Lettuce (Lactuca sativa) productivity influenced by microbial inocula under nitrogen-limited conditions in aquaponics.

The demand for food will outpace productivity of conventional agriculture due to projected growth of the human population, concomitant with shrinkage of arable land, increasing scarcity of freshwater, and a rapidly changing climate. While aquaponics has potential to sustainably supplement food production with minimal environmental impact, there is a need to better characterize the complex interplay between the various components (fish, plant, microbiome) of these systems to optimize scale up and productivity. Here, we investigated how the commonly-implemented practice of continued microbial community transfer from pre-existing systems might promote or impede productivity of aquaponics. Specifically, we monitored plant growth phenotypes, water chemistry, and microbiome composition of rhizospheres, biofilters, and fish feces over 61-days of lettuce (Lactuca sativa var. crispa) growth in nitrogen-limited aquaponic systems inoculated with bacteria that were either commercially sourced or originating from a pre-existing aquaponic system. Lettuce above- and below-ground growth were significantly reduced across replicates treated with a pre-existing aquaponic system inoculum when compared to replicates treated with a commercial inoculum. Reduced productivity was associated with enrichment in specific bacterial genera in plant roots, including Pseudomonas, following inoculum transfer from pre-existing systems. Increased productivity was associated with enrichment of nitrogen-fixing Rahnella in roots of plants treated with the commercial inoculum. Thus, we show that inoculation from a pre-existing system, rather than from a commercial inoculum, is associated with lower yields. Further work will be necessary to test the putative mechanisms involved

Contribution of Microorganisms with the Clade II Nitrous Oxide Reductase to Suppression of Surface Emissions of Nitrous Oxide

The sources and sinks of nitrous oxide, as control emissions to the atmosphere, are generally poorly constrained for most environmental systems. Initial depth-resolved analysis of nitrous oxide flux from observation wells and the proximal surface within a nitrate contaminated aquifer system revealed high subsurface production but little escape from the surface. To better understand the environmental controls of production and emission at this site, we used a combination of isotopic, geochemical, and molecular analyses to show that chemodenitrification and bacterial denitrification are major sources of nitrous oxide in this subsurface, where low DO, low pH, and high nitrate are correlated with significant nitrous oxide production. Depth-resolved metagenomes showed that consumption of nitrous oxide near the surface was correlated with an enrichment of Clade II nitrous oxide reducers, consistent with a growing appreciation of their importance in controlling release of nitrous oxide to the atmosphere. Our work also provides evidence for the reduction of nitrous oxide at a pH of 4, well below the generally accepted limit of pH 5

Cloning Should Be Simple: <i>Escherichia coli</i> DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies

<div><p>Numerous DNA assembly technologies exist for generating plasmids for biological studies. Many procedures require complex <i>in vitro</i> or <i>in vivo</i> assembly reactions followed by plasmid propagation in recombination-impaired <i>Escherichia coli</i> strains such as DH5α, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5α retains sufficient recombinase activity to assemble up to six double-stranded DNA fragments ranging in size from 150 bp to at least 7 kb into plasmids <i>in vivo</i>. This process also requires surprisingly small amounts of DNA, potentially obviating the need for upstream assembly processes associated with most common applications of DNA assembly. We demonstrate the application of this process in cloning of various DNA fragments including synthetic genes, preparation of knockout constructs, and incorporation of guide RNA sequences in constructs for clustered regularly interspaced short palindromic repeats (CRISPR) genome editing. This consolidated process for assembly and amplification in a widely available strain of <i>E</i>. <i>coli</i> may enable productivity gain across disciplines involving recombinant DNA work.</p></div

pUC19-<i>lacZα</i> assembly assay.

<p>(<b>A</b>) Two fragments were PCR-amplified from the pUC19 vector to create an efficient screen for DNA assembly capability. The smaller “insert” fragment contained the coding sequence of the <i>lacZα</i> gene starting at position five and some downstream vector sequence. The larger “vector” fragment contained the rest of the plasmid, including the Amp resistance gene (<i>bla</i>) and the origin of replication. The fragments shared 50-bp homology at both ends. (<b>B</b>) Blue colony formation as a function of DNA concentration. Very few white colonies were observed on any of the plates (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137466#pone.0137466.s009" target="_blank">S3 Table</a>). Small numbers of blue colonies present in the vector-only transformations are indicative of the small amount of contaminating circular template pUC19 used in PCR-mediated linearization of the vector and undigested during DpnI treatment. Insert-to-vector molar ratio was maintained at 5:1, and 25 μl of cells were used, corresponding to ¼ of the recommended volume. (<b>C</b>) Effect of insert-to-vector molar ratio on assembly efficiency. The vector DNA quantity was maintained at 0.5 ng. Error bars indicate standard deviation from two independent sets of experiments.</p

<i>In vivo</i> DNA assembly and cloning in <i>E</i>. <i>coli</i> DH5α.

<p>(<b>A</b>) <i>E</i>. <i>coli</i> DH5α-mediated DNA assembly involves only two basic steps: 1) preparation of fragments with homologous ends and 2) introduction of the fragments into competent cells. This approach minimizes the time and reagents required for DNA assembly in comparison to other common methods, which contain a separate assembly step before the introduction of the constructed plasmid into a recombination-impaired cloning strain such as DH5α (<b>B-D</b>). Assembly is typically carried out either with added enzymes <i>in vitro</i> (<b>B</b>), or <i>in vivo</i>, using as a host <i>S</i>. <i>cerevisiae</i> (<b>C</b>) or specialized <i>E</i>. <i>coli</i> strains expressing the λ Red or RecET phage-based systems (<b>D</b>).</p

Knockout cassette assembly for the deletion of GSU 1371 from <i>Geobacter sulfurreducens</i>.

<p>(<b>A</b>) The GSU 1371 knockout construct was assembled from four fragments including 0.5-kb sequences upstream and downstream of GSU 1371, the kanamycin cassette (amplified from pET28a), and the PCR-linearized pBR322 vector. All adjacent fragments shared 50-bp end homology. In five- and six-fragment assembly experiments, a fifth site (5-F) and both the fifth and sixth sites (5-F and 6-F), respectively, were used to make additional junctions with homology for assembly. (<b>B</b>) Assembly efficiency as a function of DNA concentration was examined using plates containing both Kan and Amp. Colony PCR confirmed the correct insertion in 30/30 transformants tested (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137466#pone.0137466.s004" target="_blank">S4 Fig</a>). (<b>C</b>) The size of the fragments upstream and downstream of GSU 1371 was varied to test the effect on DNA assembly efficiency. Reducing the length of fragments to 250 bp and less resulted in a substantially lower number of colonies. Molar ratio of insert fragments-to-vector was maintained at 5:1 in all experiments.</p

Comparative Proteomic Analysis of Desulfotomaculum reducens MI-1: Insights into the Metabolic Versatility of a Gram-positive Sulfate and Metal-reducing Bacterium

The proteomes of the metabolically versatile and poorly characterized Gram-positive bacterium Desulfotomaculum reducens MI-1 were compared across four cultivation conditions including sulfate reduction, soluble Fe(III) reduction, insoluble Fe(III) reduction, and pyruvate fermentation. Collectively across conditions, we observed at high confidence ~38% of genome-encoded proteins. Here, we focus on proteins that display significant differential abundance on conditions tested. To the best of our knowledge, this is the first full-proteome study focused on a Gram-positive organism grown either on sulfate or metal-reducing conditions. Several proteins with uncharacterized function encoded within heterodisulfide reductase (hdr)-containing loci were upregulated on either sulfate (Dred_0633-4, Dred_0689-90, and Dred_1325-30) or Fe(III)-citrate-reducing conditions (Dred_0432-3 and Dred_1778-84). Two of these hdr-containing loci display homology to recently described flavin-based electron bifurcation (FBEB) pathways (Dred_1325-30 and Dred_1778-84). Additionally, we propose that a cluster of proteins, which is homologous to a described FBEB lactate dehydrogenase (LDH) complex, is performing lactate oxidation in D. reducens (Dred_0367-9). Analysis of the putative sulfate reduction machinery in D. reducens revealed that most of these proteins are constitutively expressed across cultivation conditions tested. In addition, peptides from the single multiheme c-type cytochrome (MHC) in the genome were exclusively observed on the insoluble Fe(III) condition, suggesting that this MHC may play a role in reduction of insoluble metals

Systems biology approaches towards predictive microbial ecology.

Through complex interspecies interactions, microbial processes drive nutrient cycling and biogeochemistry. However, we still struggle to predict specifically which organisms, communities, and biotic and abiotic processes are determining ecosystem function and how environmental changes will alter their roles and stability. While the tools to create such a predictive microbial ecology capability exist, cross-disciplinary integration of high-resolution field measurements, detailed laboratory studies, and computation is essential. In this perspective, we emphasize the importance of pursuing a multiscale, systems approach to iteratively link ecological processes measured in the field to testable hypotheses that drive high-throughput laboratory experimentation. Mechanistic understanding of microbial processes gained in controlled lab systems will lead to the development of theory that can be tested back in the field. Using