Omri M. Finkel

Geographical Location Determines the Population Structure in Phyllosphere Microbial Communities of a Salt-Excreting Desert Tree

ABSTRACT The leaf surfaces of Tamarix, a salt-secreting desert tree, harbor a diverse community of microbial epiphytes. This ecosystem presents a unique combination of ecological characteristics and imposes a set of extreme stress conditions. The composition of the microbial community along ecological gradients was studied from analyses of microbial richness and diversity in the phyllosphere of three Tamarix species in the Mediterranean and Dead Sea regions in Israel and in two locations in the United States. Over 200,000 sequences of the 16S V6 and 18S V9 hypervariable regions revealed a diverse community, with 788 bacterial and 64 eukaryotic genera but only one archaeal genus. Both geographic location and tree species were determinants of microbial community structures, with the former being more dominant. Tree leaves of all three species in the Mediterranean region were dominated by Halomonas and Halobacteria, whereas trees from the Dead Sea area were dominated by Actinomycetales and Bacillales. Our findings demonstrate that microbial phyllosphere communities on different Tamarix species are highly similar in the same locale, whereas trees of the same species that grow in different climatic regions host distinct microbial communities.

Distance-Decay Relationships Partially Determine Diversity Patterns of Phyllosphere Bacteria on Tamrix Trees across the Sonoran Desert

ABSTRACT Dispersal limitation in phyllosphere communities was measured on the leaf surfaces of salt-excreting Tamarix trees, which offer unique, discrete habitats for microbial assemblages. We employed 16S rRNA gene pyrosequencing to measure bacterial community dissimilarity on leaves of spatially dispersed Tamarix specimens in sites with uniform climatic conditions across the Sonoran Desert in the Southwestern United States. Our analyses revealed diverse bacterial communities with four dominant phyla that exhibited differential effects of environmental and geographic variables. Geographical distance was the most important parameter that affected community composition, particularly that of betaproteobacteria, which displayed a statistically significant, distance-decay relationship.

Microbial rhodopsins on leaf surfaces of terrestrial plants

The above-ground surfaces of terrestrial plants, the phyllosphere, comprise the main interface between the terrestrial biosphere and solar radiation. It is estimated to host up to 10(26) microbial cells that may intercept part of the photon flux impinging on the leaves. Based on 454-pyrosequencing-generated metagenome data, we report on the existence of diverse microbial rhodopsins in five distinct phyllospheres from tamarisk (Tamarix nilotica), soybean (Glycine max), Arabidopsis (Arabidopsis thaliana), clover (Trifolium repens) and rice (Oryza sativa). Our findings, for the first time describing microbial rhodopsins from non-aquatic habitats, point towards the potential coexistence of microbial rhodopsin-based phototrophy and plant chlorophyll-based photosynthesis, with the different pigments absorbing non-overlapping fractions of the light spectrum.

Coordinated transporter activity shapes high-affinity iron acquisition in cyanobacteria

Iron bioavailability limits biological activity in many aquatic and terrestrial environments. Broad scale genomic meta-analyses indicated that within a single organism, multiple iron transporters may contribute to iron acquisition. Here, we present a functional characterization of a cyanobacterial iron transport pathway that utilizes concerted transporter activities. Cyanobacteria are significant contributors to global primary productivity with high iron demands. Certain cyanobacterial species employ a siderophore-mediated uptake strategy; however, many strains possess neither siderophore biosynthesis nor siderophore transport genes. The unicellular, planktonic, freshwater cyanobacterium, Synechocystis sp. PCC 6803, employs an alternative to siderophore-based uptake-reduction of Fe(III) species before transport through the plasma membrane. In this study, we combine short-term radioactive iron uptake and reduction assays with a range of disruption mutants to generate a working model for iron reduction and uptake in Synechocystis sp. PCC 6803. We found that the Fe(II) transporter, FeoB, is the major iron transporter in this organism. In addition, we uncovered a link between a respiratory terminal oxidase (Alternate Respiratory Terminal Oxidase) and iron reduction - suggesting a coupling between these two electron transfer reactions. Furthermore, quantitative RNA transcript analysis identified a function for subunits of the Fe(III) transporter, FutABC, in modulating reductive iron uptake. Collectively, our results provide a molecular basis for a tightly coordinated, high-affinity iron transport system.

Global abundance of microbial rhodopsins.

Photochemical reaction centers and rhodopsins are the only phototrophic mechanisms known to have evolved on Earth. The minimal cost of bearing a rhodopsin-based phototrophic mechanism in comparison to maintaining a photochemical reaction center suggests that rhodopsin is the more abundant of the two. We tested this hypothesis by conducting a global abundance calculation of phototrophic mechanisms from 116 marine and terrestrial microbial metagenomes. On average, 48% of the cells from which these metagenomes were generated harbored a rhodopsin gene, exceeding the reaction center abundance by threefold. Evidence from metatranscriptomic data suggests that this genomic potential is realized to a substantial extent, at least for the small-sized (>0.8 μm) of microbial fractions.

Bacterial anoxygenic photosynthesis on plant leaf surfaces.

The aerial surface of plants, the phyllosphere, is colonized by numerous bacteria displaying diverse metabolic properties that enable their survival in this specific habitat. Recently, we reported on the presence of microbial rhodopsin harbouring bacteria on the top of leaf surfaces. Here, we report on the presence of additional bacterial populations capable of harvesting light as a means of supplementing their metabolic requirements. An analysis of six phyllosphere metagenomes revealed the presence of a diverse community of anoxygenic phototrophic bacteria, including the previously reported methylobacteria, as well as other known and unknown phototrophs. The presence of anoxygenic phototrophic bacteria was also confirmed in situ by infrared epifluorescence microscopy. The microscopic enumeration correlated with estimates based on metagenomic analyses, confirming both the presence and high abundance of these microorganisms in the phyllosphere. Our data suggest that the phyllosphere contains a phylogenetically diverse assemblage of phototrophic species, including some yet undescribed bacterial clades that appear to be phyllosphere-unique.

Biogeographical diversity of leaf-associated microbial communities from salt-secreting Tamarix trees of the Dead Sea region.

The leaves of Tamarix, a salt-secreting desert tree, form an extreme niche that harbors a unique microbial community. In view of the global distribution of this tree, its island-like phyllosphere is highly suitable for studying microbial diversity along geographical gradients. Here we present an analysis of microbial community diversity using leaf surface samples collected at six different sites, on both sides of the Dead Sea, over a period of one year. Biodiversity analysis of denaturing gradient gel electrophoresis (DGGE) patterns of the bacterial 16S rRNA gene revealed a significant degree of bacterial community similarity within trees sampled at the same site, much higher than the similarity between trees from different geographical locations. Statistical analysis indicated that the degree of similarity was negatively correlated with the distance between sampling sites, and that a weak correlation existed between diversity and leaf pH.

Convergent patterns in the evolution of mealybug symbioses involving different intrabacterial symbionts

Mealybugs (Insecta: Hemiptera: Pseudococcidae) maintain obligatory relationships with bacterial symbionts, which provide essential nutrients to their insect hosts. Most pseudococcinae mealybugs harbor a unique symbiosis setup with enlarged betaproteobacterial symbionts (‘Candidatus Tremblaya princeps’), which themselves contain gammaproteobacterial symbionts. Here we investigated the symbiosis of the manna mealybug, Trabutina mannipara, using a metagenomic approach. Phylogenetic analyses revealed that the intrabacterial symbiont of T. mannipara represents a novel lineage within the Gammaproteobacteria, for which we propose the tentative name ‘Candidatus Trabutinella endobia’. Combining our results with previous data available for the nested symbiosis of the citrus mealybug Planococcus citri, we show that synthesis of essential amino acids and vitamins and translation-related functions partition between the symbiotic partners in a highly similar manner in the two systems, despite the distinct evolutionary origin of the intrabacterial symbionts. Bacterial genes found in both mealybug genomes and complementing missing functions in both symbioses were likely integrated in ancestral mealybugs before T. mannipara and P. citri diversified. The high level of correspondence between the two mealybug systems and their highly intertwined metabolic pathways are unprecedented. Our work contributes to a better understanding of the only known intracellular symbiosis between two bacteria and suggests that the evolution of this unique symbiosis included the replacement of intrabacterial symbionts in ancestral mealybugs.

Diverse Microhabitats Experienced by Halomonas variabilis on Salt-Secreting Leaves

ABSTRACT The leaf surfaces of the salt-excreting tree Tamarix aphylla harbor a wide diversity of halophilic microorganisms, including Halomonas sp., but little is known of the factors that shape community composition in this extreme habitat. We isolated a strain of Halomonas variabilis from the leaf surface of T. aphylla and used it to determine the heterogeneity of salt concentrations experienced by bacteria in this environment. This halophilic strain was transformed with a proU::gfp reporter gene fusion, the fluorescence of which was responsive to NaCl concentrations up to 200 g liter−1. These bioreporting cells were applied to T. aphylla leaves and were subsequently recovered from dew droplets adhering to the leaf surface. Although cells from within a given dew droplet exhibited similar green fluorescent protein fluorescence, the fluorescence intensity varied between droplets and was correlated with the salt concentration measured in each drop. Growth of H. variabilis was observed in all droplets, regardless of the salt concentration. However, cells found in desiccated microniches between dew drops were low in abundance and generally dead. Other bacteria recovered from T. aphylla displayed higher desiccation tolerance than H. variabilis, both in culture and on inoculated plants, despite having lower osmotic tolerance. Thus, the Tamarix leaf surface can be described as a salty desert with occasional oases where water droplets form under humid conditions. While halotolerant bacteria such as Halomonas grow in high concentrations of salt in such wet microniches, other organisms are better suited to survive desiccation in sites that are not wetted.

Metagenomic Signatures of Bacterial Adaptation to Life in the Phyllosphere of a Salt-Secreting Desert Tree

ABSTRACT The leaves of Tamarix aphylla, a globally distributed, salt-secreting desert tree, are dotted with alkaline droplets of high salinity. To successfully inhabit these organic carbon-rich droplets, bacteria need to be adapted to multiple stress factors, including high salinity, high alkalinity, high UV radiation, and periodic desiccation. To identify genes that are important for survival in this harsh habitat, microbial community DNA was extracted from the leaf surfaces of 10 Tamarix aphylla trees along a 350-km longitudinal gradient. Shotgun metagenomic sequencing, contig assembly, and binning yielded 17 genome bins, six of which were >80% complete. These genomic bins, representing three phyla (Proteobacteria, Bacteroidetes, and Firmicutes), were closely related to halophilic and alkaliphilic taxa isolated from aquatic and soil environments. Comparison of these genomic bins to the genomes of their closest relatives revealed functional traits characteristic of bacterial populations inhabiting the Tamarix phyllosphere, independent of their taxonomic affiliation. These functions, most notably light-sensing genes, are postulated to represent important adaptations toward colonization of this habitat. IMPORTANCE Plant leaves are an extensive and diverse microbial habitat, forming the main interface between solar energy and the terrestrial biosphere. There are hundreds of thousands of plant species in the world, exhibiting a wide range of morphologies, leaf surface chemistries, and ecological ranges. In order to understand the core adaptations of microorganisms to this habitat, it is important to diversify the type of leaves that are studied. This study provides an analysis of the genomic content of the most abundant bacterial inhabitants of the globally distributed, salt-secreting desert tree Tamarix aphylla. Draft genomes of these bacteria were assembled, using the culture-independent technique of assembly and binning of metagenomic data. Analysis of the genomes reveals traits that are important for survival in this habitat, most notably, light-sensing and light utilization genes.