Yvette M. Piceno

Deciphering the rhizosphere microbiome for disease-suppressive bacteria.

A common plant pathogen induces the growth of disease-suppressive microbes in local soil communities. Disease-suppressive soils are exceptional ecosystems in which crop plants suffer less from specific soil-borne pathogens than expected owing to the activities of other soil microorganisms. For most disease-suppressive soils, the microbes and mechanisms involved in pathogen control are unknown. By coupling PhyloChip-based metagenomics of the rhizosphere microbiome with culture-dependent functional analyses, we identified key bacterial taxa and genes involved in suppression of a fungal root pathogen. More than 33,000 bacterial and archaeal species were detected, with Proteobacteria, Firmicutes, and Actinobacteria consistently associated with disease suppression. Members of the γ-Proteobacteria were shown to have disease-suppressive activity governed by nonribosomal peptide synthetases. Our data indicate that upon attack by a fungal root pathogen, plants can exploit microbial consortia from soil for protection against infections.

Urban aerosols harbor diverse and dynamic bacterial populations

Considering the importance of its potential implications for human health, agricultural productivity, and ecosystem stability, surprisingly little is known regarding the composition or dynamics of the atmospheres microbial inhabitants. Using a custom high-density DNA microarray, we detected and monitored bacterial populations in two U.S. cities over 17 weeks. These urban aerosols contained at least 1,800 diverse bacterial types, a richness approaching that of some soil bacterial communities. We also reveal the consistent presence of bacterial families with pathogenic members including environmental relatives of select agents of bioterrorism significance. Finally, using multivariate regression techniques, we demonstrate that temporal and meteorological influences can be stronger factors than location in shaping the biological composition of the air we breathe.

High-Density Universal 16S rRNA Microarray Analysis Reveals Broader Diversity than Typical Clone Library When Sampling the Environment

Molecular approaches aimed at detection of a broad-range of prokaryotes in the environment routinely rely on classifying heterogeneous 16S rRNA genes amplified by polymerase chain reaction (PCR) using primers with broad specificity. The general method of sampling and categorizing DNA has been to clone then sequence the PCR products. However, the number of clones required to adequately catalog the majority of taxa in a sample is unwieldy. Alternatively, hybridizing target sequences to a universal 16S rRNA gene microarray may provide a more rapid and comprehensive view of prokaryotic community composition. This study investigated the breadth and accuracy of a microarray in detecting diverse 16S rRNA gene sequence types compared to clone-and-sequencing using three environmental samples: urban aerosol, subsurface soil, and subsurface water. PCR products generated from universal 16S rRNA gene-targeted primers were classified by using either the clone-and-sequence method or by hybridization to a novel high-density microarray of 297,851 probes complementary to 842 prokaryotic subfamilies. The three clone libraries comprised 1391 high-quality sequences. Approximately 8% of the clones could not be placed into a known subfamily and were considered novel. The microarray results confirmed the majority of clone-detected subfamilies and additionally demonstrated greater amplicon diversity extending into phyla not observed by the cloning method. Sequences matching operational taxonomic units within the phyla Nitrospira, Planctomycetes, and TM7, which were uniquely detected by the array, were verified with specific primers and subsequent amplicon sequencing. Subfamily richness detected by the array corresponded well with nonparametric richness predictions extrapolated from clone libraries except in the water community where clone-based richness predictions were greatly exceeded. It was concluded that although the microarray is unreliable in identifying novel prokaryotic taxa, it reveals greater diversity in environmental samples than sequencing a typically sized clone library. Furthermore, the microarray allowed samples to be rapidly evaluated with replication, a significant advantage in studies of microbial ecology.

Chronic kidney disease alters intestinal microbial flora

The population of microbes (microbiome) in the intestine is a symbiotic ecosystem conferring trophic and protective functions. Since the biochemical environment shapes the structure and function of the microbiome, we tested whether uremia and/or dietary and pharmacologic interventions in chronic kidney disease alters the microbiome. To identify different microbial populations, microbial DNA was isolated from the stools of 24 patients with end-stage renal disease (ESRD) and 12 healthy persons, and analyzed by phylogenetic microarray. There were marked differences in the abundance of 190 bacterial operational taxonomic units (OTUs) between the ESRD and control groups. OTUs from Brachybacterium, Catenibacterium, Enterobacteriaceae, Halomonadaceae, Moraxellaceae, Nesterenkonia, Polyangiaceae, Pseudomonadaceae, and Thiothrix families were markedly increased in patients with ESRD. To isolate the effect of uremia from inter-individual variations, comorbid conditions, and dietary and medicinal interventions, rats were studied 8 weeks post 5/6 nephrectomy or sham operation. This showed a significant difference in the abundance of 175 bacterial OTUs between the uremic and control animals, most notably as decreases in the Lactobacillaceae and Prevotellaceae families. Thus, uremia profoundly alters the composition of the gut microbiome. The biological impact of this phenomenon is unknown and awaits further investigation.

Bacterial diversity and White Plague Disease-associated community changes in the Caribbean coral Montastraea faveolata

Increasing evidence confirms the crucial role bacteria and archaea play within the coral holobiont, that is, the coral host and its associated microbial community. The bacterial component constitutes a community of high diversity, which appears to change in structure in response to disease events. In this study, we highlight the limitation of 16S rRNA gene (16S rDNA) clone library sequencing as the sole method to comprehensively describe coral-associated communities. This limitation was addressed by combining a high-density 16S rRNA gene microarray with, clone library sequencing as a novel approach to study bacterial communities in healthy versus diseased corals. We determined an increase in diversity as well as a significant shift in community structure in Montastraea faveolata colonies displaying phenotypic signs of White Plague Disease type II (WPD-II). An accumulation of species that belong to families that include known coral pathogens (Alteromonadaceae, Vibrionaceae), bacteria previously isolated from diseased, stressed or injured marine invertebrates (for example, Rhodobacteraceae), and other species (for example, Campylobacteraceae) was observed. Some of these species were also present in healthy tissue samples, but the putative primary pathogen, Aurantimonas corallicida, was not detected in any sample by either method. Although an ecological succession of bacteria during disease progression after causation by a primary agent represents a possible explanation for our observations, we also discuss the possibility that a disease of yet to be determined etiology may have affected M. faveolata colonies and resulted in (or be a result of) an increase in opportunistic pathogens.

Bacterial Community Structure in Geographically Distributed Biological Wastewater Treatment Reactors

Current knowledge of the microbial communities within biological wastewater treatment reactors is incomplete due to limitations of traditional culture-based techniques and despite the emergence of recently applied molecular techniques. Here we demonstrate the application of high-density microarrays targeting universal 16S rRNA genes to evaluate microbial community composition in five biological wastewater treatment reactors in China and the United States. Results suggest a surprisingly consistent composition of microbial community structure among all five reactors. All investigated communities contained a core of bacterial phyla (53-82% of 2119 taxa identified) with almost identical compositions (as determined by colinearity analysis). These core species were distributed widely in terms of abundance but their proportions were virtually the same in all samples. Proteobacteria was the largest phylum and Firmicutes, Actinobacteria, Bacteroidetes were the subdominant phyla. The diversity among the samples can be attributed solely to a group of operational taxonomic units (OTUs) that were detected only in specific samples. Typically, these organisms ranked somewhat lower in terms of abundance but a few were present is much higher proportions.

Molecular analysis of diazotroph diversity in the rhizosphere of the smooth cordgrass, Spartina alterniflora.

ABSTRACT N2 fixation by diazotrophic bacteria associated with the roots of the smooth cordgrass, Spartina alterniflora, is an important source of new nitrogen in many salt marsh ecosystems. However, the diversity and phylogenetic affiliations of these rhizosphere diazotrophs are unknown. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified nifH sequence segments was used in previous studies to examine the stability and dynamics of the Spartina rhizosphere diazotroph assemblages in the North Inlet salt marsh, near Georgetown, S.C. In this study, plugs were taken from gel bands from representative DGGE gels, thenifH amplimers were recovered and cloned, and their sequences were determined. A total of 59 sequences were recovered, and the amino acid sequences predicted from them were aligned with sequences from known and unknown diazotrophs in order to determine the types of organisms present in the Spartina rhizosphere. We recovered numerous sequences from diazotrophs in the γ subdivision of the division Proteobacteria(γ-Proteobacteria) and from various anaerobic diazotrophs. Diazotrophs in the α-Proteobacteria were poorly represented. None of the Spartina rhizosphere DGGE band sequences were identical to any known or previously recovered environmental nifH sequences. TheSpartina rhizosphere diazotroph assemblage is very diverse and apparently consists mainly of unknown organisms.

Succession of Hydrocarbon-Degrading Bacteria in the Aftermath of the Deepwater Horizon Oil Spill in the Gulf of Mexico

The Deepwater Horizon oil spill produced large subsurface plumes of dispersed oil and gas in the Gulf of Mexico that stimulated growth of psychrophilic, hydrocarbon degrading bacteria. We tracked succession of plume bacteria before, during and after the 83-day spill to determine the microbial response and biodegradation potential throughout the incident. Dominant bacteria shifted substantially over time and were dependent on relative quantities of different hydrocarbon fractions. Unmitigated flow from the wellhead early in the spill resulted in the highest proportions of n-alkanes and cycloalkanes at depth and corresponded with dominance by Oceanospirillaceae and Pseudomonas. Once partial capture of oil and gas began 43 days into the spill, petroleum hydrocarbons decreased, the fraction of aromatic hydrocarbons increased, and Colwellia, Cycloclasticus, and Pseudoalteromonas increased in dominance. Enrichment of Methylomonas coincided with positive shifts in the δ(13)C values of methane in the plume and indicated significant methane oxidation occurred earlier than previously reported. Anomalous oxygen depressions persisted at plume depths for over six weeks after well shut-in and were likely caused by common marine heterotrophs associated with degradation of high-molecular-weight organic matter, including Methylophaga. Multiple hydrocarbon-degrading bacteria operated simultaneously throughout the spill, but their relative importance was controlled by changes in hydrocarbon supply.

Environmental microarray analyses of Antarctic soil microbial communities

Antarctic ecosystems are fascinating in their limited trophic complexity, with decomposition and nutrient cycling functions being dominated by microbial activities. Not only are Antarctic habitats exposed to extreme environmental conditions, the Antarctic Peninsula is also experiencing unequalled effects of global warming. Owing to their uniqueness and the potential impact of global warming on these pristine systems, there is considerable interest in determining the structure and function of microbial communities in the Antarctic. We therefore utilized a recently designed 16S rRNA gene microarray, the PhyloChip, which targets 8741 bacterial and archaeal taxa, to interrogate microbial communities inhabiting densely vegetated and bare fell-field soils along a latitudinal gradient ranging from 51 °S (Falkland Islands) to 72 °S (Coal Nunatak). Results indicated a clear decrease in diversity with increasing latitude, with the two southernmost sites harboring the most distinct Bacterial and Archaeal communities. The microarray approach proved more sensitive in detecting the breadth of microbial diversity than polymerase chain reaction-based bacterial 16S rRNA gene libraries of modest size (∼190 clones per library). Furthermore, the relative signal intensities summed for phyla and families on the PhyloChip were significantly correlated with the relative occurrence of these taxa in clone libraries. PhyloChip data were also compared with functional gene microarray data obtained earlier, highlighting numerous significant relationships and providing evidence for a strong link between community composition and functional gene distribution in Antarctic soils. Integration of these PhyloChip data with other complementary methods provides an unprecedented understanding of the microbial diversity and community structure of terrestrial Antarctic habitats.

PhyloChip hybridization uncovered an enormous bacterial diversity in the rhizosphere of different potato cultivars: many common and few cultivar-dependent taxa

The phylogenetic composition of bacterial communities in the rhizosphere of three potato cultivars grown at two distant field sites was analysed. Ribosomal gene fragments amplified from total community DNA were hybridized to PhyloChips. A total of 2432 operational taxonomic units (OTUs) were detected by the PhyloChips, of which 65% were found in the rhizosphere of all cultivars at both field sites. From all detected OTUs, 9% revealed a cultivar-dependent abundance at the one or the other field site and 4% at both sites. Differential abundance on the three cultivars was mainly observed for OTUs belonging to the Pseudomonadales, Actinomycetales and Enterobacteriales. More than 40% of OTUs belonging to Bradyrhizobiales, Sphingomonadales, Burkholderiales, Rhodocyclales, Xanthomonadales and Actinomycetales differed significantly in their abundance between the sites. A sequence analysis of six 16S rRNA gene clone libraries corresponded well with the taxonomic community structure evidenced by the PhyloChip hybridization. Most ribotypes matched OTUs detected by the PhyloChip. Those OTUs that responded to the potato cultivar at both field sites might be of interest in view of cultivar-specific effects on bacterial biocontrol strains and pathogens.