2-n-Pentyl-4-Quinolinol Produced by a Marine Alteromonas sp. and Its Potential Ecological and Biogeochemical Roles

Bacterium-bacterium interactions occur at intimate spatial scales on the order of micrometers, but our knowledge of interactions at this level is rudimentary. Antagonism is a potential interaction in such microenvironments. To study the ecological role of antibiosis, we developed a model system involving an antibiotic-producing isolate (SWAT5) derived from a marine particle and its dominant antibiotic product, 2-n-pentyl-4-quinolinol (PQ). This system was used to address questions about the significance of this antibiotic for microbial ecology and carbon cycling on particles. We characterized the chemical and inhibitory properties of PQ in relation to the mechanisms used by particle-associated bacteria in interacting with particles and with other attached bacteria. PQ was produced by SWAT5 only on surfaces. When SWAT5 was grown in polysaccharide matrices, PQ diffused within the matrices but not into the surrounding seawater. SWAT5 might thus be able to generate a localized zone of high antibiotic concentration on particles suspended or sinking through seawater. Target bacterial respiration was most sensitive to PQ (75 nM), while inhibition of DNA synthesis, protein synthesis, and bacterial motility required higher (micromolar) PQ levels. The presence of PQ altered the composition of the bacterial community that colonized and developed in a model particle system. PQ also inhibited Synechococcus and phytoplankton growth. Our results suggest that antibiosis may significantly influence community composition and activities of attached bacterial and thus regulate the biogeochemical fate of particulate organic matter in the ocean.     

Effects of Inorganic Particles on Metabolism by a Periphytic Marine Bacterium

Measurements were made of adsorption of a periphytic marine bacterium, glucose, and glutamic acid to inorganic particles in seawater and defined bacterial growth medium. Measurements of the metabolism of bacteria were made in the presence and absence of particles by microcalorimetry and radiorespirometry. It was found that hydroxyapatite adsorbs glutamic acid, but not glucose, from the experimental medium. It was also found that hydroxyapatite adsorbs essentially all of the bacteria from the medium when the bacterial concentration is approximately 6 × 10⁵ bacteria per ml. If the bacterial concentration is approximately 6 × 10⁷, then only a small fraction of cells become attached. It was therefore possible to select bacterial concentrations and organic nutrients so that bacterial attachment, organic nutrient adsorption, or both would occur in different experiments. In this experimental system the metabolism by attached and nonattached bacteria of adsorbing and nonadsorbing organic nutrients was measured. The results show that bacterial activity in this model system was not enhanced by the particles, regardless of whether the bacteria, the organic nutrient, or both were associated with the surface. In fact, the respiratory activity of the attached bacteria was diminished in comparison with that of free bacteria.     

Bacterial Colonization and Ectoenzymatic Activity in Phytoplankton-Derived Model Particles: Cleavage of Peptides and Uptake of Amino Acids

Abstract Phytoplankton-derived model particles were created in laboratory from a mixture of autoclaved diatom cultures. These particles were colonized by a marine bacterial community and incubated in rolling tanks in order to examine the relationship between aminopeptidase activity and leucine uptake. Bacteria inhabiting particles and ambient water were characterized for abundance, biovolume, aminopeptidase activity, leucine uptake, and growth rate. Particles were a less favorable habitat than ambient water for bacterial growth since growth rates of particle-attached bacteria were similar or even lower than those of free-living bacteria. During the first ∼100 h of the particle decomposition process, there were not statistically significant differences in the aminopeptidase activity:leucine uptake ratio between attached and free-living bacteria. From ∼100 h to ∼200 h, this ratio was higher for attached bacteria than for free-living bacteria. This indicates an uncoupling of aminopeptidase activity and leucine uptake. During this period, attached and free-living bacteria showed similar hydrolytic activities on a cell-specific basis. In the free-living bacterial community, variations in aminopeptidase activity per cell were associated with variations in leucine uptake per cell and growth rates. However, in the attached bacterial community, when leucine uptake and growth rates decreased, aminopeptidase activity remained constant. Thus, after ∼100 h, particle-attached bacteria were not taking advantage of their high aminopeptidase activity; consequently the hydrolysed amino acids were released into the ambient water, supporting the growth of free-living bacteria. These results demonstrate that over the particle decomposition process, the relationship between hydrolysis and uptake of the protein fraction shows different patterns of variation for attached and free-living bacterial communities. However, in our experiments, this uncoupling was not based on a hyperproduction of enzymes by attached bacteria, but on lower uptake rates when compared to the free-living bacteria. Received: 4 February 1997; Accepted: 9 May 1997     

Links between Phytoplankton and Bacterial Community Dynamics in a Coastal Marine Environment

Bacteria and phytoplankton dynamics are thought to be closely linked in coastal marine environments, with correlations frequently observed between bacterial and phytoplankton biomass. In contrast, little is known about how these communities interact with each other at the species composition level. The purpose of the current study was to analyze bacterial community dynamics in a productive, coastal ecosystem and to determine whether they were related to phytoplankton community dynamics. Near-surface seawater samples were collected in February, May, July, and September 2000 from several stations in the Bay of Fundy. Savin et al. (M.C. Savin et al., Microb Ecol 48: 51-65) analyzed the phytoplankton community in simultaneously collected samples. The attached and free-living bacterial communities were collected by successive filtration onto 5 m and 0.22 m pore-size filters, respectively. DNA was extracted from filters and bacterial 16S rRNA gene fragments were amplified and analyzed by denaturing gradient gel electrophoresis (DGGE). DGGE revealed that diversity and temporal variability were lower in the free-living than the attached bacterial community. Both attached and free-living communities were dominated by members of the Roseobacter and Cytophaga groups. Correspondence analysis (CA) ordination diagrams showed similar patterns for the phytoplankton and attached bacterial communities, indicating that shifts in the species composition of these communities were linked. Similarly, canonical CA revealed that the diversity, abundance, and percentage of diatoms in the phytoplankton community accounted for a significant amount of the variability in the attached bacterial community composition. In contrast, ordination analyses did not reveal an association between free-living bacteria and phytoplankton. These results suggest that there are specific interactions between phytoplankton and the bacteria attached to them, and that these interactions influence the composition of both communities.     

Impact of hydrology on free-living and particle-associated microorganisms in a river floodplain system (Danube, Austria)

1. A floodplain segment of the Danube River downstream of Vienna was studied during the hydrologically most dynamic phase (spring–summer) to evaluate the significance of connection between the main channel and the floodplain segment for particle abundance and quality as well as for bacterial and viral parameters, both free‐living and attached to particles. 2. Hydrological connectivity between the main channel and its floodplain segment (expressed as water age) influenced particle abundance and quality. Polysaccharide‐containing particles [Alcian positive‐stained particles (ASP)] and protein‐containing particles [Coomassie positive‐stained particles (CSP)] each contributed a substantial fraction to total suspended solids and were both positively related to water age. ASP were about twice as abundant as CSP. 3. Water age influenced bacterial and viral abundance and the bacterioplankton productivity in the surrounding water. Free‐living bacterial abundance and their bacterial secondary production (BSP) increased continuously with water age, best described by a linear regression. Water age also significantly impacted BSP and per cell BSP of bacteria attached to particles. The abundance of attached bacteria and viruses was not influenced by water age. 4. Bacteria and viruses on particles were influenced by particle quality. Their abundance on particles was closely related to particle size. Particle‐attached bacteria accounted on average for 30.34% (± 3.09) of the total bacterial abundance. A variable and occasionally significant proportion of viruses, between 0.43% and 35.06%, were associated with particulate material. 5. Bacteria attached to particles were significantly more productive than their free‐living counterparts. Their per‐cell activity was on average 8.6 times higher than that of the free‐living fraction. 6. Hydrological connectivity between the Danube River and its floodplain is crucial not only for the exchange of water, sediment and nutrients, but also for microbiota, thus influencing microbial life, distribution and activity.     

Studies on bacteria-like particles sampled from the stratosphere

Bacteria-like particles recovered from the stratosphere and deposited on cellulose acetate membranes have been analysed to confirm their bacterial nature. One particle appeared to be attached to an inorganic particle apparently by mucoid material typically produced by bacteria. A filamentous structure, morphologically similar to a fungal hypha, was also observed. EDS analysis showed that the particles were all non-mineral and therefore could be biological in nature. However, the composition of several clumps of nanobacteria-sized particles were found, by SIMS analysis, to be inconsistent with that of bacteria. The results show that it is dangerous to assume that bacteria-like particles seen under scanning electron microscopy are necessarily bacteria.     

Bacterial Community Structure of Biofilms on Artificial Surfaces in an Estuary

This study examined bacterial community structure of biofilms on stainless steel and polycarbonate in seawater from the Delaware Bay. Free-living bacteria in the surrounding seawater were compared to the attached bacteria during the first few weeks of biofilm growth. Surfaces exposed to seawater were analyzed by using 16S rDNA libraries, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE). Community structure of the free-living bacterial community was different from that of the attached bacteria according to FISH and DGGE. In particular, alpha-proteobacteria dominated the attached communities. Libraries of 16S rRNA genes revealed that representatives of the Rhodobacterales clade were the most abundant members of biofilm communities. Changes in community structure during biofilm growth were also examined by DGGE analysis. We hypothesized that bacterial communities on dissimilar surfaces would initially differ and become more similar over time. In contrast, the compositions of stainless steel and polycarbonate biofilms were initially the same, but differed after about 1 week of biofilm growth. These data suggest that the relationship between surface properties and biofilm community structure changes as biofilms grow on surfaces such as stainless steel and polycarbonate in estuarine water.     

Community composition and activity of prokaryotes associated to detrital particles in two contrasting lake ecosystems

The composition, distribution and extracellular enzyme activities of bacteria attached to small (2-50 microm in size) transparent exopolymer and Coomassie-stained proteinaceous particles (TEP and CSP) were examined in two lakes of different trophic status located in the Massif Central of France. TEP concentrations (10(4)-10(6) particle per L) were significantly higher in the more productive lake and were significantly related to chlorophyll a concentrations. The majority of TEP and CSP were colonized by bacteria that constituted 2.6% and 7.4% of the total 4',6-diamidino-2-phenylindole-stained bacteria in lakes Pavin and Aydat, respectively. In both lakes, the composition of particle-associated bacteria was different from that of free-living bacteria, the Betaproteobacteria and Bacteroidetes (i.e. former Cytophaga-Flavobacteria group) being the dominant groups on particles. We also found that 2-5 microm TEP were more colonized than 2-5 microm CSP in the two lakes, and that TEP colonization was higher in the less productive lake. Measurements of Leucine aminopeptidase and alpha-glucosidase activities in fractionated lake water (0.2-1.2, 1.2-5 and >5 microm fractions) indicated that proteolytic activity was always higher and that particle-associated bacteria have higher enzymatic activities than free-living bacteria. The glycolytic activities in the 1.2-5 and >5 microm fractions were related to the abundance of TEP. We conclude that small freshwater detrital organic particles constitute microhabitats with high bacterial activities in pelagic environments and, undoubtedly, present significant ecological implications for the prokaryotic community structure and function in aquatic ecosystems.     

Studies on bacteria-like particles sampled from the stratosphere

Bacteria-like particles recovered from the stratosphere and deposited on cellulose acetate membranes have been analysed to confirm their bacterial nature. One particle appeared to be attached to an inorganic particle apparently by mucoid material typically produced by bacteria. A filamentous structure, morphologically similar to a fungal hypha, was also observed. EDS analysis showed that the particles were all non-mineral and therefore could be biological in nature. However, the composition of several clumps of nanobacteria-sized particles were found, by SIMS analysis, to be inconsistent with that of bacteria. The results show that it is dangerous to assume that bacteria-like particles seen under scanning electron microscopy are necessarily bacteria.     

Studies on bacteria-like particles sampled from the stratosphere

Bacteria-like particles recovered from the stratosphere and deposited on cellulose acetate membranes have been analysed to confirm their bacterial nature. One particle appeared to be attached to an inorganic particle apparently by mucoid material typically produced by bacteria. A filamentous structure, morphologically similar to a fungal hypha, was also observed. EDS analysis showed that the particles were all non-mineral and therefore could be biological in nature. However, the composition of several clumps of nanobacteria-sized particles were found, by SIMS analysis, to be inconsistent with that of bacteria. The results show that it is dangerous to assume that bacteria-like particles seen under scanning electron microscopy are necessarily bacteria.     

马里亚纳海沟共培养细菌多样性动态变化及潜在微生物互作关系

马里亚纳海沟是世界已知最深的海沟,其寡营养、高压、低温、低氧等极端的深海环境孕育出独特的细菌群落结构及多样性特征。选取寡营养培养基对马里亚纳海沟海水及表层沉积物分别进行液体共培养,并在不同培养阶段取样进行高通量测序,分析细菌群落结构组成及其多样性的动态变化,探讨微生物之间可能的互作关系。研究结果表明：液体共培养样品中一共检测到19个门、34个纲、76个目、131个科、227个属的细菌,其中变形菌门（Proteobacteria）和拟杆菌门（Bacteroidetes）为优势菌群,其次为厚壁菌门（Firmicutes）;与其他样品相比,1000米海水样品中细菌群落的多样性最高,并且蓝细菌门（Cyanobacteria）具有更高的相对丰度。共培养样品中细菌丰富度、多样性、群落结构均随培养时间而改变,其中共培养中期样品的细菌多样性较高;表层沉积物样本中,盐单胞菌属（Halomonas）可能由于较强的竞争能力在共培养后期占据优势地位。基因功能预测与代谢通路富集结果显示,随着共培养时间的增加,微生物生长相关的代谢通路丰度明显下降,而与互作相关的代谢通路丰度明显增加。共培养样品检测到的细菌多样性远高于单独分离培养的多样性,仅有少量菌属为单独分离培养与共培养样品均检测到的共有属。综上所述,马里亚纳海沟细菌群落中存在竞争、互利共生的相互作用,共培养法有利于揭示细菌间的互作关系。研究为深渊及深海等极端环境下微生物生态系统组成及维持奠定了理论基础,也为进一步研究极端微生物的生存策略提供了科学指导。     

Bacterial colonization of particles: growth and interactions

Marine particles in the ocean are exposed to diverse bacterial communities, and colonization and growth of attached bacteria are important processes in the degradation and transformation of the particles. In an earlier study, we showed that the initial colonization of model particles by individual bacterial strains isolated from marine aggregates was a function of attachment and detachment. In the present study, we have investigated how this colonization process was further affected by growth and interspecific interactions among the bacteria. Long-term incubation experiments showed that growth dominated over attachment and detachment after a few hours in controlling the bacterial population density on agar particles. In the absence of grazing mortality, this growth led to an equilibrium population density consistent with the theoretical limit due to oxygen diffusion. Interspecific interaction experiments showed that the presence of some bacterial strains ("residents") on the agar particles either increased or decreased the colonization rate of other strains ("newcomers"). Comparison between an antibiotic-producing strain and its antibiotic-free mutant showed no inhibitory effect on the newcomers due to antibiotic production. On the contrary, hydrolytic activity of the antibiotic-producing strain appeared to benefit the newcomers and enhance their colonization rate. These results show that growth- and species-specific interactions have to be taken into account to adequately describe bacterial colonization of marine particles. Changes in colonization pattern due to such small-scale processes may have profound effects on the transformation and fluxes of particulate matter in the ocean.     

Action of Penicillin G on Endosymbiote Lambda Particles of Paramecium aurelia

The kinetics of loss from the cytoplasm and changes in ultrastructure of symbiont lambda particles after treatment of axenically cultivated lambda-bearing Paramecium aurelia with penicillin G was investigated. Low concentrations (1 to 2 unit/ml) of the antibiotic caused many particles within the cell to become filamentous; high concentrations (2,000 unit/ml) caused lysis of the particles without noticeably affecting the protozoan. The ED(50) value (2 to 3 unit/ml) was within the range of values found to cause lysis of many gram-negative bacteria. Rapidly dividing lambda were more vulnerable to the action of the antibiotic than slowly dividing particles. Nondividing particles were not affected by exposure to the antibiotic. Ultrastructural changes observed in lambda during lysis by penicillin G were consistent with the view that penicillin interferes with the synthesis of a vital component of the cell envelope of the particle, possibly a peptidoglycan similar to that found in the cell walls of bacteria. The deoxyribonucleic acid of lambda was dispersed throughout the particle as electron dense fibers enclosed within electron transparent areas. The cell envelope appeared to consist of at least two morphologically distinguishable layers, an inner layer homologous to the plasma membrane of bacteria and an outer layer homologous to the bacterial cell wall. Lambda may be regarded as a randomly distributed population of bacteria growing and dividing synchronously within the collective cytoplasm of its protozoan host.     

Iron effects on colonization behavior, motility, and enzymatic activity of marine bacteria

Iron availability in the ocean has been shown to affect the growth and production of phytoplankton and free-living bacteria. A large fraction of marine bacteria are specialized in colonizing and living on particles and aggregates, but the effects of iron limitation on these bacteria are not fully known. We conducted laboratory experiments to study the effects of iron availability on particle colonization behavior, motility, and enzymatic activities of 4 strains of marine bacteria. Iron depletion reduced the bacterial particle colonization rate by 1.7%-43.1%, which could be attributed to reduced swimming speeds in 2 of the 4 strains. Protease activity was not affected by iron availability. However, attached bacteria did show higher protease activities than their free counterparts. Our results suggest that iron limitation in the ocean could in some cases reduce bacteria-particle interactions by reducing bacterial motility and colonization rate.     

Bacterial Colonization of Particles: Growth and Interactions

Marine particles in the ocean are exposed to diverse bacterial communities, and colonization and growth of attached bacteria are important processes in the degradation and transformation of the particles. In an earlier study, we showed that the initial colonization of model particles by individual bacterial strains isolated from marine aggregates was a function of attachment and detachment. In the present study, we have investigated how this colonization process was further affected by growth and interspecific interactions among the bacteria. Long-term incubation experiments showed that growth dominated over attachment and detachment after a few hours in controlling the bacterial population density on agar particles. In the absence of grazing mortality, this growth led to an equilibrium population density consistent with the theoretical limit due to oxygen diffusion. Interspecific interaction experiments showed that the presence of some bacterial strains (“residents”) on the agar particles either increased or decreased the colonization rate of other strains (“newcomers”). Comparison between an antibiotic-producing strain and its antibiotic-free mutant showed no inhibitory effect on the newcomers due to antibiotic production. On the contrary, hydrolytic activity of the antibiotic-producing strain appeared to benefit the newcomers and enhance their colonization rate. These results show that growth- and species-specific interactions have to be taken into account to adequately describe bacterial colonization of marine particles. Changes in colonization pattern due to such small-scale processes may have profound effects on the transformation and fluxes of particulate matter in the ocean.     

Dynamics of bacterial community composition and activity during a mesocosm diatom bloom

Bacterial community composition, enzymatic activities, and carbon dynamics were examined during diatom blooms in four 200-liter laboratory seawater mesocosms. The objective was to determine whether the dramatic shifts in growth rates and ectoenzyme activities, which are commonly observed during the course of phytoplankton blooms and their subsequent demise, could result from shifts in bacterial community composition. Nutrient enrichment of metazoan-free seawater resulted in diatom blooms dominated by a Thalassiosira sp., which peaked 9 days after enrichment ( approximately 24 microg of chlorophyll a liter(-1)). At this time bacterial abundance abruptly decreased from 2.8 x 10(6) to 0.75 x 10(6) ml(-1), and an analysis of bacterial community composition, by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments, revealed the disappearance of three dominant phylotypes. Increased viral and flagellate abundances suggested that both lysis and grazing could have played a role in the observed phylotype-specific mortality. Subsequently, new phylotypes appeared and bacterial production, abundance, and enzyme activities shifted from being predominantly associated with the <1.0-microm size fraction towards the >1.0-microm size fraction, indicating a pronounced microbial colonization of particles. Sequencing of DGGE bands suggested that the observed rapid and extensive colonization of particulate matter was mainly by specialized alpha-Proteobacteria- and Cytophagales-related phylotypes. These particle-associated bacteria had high growth rates as well as high cell-specific aminopeptidase, beta-glucosidase, and lipase activities. Rate measurements as well as bacterial population dynamics were almost identical among the mesocosms indicating that the observed bacterial community dynamics were systematic and repeatable responses to the manipulated conditions.     

Cytosporone E: racemic synthesis and preliminary antibacterial testing

The antibiotic cytosporone E (isolated from the broth of the endophytic fungi CR 200 (Cytospora sp.) and CR 146 (Diaporthe sp.)) was synthesized as a racemic mixture. The key step in the synthesis is the Meyers ortho-alkylation of a chiral aromatic oxazoline. Preliminary antibiotic activity shows antibiosis against Gram-positive bacteria but not Gram-negative bacteria as previously reported.     

Pelagic Bacteria–Particle Interactions and Community-Specific Growth Rates in Four Lakes along a Trophic Gradient

Abstract The relationships between bacterial concentration, bacterial production, and cell-specific activity of both free and attached bacteria and environmental factors such as suspended solids, nutrients, and temperature were examined in four lakes, two in New Zealand and two in Switzerland. Estimates of cell-specific production were obtained by microautoradiographic counts of [³H]thymidine-labeled cells. Bacteria attached to particles accounted for only 1.3 to 11.6% of the total bacterial abundance, but showed overall 20-fold higher specific growth rates and were relatively more active than their free counterparts. On average, 80 to 100% of epibacteria were attached to organic particles. The abundance and production of free and attached bacteria were positively correlated; however, relationships between these fractions and some environmental variables differed. Cell-specific activities of active bacteria were not equivalent to mean cellular activities of the entire bacterial community and differed in their relationship to trophic state. [³H]Thymidine-positive bacteria were more tightly linked to chlorophyll a than were total bacteria. Our findings indicate that production by attached bacteria, fueled by phytoplankton carbon, supplies ``new' free bacteria to the bacterial community. Our results support the idea that particulate organic matter acts as a source of dissolved nutrients to free bacteria. Bottom-up control of bacterial biomass, as shown by regressions of biomass vs production, appeared to be stronger in two ultraoligotrophic lakes than in two more eutrophic ones. Received: 17 April 1998; Accepted: 24 August 1998     

Diel and Seasonal Variations in Abundance,Activity, and Community Structure of Particle-Attached and Free-Living Bacteria in NW Mediterranean Sea

Diel and seasonal variations in abundance, activity, and structure of particle-attached vs free-living bacterial communities were investigated in offshore NW Mediterranean Sea (0–1000 m). Attached bacteria were always less abundant and less diverse but generally more active than free-living bacteria. The most important finding of this study was that the activity of attached bacteria showed pronounced diel variations in the upper mixed water column with higher activities at night. Under mesotrophic conditions, the contribution of attached bacteria to total bacterial activity increased from less than 10% at day time to 83% at night time. At high chlorophyll a concentration, the highest cell-specific activities and contribution to total bacterial activity were due to free-living bacteria at day and to attached bacteria at night. Under summer oligotrophic conditions, free-living bacteria dominated and contributed to the most important part of the bacterial activity at both day and night, whereas attached bacteria were much less abundant but presented the highest cell-specific activities. These diel and seasonal variations in activities were concomitant to changes in bacterial community structure, mainly in the upper layer. The number of attached ribotypes was fairly constant suggesting that particles are colonized by a relatively limited number of ubiquitous ribotypes. Most of these ribotypes were also free-living ribotypes suggesting that attached bacteria probably originate from colonization of newly formed particles by free-living bacteria in the upper layer. These results reinforce the biogeochemical role of attached bacteria in the cycling of particulate organic carbon in the NW Mediterranean Sea and the importance of diel variability in these processes.     

Host–microbe interactions as a driver of acclimation to salinity gradients in brown algal cultures

Like most eukaryotes, brown algae live in association with bacterial communities that frequently have beneficial effects on their development. Ectocarpus is a genus of small filamentous brown algae, which comprises a strain that has recently colonized freshwater, a rare transition in this lineage. We generated an inventory of bacteria in Ectocarpus cultures and examined the effect they have on acclimation to an environmental change, that is, the transition from seawater to freshwater medium. Our results demonstrate that Ectocarpus depends on bacteria for this transition: cultures that have been deprived of their associated microbiome do not survive a transfer to freshwater, but restoring their microflora also restores the capacity to acclimate to this change. Furthermore, the transition between the two culture media strongly affects the bacterial community composition. Examining a range of other closely related algal strains, we observed that the presence of two bacterial operational taxonomic units correlated significantly with an increase in low salinity tolerance of the algal culture. Despite differences in the community composition, no indications were found for functional differences in the bacterial metagenomes predicted to be associated with algae in the salinities tested, suggesting functional redundancy in the associated bacterial community. Our study provides an example of how microbial communities may impact the acclimation and physiological response of algae to different environments, and thus possibly act as facilitators of speciation. It paves the way for functional examinations of the underlying host–microbe interactions, both in controlled laboratory and natural conditions.