Isolation of Optically Targeted Single Bacteria by Application of Fluidic Force Microscopy to Aerobic Anoxygenic Phototrophs from the Phyllosphere

Stiefel, Philipp; Zambelli, Tomaso; Vorholt, Julia A.

doi:10.1128/aem.01087-13

Cited by 46 publications

(37 citation statements)

References 75 publications

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“…One such trait, as is apparent from the ubiquity of light-sensing and light protection genes, is the exposure to solar radiation. The ability to sense and utilize light by phyllosphere microbes is emerging as a general trend (39)(40)(41). Indeed, the prevalence of light-sensing and light utilization mechanisms, such as rhodopsin, PYP, and, notably, anoxygenic photosystem genes, reiterates the contention that light is an important resource and signal in the leaf surface habitat.…”

Section: Discussionmentioning

confidence: 99%

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

Finkel

Delmont

Post

et al. 2016

Appl Environ Microbiol

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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. IMPORTANCEPlant 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.

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Section: Discussionmentioning

confidence: 99%

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

Finkel

Delmont

Post

et al. 2016

Appl Environ Microbiol

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“…These observations have prompted many in the microbial ecology community to disregard community analyses that examine only those community members that are culturable. Unfortunately, only a few other studies have addressed the culturability of phyllosphere bacteria (22,23,39). The low incidence of culturability is particularly noteworthy in soil, where the apparent presence of a heterogeneous mixture of microenvironments with distinct chemical and physical restrictions presumably select for bacteria with distinct features capable of exploiting only localized regions of the soil (19).…”

Section: Discussionmentioning

confidence: 99%

“…However, this study supports the findings of a few recent studies that reveal that not all habitats harbor bacteria that are similarly difficult to culture, since cultured representatives of 34 out of 40 relatively abundant (Ͼ0.2%) OTU on leaves could be recovered. Although this was surprising, it was not unprecedented; a recent study of phyllosphere bacteria using fluidic force microscopy demonstrated that 69 of 100 randomly chosen cells of clover epiphytes recovered from leaves were capable of growth in a lownutrient medium (22), and high-throughput sequencing efforts in Arabidopsis similarly found nearly 50% of the relatively abundant (Ͼ0.1%) OTU determined by direct DNA sequencing to also be represented in a library of cultured strains from these same samples (23). Furthermore, a previous study on the apple phyllosphere sequenced 300 clones and found that 85% of them corresponded to known cultured OTUs, even though 4=,6-diamidino-2-phenylindole (DAPI) staining would have indicated that less than 1% of the cells were enumerated by culturing (39).…”

Section: Discussionmentioning

confidence: 99%

“…However, agar surfaces likely approximate leaf surface conditions more than other environments, such as heterogeneous soil particles. While quantitative PCR-based methods suggest that as little as 0.1 to 5% of the bacteria on leaves are cultivable, a proportion similar to that in most other habitats (20), frequencies of 50% and higher have been recently described when more sophisticated methods are employed (21)(22)(23). Therefore, as part of our investigation into the diversity of biosurfactant producers, we also focused on identifying those bacterial taxa that might not be interrogated for lack of their culturability.…”

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confidence: 99%

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High-Level Culturability of Epiphytic Bacteria and Frequency of Biosurfactant Producers on Leaves

Burch

Paulina

Sbodio

et al. 2016

Appl Environ Microbiol

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To better characterize the bacterial community members capable of biosurfactant production on leaves, we distinguished culturable biosurfactant-producing bacteria from nonproducers and used community sequencing to compare the composition of these distinct cultured populations with that from DNA directly recovered from leaves. Communities on spinach, romaine, and head lettuce leaves were compared with communities from adjacent samples of soil and irrigation source water. Soil communities were poorly described by culturing, with recovery of cultured representatives from only 21% of the prevalent operational taxonomic units (OTUs) (>0.2% reads) identified. The dominant biosurfactant producers cultured from soil included bacilli and pseudomonads. In contrast, the cultured communities from leaves are highly representative of the culture-independent communities, with over 85% of the prevalent OTUs recovered. The dominant taxa of surfactant producers from leaves were pseudomonads as well as members of the infrequently studied genus Chryseobacterium. The proportions of bacteria cultured from head lettuce and romaine leaves that produce biosurfactants were directly correlated with the culture-independent proportion of pseudomonads in a given sample, whereas spinach harbored a wider diversity of biosurfactant producers. A subset of the culturable bacteria in irrigation water also became enriched on romaine leaves that were irrigated overhead. Although our study was designed to identify surfactant producers on plants, we also provide evidence that most bacteria in some habitats, such as agronomic plant surfaces, are culturable, and these communities can be readily investigated and described by more classical culturing methods. IMPORTANCEThe importance of biosurfactant production to the bacteria that live on waxy leaf surfaces as well as their ability to be accurately assessed using culture-based methodologies was determined by interrogating epiphytic populations by both culture-dependent and culture-independent methods. Biosurfactant production was much more frequently observed in cultured communities on leaves than in other nearby habitats, such as soil and water, suggesting that this trait is important to life on a leaf by altering either the leaf itself or the interaction of bacteria with water. While pseudomonads were the most common biosurfactant producers isolated, this habitat also selects for taxa, such as Chryseobacterium, for which this trait was previously unrecognized. The finding that most epiphytic bacterial taxa were culturable validates strategies using more classical culturing methodologies for their study in this habitat. The phyllosphere is recognized to be a selective habitat in which epiphytic bacteria must be able to access limited and spatially heterogeneous nutrient supplies and endure frequent fluctuations in moisture availability on a water-repellent surface (1, 2). Recent culture-independent community analyses have confirmed the apparent selectivity of leaf surfaces, finding that leaf sur...

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“…Besides oxidation of organic carbon, utilization of sunlight may act as accessory mode of energy conservation for phyllosphere bacteria (53,54). The M. extorquens PA1 genome encodes for proteins of aerobic anoxygenic photosynthesis and photosynthetic cytochrome PufC (Mext_2738) was among the most up-regulated proteins in planta (Table I).…”

Section: Swath Assaymentioning

confidence: 99%

Systems-level Proteomics of Two Ubiquitous Leaf Commensals Reveals Complementary Adaptive Traits for Phyllosphere Colonization

Müller

Schubert

Röst

et al. 2016

Molecular & Cellular Proteomics

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Plants are colonized by a diverse community of microorganisms, the plant microbiota, exhibiting a defined and conserved taxonomic structure. Niche separation based on spatial segregation and complementary adaptation strategies likely forms the basis for coexistence of the various microorganisms in the plant environment. To gain insights into organism-specific adaptations on a molecular level, we selected two exemplary community members of the core leaf microbiota and profiled their proteomes upon Arabidopsis phyllosphere colonization. The highly quantitative mass spectrometric technique SWATH MS was used and allowed for the analysis of over two thousand proteins spanning more than three orders of magnitude in abundance for each of the model strains. The data suggest that Sphingomonas melonis utilizes amino acids and hydrocarbon compounds during colonization of leaves whereas Methylobacterium extorquens relies on methanol metabolism in addition to oxalate metabolism, aerobic anoxygenic photosynthesis and alkanesulfonate utilization. Comparative genomic analyses indicates that utilization of oxalate and alkanesulfonates is widespread among leaf microbiota members whereas, aerobic anoxygenic photosynthesis is almost exclusively found in Methylobacteria. Despite the apparent niche separation between these two strains we also found a relatively small subset of proteins to be coregulated, indicating common mechanisms, underlying successful leaf colonization. Overall, our results reveal for two ubiquitous phyllosphere commensals speciesspecific adaptations to the host environment and provide evidence for niche separation within the plant microbiota. Molecular & Cellular Proteomics 15: 10.1074/mcp.M116.058164, 3256-3269, 2016.

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Isolation of Optically Targeted Single Bacteria by Application of Fluidic Force Microscopy to Aerobic Anoxygenic Phototrophs from the Phyllosphere

Cited by 46 publications

References 75 publications

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

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

High-Level Culturability of Epiphytic Bacteria and Frequency of Biosurfactant Producers on Leaves

Systems-level Proteomics of Two Ubiquitous Leaf Commensals Reveals Complementary Adaptive Traits for Phyllosphere Colonization

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