Water Quality Assessment Bias Associated with Long-Screened Wells Screened across Aquifers with High Nitrate and Arsenic Concentrations.

Semi-arid regions with little surface water commonly experience rapid water table decline rates. To hedge against the falling water table, production wells in central Mexico are commonly installed to depths of several hundred meters below the present water table and constructed as open boreholes or perforated casings across their entire length. Such wells represent highly conductive pathways leading to non-negligible flow across chemically distinct layers of an aquifer-a phenomenon known as ambient flow. The objectives of this study were to estimate the rate of ambient flow in seven production wells utilizing an end-member mixing model that is constrained by the observed transient chemical composition of produced water. The end-member chemical composition of the upper and lower layers of an urban aquifer that overlies geothermal heat is estimated to anticipate the future quality of this sole source of water for a rapidly growing urban area. The comprehensive water chemistry produced by seven continuously perforated municipal production wells, spanning three geologically unique zones across the city of San Miguel de Allende in Guanajuato State, was monitored during one day of pumping. The concentration of conservative constituents gradually converged on steady-state values. The model indicates that, relative to the lower aquifer, the upper aquifer generally has higher specific conductance (SC), chloride (Cl), nitrate (NO3), calcium (Ca), barium (Ba) and magnesium (Mg). The lower aquifer generally has a higher temperature, sodium (Na), boron (B), arsenic (As) and radon (Rn). Ambient flow ranged from 33.1 L/min to 225.7 L/min across the seven wells, but this rate for a given well varied depending on which tracer was used. This new 3D understanding of the chemical stratification of the aquifer suggests that as water tables continue to fall, concentrations of geothermally associated contaminants of concern will increase in the near future, potentially jeopardizing the safety of municipal drinking water.

Microbial community composition across a coastal hydrological system affected by submarine groundwater discharge (SGD).

Mobile Bay, the fourth largest estuary in the USA located in the northern Gulf of Mexico, is known for extreme hypoxia in the water column during dry season caused by NH4+-rich and anoxic submarine groundwater discharge (SGD). Nutrient dynamics in the coastal ecosystem point to potentially elevated microbial activities; however, little is known about microbial community composition and their functional roles in this area. In this study, we investigated microbial community composition, distribution, and metabolic prediction along the coastal hydrological compartment of Mobile Bay using 16S rRNA gene sequencing. We collected microbial samples from surface (river and bay water) and subsurface water (groundwater and coastal pore water from two SGD sites with peat and sandy lithology, respectively). Salinity was identified as the primary factor affecting the distribution of microbial communities across surface water samples, while DON and PO43- were the major predictor of community shift within subsurface water samples. Higher microbial diversity was found in coastal pore water in comparison to surface water samples. Gammaproteobacteria, Bacteroidia, and Oxyphotobacteria dominated the bacterial community. Among the archaea, methanogens were prevalent in the peat-dominated SGD site, while the sandy SGD site was characterized by a higher proportion of ammonia-oxidizing archaea. Cyanobium PCC-6307 and unclassified Thermodesulfovibrionia were identified as dominant taxa strongly associated with trends in environmental parameters in surface and subsurface samples, respectively. Microbial communities found in the groundwater and peat layer consisted of taxa known for denitrification and dissimilatory nitrate reduction to ammonium (DNRA). This finding suggested that microbial communities might also play a significant role in mediating nitrogen transformation in the SGD flow path and in affecting the chemical composition of SGD discharging to the water column. Given the ecological importance of microorganisms, further studies at higher taxonomic and functional resolution are needed to accurately predict chemical biotransformation processes along the coastal hydrological continuum, which influence water quality and environmental condition in Mobile Bay.

Uncovering the biosynthetic potential of rare metagenomic DNA using co-occurrence network analysis of targeted sequences.

Sequencing of DNA extracted from environmental samples can provide key insights into the biosynthetic potential of uncultured bacteria. However, the high complexity of soil metagenomes, which can contain thousands of bacterial species per gram of soil, imposes significant challenges to explore secondary metabolites potentially produced by rare members of the soil microbiome. Here, we develop a targeted sequencing workflow termed CONKAT-seq (co-occurrence network analysis of targeted sequences) that detects physically clustered biosynthetic domains, a hallmark of bacterial secondary metabolism. Following targeted amplification of conserved biosynthetic domains in a highly partitioned metagenomic library, CONKAT-seq evaluates amplicon co-occurrence patterns across library subpools to identify chromosomally clustered domains. We show that a single soil sample can contain more than a thousand uncharacterized biosynthetic gene clusters, most of which originate from low frequency genomes which are practically inaccessible through untargeted sequencing. CONKAT-seq allows scalable exploration of largely untapped biosynthetic diversity across multiple soils, and can guide the discovery of novel secondary metabolites from rare members of the soil microbiome.

Atolypenes, Tricyclic Bacterial Sesterterpenes Discovered Using a Multiplexed In Vitro Cas9-TAR Gene Cluster Refactoring Approach.

Most natural product biosynthetic gene clusters identified in bacterial genomic and metagenomic sequencing efforts are silent under laboratory growth conditions. Here, we describe a scalable biosynthetic gene cluster activation method wherein the gene clusters are disassembled at interoperonic regions in vitro using CRISPR/Cas9 and then reassembled with PCR-amplified, short DNAs, carrying synthetic promoters, using transformation assisted recombination (TAR) in yeast. This simple, cost-effective, and scalable method allows for the simultaneous generation of combinatorial libraries of refactored gene clusters, eliminating the need to understand the transcriptional hierarchy of the silent genes. In two test cases, this in vitro disassembly-TAR reassembly method was used to create collections of promoter-replaced gene clusters that were tested in parallel to identify versions that enabled secondary metabolite production. Activation of the atolypene ( ato) gene cluster led to the characterization of two unprecedented bacterial cyclic sesterterpenes, atolypene A (1) and B (2), which are moderately cytotoxic to human cancer cell lines. This streamlined in vitro disassembly- in vivo reassembly method offers a simplified approach for silent gene cluster refactoring that should facilitate the discovery of natural products from silent gene clusters cloned from either metagenomes or cultured bacteria.

Rifamycin congeners kanglemycins are active against rifampicin-resistant bacteria via a distinct mechanism.

Rifamycin antibiotics (Rifs) target bacterial RNA polymerases (RNAPs) and are widely used to treat infections including tuberculosis. The utility of these compounds is threatened by the increasing incidence of resistance (RifR). As resistance mechanisms found in clinical settings may also occur in natural environments, here we postulated that bacteria could have evolved to produce rifamycin congeners active against clinically relevant resistance phenotypes. We survey soil metagenomes and identify a tailoring enzyme-rich family of gene clusters encoding biosynthesis of rifamycin congeners (kanglemycins, Kangs) with potent in vivo and in vitro activity against the most common clinically relevant RifR mutations. Our structural and mechanistic analyses reveal the basis for Kang inhibition of RifR RNAP. Unlike Rifs, Kangs function through a mechanism that includes interfering with 5'-initiating substrate binding. Our results suggest that examining soil microbiomes for new analogues of clinically used antibiotics may uncover metabolites capable of circumventing clinically important resistance mechanisms.

Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters.

Large-scale sequencing of prokaryotic (meta)genomic DNA suggests that most bacterial natural product gene clusters are not expressed under common laboratory culture conditions. Silent gene clusters represent a promising resource for natural product discovery and the development of a new generation of therapeutics. Unfortunately, the characterization of molecules encoded by these clusters is hampered owing to our inability to express these gene clusters in the laboratory. To address this bottleneck, we have developed a promoter-engineering platform to transcriptionally activate silent gene clusters in a model heterologous host. Our approach uses yeast homologous recombination, an auxotrophy complementation-based yeast selection system and sequence orthogonal promoter cassettes to exchange all native promoters in silent gene clusters with constitutively active promoters. As part of this platform, we constructed and validated a set of bidirectional promoter cassettes consisting of orthogonal promoter sequences, Streptomyces ribosome binding sites, and yeast selectable marker genes. Using these tools we demonstrate the ability to simultaneously insert multiple promoter cassettes into a gene cluster, thereby expediting the reengineering process. We apply this method to model active and silent gene clusters (rebeccamycin and tetarimycin) and to the silent, cryptic pseudogene-containing, environmental DNA-derived Lzr gene cluster. Complete promoter refactoring and targeted gene exchange in this "dead" cluster led to the discovery of potent indolotryptoline antiproliferative agents, lazarimides A and B. This potentially scalable and cost-effective promoter reengineering platform should streamline the discovery of natural products from silent natural product biosynthetic gene clusters.

Multiplexed metagenome mining using short DNA sequence tags facilitates targeted discovery of epoxyketone proteasome inhibitors.

In molecular evolutionary analyses, short DNA sequences are used to infer phylogenetic relationships among species. Here we apply this principle to the study of bacterial biosynthesis, enabling the targeted isolation of previously unidentified natural products directly from complex metagenomes. Our approach uses short natural product sequence tags derived from conserved biosynthetic motifs to profile biosynthetic diversity in the environment and then guide the recovery of gene clusters from metagenomic libraries. The methodology is conceptually simple, requires only a small investment in sequencing, and is not computationally demanding. To demonstrate the power of this approach to natural product discovery we conducted a computational search for epoxyketone proteasome inhibitors within 185 globally distributed soil metagenomes. This led to the identification of 99 unique epoxyketone sequence tags, falling into 6 phylogenetically distinct clades. Complete gene clusters associated with nine unique tags were recovered from four saturating soil metagenomic libraries. Using heterologous expression methodologies, seven potent epoxyketone proteasome inhibitors (clarepoxcins A-E and landepoxcins A and B) were produced from these pathways, including compounds with different warhead structures and a naturally occurring halohydrin prodrug. This study provides a template for the targeted expansion of bacterially derived natural products using the global metagenome.

"Plug-and-go" strategy to manipulate streptavidin valencies.

The streptavidin-biotin set is one of the most widely utilized conjugation pairs in biotechnological applications. The tetravalent nature of streptavidin and its homologues, however, tends to result in such undesirable complications as cross-linking or ill-defined stoichiometry. Here, we describe a mutagenesis-free strategy to manipulate the valencies of wild-type streptavidin that only requires commercially available reagents. The basic idea is simple: one obtains the desired streptavidin valency by blocking off unwanted binding sites using ancillary biotin ("plug"); this way, the extraordinary fM-biotin-binding affinity is fully retained for the remaining sites in streptavidin. In the present implementation, the ancillary biotin is attached to an auxiliary separation handle, negatively charged DNA or His-tagged protein, via a photochemically or enzymatically cleavable linker. Mixing streptavidin with the ancillary biotin construct produces a distribution of streptavidin valencies. The subsequent chromatographic separation readily isolates the construct of desired streptavidin valency, and the auxiliary handles are easily removed afterward ("go"). We demonstrate how this "plug-and-go" strategy allows a precise control for the compositions of streptavidin-biotin conjugates at the single-molecule level. This low-entry-barrier protocol could further expand the application scope of the streptavidin technology.

Ligand synthesis and passivation for silver and large gold nanoparticles for single-particle-based sensing and spectroscopy.

Silver and large gold nanoparticles are more efficient scatterers than smaller particles, which can be advantageous for a variety of single-particle-based sensing and spectroscopic applications. The increased susceptibility to surface oxidation and the larger surface area of these particles, however, present challenges to colloid stability and controllable bio-conjugation strategies. In this chapter, ligand syntheses and particle passivation procedures for yielding stable and bio-conjugatable colloids of silver and large gold nanoparticles are described.

Observation of spectral anisotropy of gold nanoparticles.

Metallic nanoparticles synthesized by solution-phase chemistry usually exhibit various polygonal morphologies. The shape is known to have a great impact on a nanoparticle's optical properties, for instance, the surface plasmon resonance frequency. It remains unclear, however, whether the scattering spectrum of nanoparticles is generally anisotropic in the far field as a result. This simple question turns out to be extremely challenging to address because of the particle-to-particle shape inhomogeneity in a bulk sample, and the high sensitivity of surface plasmon resonance to local environments. We report the observation of scattering angle-dependent spectra using a newly developed single-particle tracking spectroscopy (SPS). Furthermore, we show that SPS has provided a way to directly visualize the rotational random walk of individual gold nanoparticles in water for the first time.

Observation of correlated emission intensity and polarization fluctuations in single CdSe/ZnS quantum dots.

Time-resolved single-nanoparticle spectroscopy has been carried out to examine the luminescence characteristics of individual CdSe/ZnS core/shell quantum dots. In particular, the possible correlations between emission intensity, lifetime, spectrum, and polarization fluctuations have been investigated. The emission polarization was found to be correlated with the luminescence intensity in a nonlinear way. The low-emissive states were found to correlate with red-shifted spectrum, increased nonradiative decay, and low degree of emission polarization. The observations are consistent with the model that charged quantum dots can be emissive.

Guiding a confocal microscope by single fluorescent nanoparticles.

Confocal optical microscopes offer unparalleled high sensitivity and three-dimensional (3D) imaging capability but require slow point-by-point scanning; they are inefficient for imaging moving objects. We propose a more efficient solution. Instead of indiscriminate scanning, we let the focus of the microscope pursue the object of interest such that no time is wasted on uninformative background, allowing us to visualize 3D trajectories of fluorescent nanoparticles in solution with millisecond temporal and ~200 nm spatial resolution.