells, while other E. huxleyi pathogens, such as bacteria belonging to the roseobacter clade, are overlooked. EhVs kill calcifying E. huxleyi by inducing production of bioactive viral-glycosphingolipids (vGSLs), which trigger algal programmed cell death (PCD). The roseobacter Phaeobacter inhibens was recently shown to interact with and kill the calcifying cell type of E. huxleyi, but the mechanism of algal death remains unelucidated. Here we demonstrate that P. inhibens kills calcifying E. huxleyi by inducing a highly specific type of PCD called apoptosis-like-PCD (AL-PCD). Host death can successfully be abolished in the presence of a pan-caspase inhibitor, which prevents the activation of caspase-like molecules. This finding differentiates P. inhibens and EhV pathogenesis of E. huxleyi, by demonstrating that bacterial-induced AL-PCD requires active caspase-like molecules, while the viral pathogen does not. This is the first demonstration of a bacterium inducing AL-PCD in an algal host as a killing mechanism.

Interactions within the microbiome alter microbial interactions with host chemical defences and affect disease in a marine holobiont.

Our understanding of diseases has been transformed by the realisation that people are holobionts, comprised of a host and its associated microbiome(s). Disease can also have devastating effects on populations of marine organisms, including dominant habitat formers such as seaweed holobionts. However, we know very little about how interactions between microorganisms within microbiomes - of humans or marine organisms - affect host health and there is no underpinning theoretical framework for exploring this. We applied ecological models of succession to bacterial communities to understand how interactions within a seaweed microbiome affect the host. We observed succession of surface microbiomes on the red seaweed Delisea pulchra in situ, following a disturbance, with communities 'recovering' to resemble undisturbed states after only 12 days. Further, if this recovery was perturbed, a bleaching disease previously described for this seaweed developed. Early successional strains of bacteria protected the host from colonisation by a pathogenic, later successional strain. Host chemical defences also prevented disease, such that within-microbiome interactions were most important when the host's chemical defences were inhibited. This is the first experimental evidence that interactions within microbiomes have important implications for host health and disease in a dominant marine habitat-forming organism.

Bacillus amyloliquefaciens ssp. plantarum F11 isolated from Algerian salty lake as a source of biosurfactants and bioactive lipopeptides.

In this study, we identified a new Bacillus strain isolated from an Algerian salty lake that produces metabolites that are active against Gram-positive and Gram-negative bacteria, as well as fungal pathogens. The draft genome sequence of the strain is presented herein. Genome sequence analysis identified the strain to be B. amyloliquefaciens subspecies plantarum F11, and showed that the strain carries the gene clusters for the production of a number of bioactive and surface-active compounds. These include the lipopeptides surfactin and fengycin, antibacterial polyketides macrolactin and bacillaene, and a putative novel lanthipeptide, among others. Through an activity-guided purification method using hydrophobic interaction chromatographic techniques, we confirmed the ability of the strain to produce fengycin lipopeptides. The identities of the isolated fengycin homologs were ascertained through tandem mass spectrometry.

Draft Genome Sequences of Bacillus cereus E41 and Bacillus anthracis F34 Isolated from Algerian Salt Lakes.

Two strains of Bacillus, B. cereus E41 and B. anthracis F34, were isolated from a salt lake in Aïn M'lila-Oum El Bouaghi, eastern Algeria, and Ain Baida-Ouargla, southern Algeria, respectively. Their genomes display genes for the production of several bioactive secondary metabolites, including polyhydroxyalkanoate, iron siderophores, lipopeptides, and bacteriocins.

A Small Number of Phylogenetically Distinct Clonal Complexes Dominate a Coastal Vibrio cholerae Population.

UNLABELLED: Vibrio cholerae is a ubiquitous aquatic microbe in temperate and tropical coastal areas. It is a diverse species, with many isolates that are harmless to humans, while others are highly pathogenic. Most notable among them are strains belonging to the pandemic O1/O139 serogroup lineage, which contains the causative agents of cholera. The environmental selective regimes that led to this diversity are key to understanding how pathogens evolve in environmental reservoirs. A local population of V. cholerae and its close relative Vibrio metoecus from a coastal pond and lagoon system was extensively sampled during two consecutive months across four size fractions (480 isolates). In stark contrast to previous studies, the observed population was highly clonal, with 60% of V. cholerae isolates falling into one of five clonal complexes, which varied in abundance in the short temporal scale sampled. V. cholerae clonal complexes had significantly different distributions across size fractions and the two environments sampled, the pond and the lagoon. Sequencing the genomes of 20 isolates representing these five V. cholerae clonal complexes revealed different evolutionary trajectories, with considerable variations in gene content with potential ecological significance. Showing genotypic differentiation and differential spatial distribution, the dominant clonal complexes are likely ecologically divergent. Temporal variation in the relative abundance of these complexes suggests that transient blooms of specific clones could dominate local diversity. IMPORTANCE: Vibrio cholerae is commonly found in coastal areas worldwide, with only a single group of this bacterium capable of causing severe cholera outbreaks. However, the potential to evolve the ability to cause disease exists in many strains of this species in its aquatic reservoir. Understanding how pathogenic bacteria evolve requires the study of their natural environments. By extensive sampling in a geographically restricted location in the United States, we found that most cells of a V. cholerae population belong to only a small number of strains. Analysis of their genome composition and spatial distribution indicates differential environmental adaptations between these strains. Other strains exist in smaller numbers, and the population was found to be temporally varied. This suggests frequent bloom and collapse cycles on a time scale of weeks. These population dynamics make it possible that more virulent strains could stochastically rise to large numbers, allowing for infection to occur.

Indole-3-Acetic Acid Is Produced by Emiliania huxleyi Coccolith-Bearing Cells and Triggers a Physiological Response in Bald Cells.

Indole-3-acetic acid (IAA) is an auxin produced by terrestrial plants which influences development through a variety of cellular mechanisms, such as altering cell orientation, organ development, fertility, and cell elongation. IAA is also produced by bacterial pathogens and symbionts of plants and algae, allowing them to manipulate growth and development of their host. They do so by either producing excess exogenous IAA or hijacking the IAA biosynthesis pathway of their host. The endogenous production of IAA by algae remains contentious. Using Emiliania huxleyi, a globally abundant marine haptophyte, we investigated the presence and potential role of IAA in algae. Homologs of genes involved in several tryptophan-dependent IAA biosynthesis pathways were identified in E. huxleyi. This suggests that this haptophyte can synthesize IAA using various precursors derived from tryptophan. Addition of L-tryptophan to E. huxleyi stimulated IAA production, which could be detected using Salkowski's reagent and GC × GC-TOFMS in the C cell type (coccolith bearing), but not in the N cell type (bald). Various concentrations of IAA were exogenously added to these two cell types to identify a physiological response in E. huxleyi. The N cell type, which did not produce IAA, was more sensitive to it, showing an increased variation in cell size, membrane permeability, and a corresponding increase in the photosynthetic potential quantum yield of Photosystem II (PSII). A roseobacter (bacteria commonly associated with E. huxleyi) Ruegeria sp. R11, previously shown to produce IAA, was co-cultured with E. huxleyi C and N cells. IAA could not be detected from these co-cultures, and even when stimulated by addition of L-tryptophan, they produced less IAA than axenic C type culture similarly induced. This suggests that IAA plays a novel role signaling between different E. huxleyi cell types, rather than between a bacteria and its algal host.

A Bacterial Pathogen Displaying Temperature-Enhanced Virulence of the Microalga Emiliania huxleyi.

Emiliania huxleyi is a globally abundant microalga that plays a significant role in biogeochemical cycles. Over the next century, sea surface temperatures are predicted to increase drastically, which will likely have significant effects on the survival and ecology of E. huxleyi. In a warming ocean, this microalga may become increasingly vulnerable to pathogens, particularly those with temperature-dependent virulence. Ruegeria is a genus of Rhodobacteraceae whose population size tracks that of E. huxleyi throughout the alga's bloom-bust lifecycle. A representative of this genus, Ruegeria sp. R11, is known to cause bleaching disease in a red macroalga at elevated temperatures. To investigate if the pathogenicity of R11 extends to microalgae, it was co-cultured with several cell types of E. huxleyi near the alga's optimum (18°C), and at an elevated temperature (25°C) known to induce virulence in R11. The algal populations were monitored using flow cytometry and pulse-amplitude modulated fluorometry. Cultures of algae without bacteria remained healthy at 18°C, but lower cell counts in control cultures at 25°C indicated some stress at the elevated temperature. Both the C (coccolith-bearing) and S (scale-bearing swarming) cell types of E. huxleyi experienced a rapid decline resulting in apparent death when co-cultured with R11 at 25°C, but had no effect on N (naked) cell type at either temperature. R11 had no initial negative impact on C and S type E. huxleyi population size or health at 18°C, but caused death in older co-cultures. This differential effect of R11 on its host at 18 and 25°C suggest it is a temperature-enhanced opportunistic pathogen of E. huxleyi. We also detected caspase-like activity in dying C type cells co-cultured with R11, which suggests that programmed cell death plays a role in the death of E. huxleyi triggered by R11 - a mechanism induced by viruses (EhVs) and implicated in E. huxleyi bloom collapse. Given that E. huxleyi has recently been shown to have acquired resistance against EhVs at elevated temperature, bacterial pathogens with temperature-dependent virulence, such as R11, may become much more important in the ecology of E. huxleyi in a warming climate.

Draft Genome Sequences of Four Bacterial Strains Isolated from a Polymicrobial Culture of Naked (N-Type) Emiliania huxleyi CCMP1516.

Strains of Sulfitobacter spp., Erythrobacter sp., and Marinobacter sp. were isolated from a polymicrobial culture of the naked (N-type) haptophyte Emiliania huxleyi strain CCMP1516. The genomes encode genes for the production of phytohormones, vitamins, and the consumption of their hosts' metabolic by-products, suggesting symbiotic interactions within this polymicrobial culture.

Draft Genome Sequences of Seven Bacterial Strains Isolated from a Polymicrobial Culture of Coccolith-Bearing (C-Type) Emiliania huxleyi M217.

Strains of Rhodobacteraceae, Sphingomonadales, Alteromonadales, and Bacteroidetes were isolated from a polymicrobial culture of the coccolith-forming (C-type) haptophyte Emiliania huxleyi strain M217. The genomes encode genes for the production of algal growth factors and the consumption of their hosts' metabolic by-products, suggesting that the polymicrobial culture harbors many symbiotic interactions.

Characterization of clinical and environmental isolates of Vibrio cidicii sp. nov., a close relative of Vibrio navarrensis.

Four Vibrio spp. isolates from the historical culture collection at the Centers for Disease Control and Prevention, obtained from human blood specimens (n=3) and river water (n=1), show characteristics distinct from those of isolates of the most closely related species, Vibrio navarrensis and Vibrio vulnificus, based on phenotypic and genotypic tests. They are specifically adapted to survival in both freshwater and seawater, being able to grow in rich media without added salts as well as salinities above that of seawater. Phenotypically, these isolates resemble V. navarrensis, their closest known relative with a validly published name, but the group of isolates is distinguished from V. navarrensis by the ability to utilize l-rhamnose. Average nucleotide identity and percent DNA-DNA hybridization values obtained from the pairwise comparisons of whole-genome sequences of these isolates to V. navarrensis range from 95.4-95.8 % and 61.9-64.3 %, respectively, suggesting that the group represents a different species. Phylogenetic analysis of the core genome, including four protein-coding housekeeping genes (pyrH, recA, rpoA and rpoB), places these four isolates into their own monophyletic clade, distinct from V. navarrensis and V. vulnificus. Based on these differences, we propose these isolates represent a novel species of the genus Vibrio, for which the name Vibrio cidicii sp. nov. is proposed; strain LMG 29267T (=CIP 111013T=2756-81T), isolated from river water, is the type strain.

Ancient origin of the biosynthesis of lignin precursors.

BACKGROUND: Lignin plays an important role in plant structural support and water transport, and is considered one of the hallmarks of land plants. The recent discovery of lignin or its precursors in various algae has raised questions on the evolution of its biosynthetic pathway, which could be much more ancient than previously thought. To determine the taxonomic distribution of the lignin biosynthesis genes, we screened all publicly available genomes of algae and their closest non-photosynthetic relatives, as well as representative land plants. We also performed phylogenetic analysis of these genes to decipher the evolution and origin(s) of lignin biosynthesis. RESULTS: Enzymes involved in making p-coumaryl alcohol, the simplest lignin monomer, are found in a variety of photosynthetic eukaryotes, including diatoms, dinoflagellates, haptophytes, cryptophytes as well as green and red algae. Phylogenetic analysis of these enzymes suggests that they are ancient and spread to some secondarily photosynthetic lineages when they acquired red and/or green algal endosymbionts. In some cases, one or more of these enzymes was likely acquired through lateral gene transfer (LGT) from bacteria. CONCLUSIONS: Genes associated with p-coumaryl alcohol biosynthesis are likely to have evolved long before the transition of photosynthetic eukaryotes to land. The original function of this lignin precursor is therefore unlikely to have been related to water transport. We suggest that it participates in the biological defense of some unicellular and multicellular algae.

A small volume bioassay to assess bacterial/phytoplankton co-culture using WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry.

Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be subsampled throughout the experiment1-7. Subsampling of large volumes can be problematic for several reasons: 1) it causes variation in the total volume and the surface area:volume ratio of the culture during the experiment; 2) pseudo-replication (i.e., replicate samples from the same treatment flask8) is often employed rather than true replicates (i.e., sampling from replicate treatments); 3) the duration of the experiment is limited by the total volume; and 4) axenic cultures or the usual bacterial microbiota are difficult to maintain during long-term experiments as contamination commonly occurs during subsampling. The use of microtiter plates enables 1 ml culture volumes to be used for each replicate, with up to 48 separate treatments within a 12.65x8.5x2.2 cm plate, thereby decreasing the experimental volume and allowing for extensive replication without subsampling any treatment. Additionally, this technique can be modified to fit a variety of experimental formats including: bacterial-algal co-cultures, algal physiology tests, and toxin screening9-11. Individual wells with an alga, bacterium and/or co-cultures can be sampled for numerous laboratory procedures including, but not limited to: WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry, microscopy, bacterial colony forming unit (cfu) counts and flow cytometry. The combination of the microtiter plate format and WATER-PAM fluorometry allows for multiple rapid measurements of photochemical yield and other photochemical parameters with low variability between samples, high reproducibility and avoids the many pitfalls of subsampling a carboy or conical flask over the course of an experiment.

Genomes and virulence factors of novel bacterial pathogens causing bleaching disease in the marine red alga Delisea pulchra.

Nautella sp. R11, a member of the marine Roseobacter clade, causes a bleaching disease in the temperate-marine red macroalga, Delisea pulchra. To begin to elucidate the molecular mechanisms underpinning the ability of Nautella sp. R11 to colonize, invade and induce bleaching of D. pulchra, we sequenced and analyzed its genome. The genome encodes several factors such as adhesion mechanisms, systems for the transport of algal metabolites, enzymes that confer resistance to oxidative stress, cytolysins, and global regulatory mechanisms that may allow for the switch of Nautella sp. R11 to a pathogenic lifestyle. Many virulence effectors common in phytopathogenic bacteria are also found in the R11 genome, such as the plant hormone indole acetic acid, cellulose fibrils, succinoglycan and nodulation protein L. Comparative genomics with non-pathogenic Roseobacter strains and a newly identified pathogen, Phaeobacter sp. LSS9, revealed a patchy distribution of putative virulence factors in all genomes, but also led to the identification of a quorum sensing (QS) dependent transcriptional regulator that was unique to pathogenic Roseobacter strains. This observation supports the model that a combination of virulence factors and QS-dependent regulatory mechanisms enables indigenous members of the host alga's epiphytic microbial community to switch to a pathogenic lifestyle, especially under environmental conditions when innate host defence mechanisms are compromised.

Molecular musings in microbial ecology and evolution.

A few major discoveries have influenced how ecologists and evolutionists study microbes. Here, in the format of an interview, we answer questions that directly relate to how these discoveries are perceived in these two branches of microbiology, and how they have impacted on both scientific thinking and methodology.The first question is "What has been the influence of the 'Universal Tree of Life' based on molecular markers?" For evolutionists, the tree was a tool to understand the past of known (cultured) organisms, mapping the invention of various physiologies on the evolutionary history of microbes. For ecologists the tree was a guide to discover the current diversity of unknown (uncultured) organisms, without much knowledge of their physiology.The second question we ask is "What was the impact of discovering frequent lateral gene transfer among microbes?" In evolutionary microbiology, frequent lateral gene transfer (LGT) made a simple description of relationships between organisms impossible, and for microbial ecologists, functions could not be easily linked to specific genotypes. Both fields initially resisted LGT, but methods or topics of inquiry were eventually changed in one to incorporate LGT in its theoretical models (evolution) and in the other to achieve its goals despite that phenomenon (ecology).The third and last question we ask is "What are the implications of the unexpected extent of diversity?" The variation in the extent of diversity between organisms invalidated the universality of species definitions based on molecular criteria, a major obstacle to the adaptation of models developed for the study of macroscopic eukaryotes to evolutionary microbiology. This issue has not overtly affected microbial ecology, as it had already abandoned species in favor of the more flexible operational taxonomic units. This field is nonetheless moving away from traditional methods to measure diversity, as they do not provide enough resolution to uncover what lies below the species level.The answers of the evolutionary microbiologist and microbial ecologist to these three questions illustrate differences in their theoretical frameworks. These differences mean that both fields can react quite distinctly to the same discovery, incorporating it with more or less difficulty in their scientific practice.