Impact of hydrologic boundaries on microbial planktonic and biofilm communities in shallow terrestrial subsurface environments.

Subsurface environments contain a large proportion of planetary microbial biomass and harbor diverse communities responsible for mediating biogeochemical cycles important to groundwater used by human society for consumption, irrigation, agriculture and industry. Within the saturated zone, capillary fringe and vadose zones, microorganisms can reside in two distinct phases (planktonic or biofilm), and significant differences in community composition, structure and activity between free-living and attached communities are commonly accepted. However, largely due to sampling constraints and the challenges of working with solid substrata, the contribution of each phase to subsurface processes is largely unresolved. Here, we synthesize current information on the diversity and activity of shallow freshwater subsurface habitats, discuss the challenges associated with sampling planktonic and biofilm communities across spatial, temporal and geological gradients, and discuss how biofilms may be constrained within shallow terrestrial subsurface aquifers. We suggest that merging traditional activity measurements and sequencing/-omics technologies with hydrological parameters important to sediment biofilm assembly and stability will help delineate key system parameters. Ultimately, integration will enhance our understanding of shallow subsurface ecophysiology in terms of bulk-flow through porous media and distinguish the respective activities of sessile microbial communities from more transient planktonic communities to ecosystem service and maintenance.

Environmental Selection, Dispersal, and Organism Interactions Shape Community Assembly in High-Throughput Enrichment Culturing.

A central goal of microbial ecology is to identify and quantify the forces that lead to observed population distributions and dynamics. However, these forces, which include environmental selection, dispersal, and organism interactions, are often difficult to assess in natural environments. Here, we present a method that links microbial community structures with selective and stochastic forces through highly replicated subsampling and enrichment of a single environmental inoculum. Specifically, groundwater from a well-studied natural aquifer was serially diluted and inoculated into nearly 1,000 aerobic and anaerobic nitrate-reducing cultures, and the final community structures were evaluated with 16S rRNA gene amplicon sequencing. We analyzed the frequency and abundance of individual operational taxonomic units (OTUs) to understand how probabilistic immigration, relative fitness differences, environmental factors, and organismal interactions contributed to divergent distributions of community structures. We further used a most probable number (MPN) method to estimate the natural condition-dependent cultivable abundance of each of the nearly 400 OTU cultivated in our study and infer the relative fitness of each. Additionally, we infer condition-specific organism interactions and discuss how this high-replicate culturing approach is essential in dissecting the interplay between overlapping ecological forces and taxon-specific attributes that underpin microbial community assembly.IMPORTANCE Through highly replicated culturing, in which inocula are subsampled from a single environmental sample, we empirically determine how selective forces, interspecific interactions, relative fitness, and probabilistic dispersal shape bacterial communities. These methods offer a novel approach to untangle not only interspecific interactions but also taxon-specific fitness differences that manifest across different cultivation conditions and lead to the selection and enrichment of specific organisms. Additionally, we provide a method for estimating the number of cultivable units of each OTU in the original sample through the MPN approach.

Rex (encoded by DVU_0916) in Desulfovibrio vulgaris Hildenborough is a repressor of sulfate adenylyl transferase and is regulated by NADH.

Although the enzymes for dissimilatory sulfate reduction by microbes have been studied, the mechanisms for transcriptional regulation of the encoding genes remain unknown. In a number of bacteria the transcriptional regulator Rex has been shown to play a key role as a repressor of genes producing proteins involved in energy conversion. In the model sulfate-reducing microbe Desulfovibrio vulgaris Hildenborough, the gene DVU_0916 was observed to resemble other known Rex proteins. Therefore, the DVU_0916 protein has been predicted to be a transcriptional repressor of genes encoding proteins that function in the process of sulfate reduction in D. vulgaris Hildenborough. Examination of the deduced DVU_0916 protein identified two domains, one a winged helix DNA-binding domain common for transcription factors, and the other a Rossman fold that could potentially interact with pyridine nucleotides. A deletion of the putative rex gene was made in D. vulgaris Hildenborough, and transcript expression studies of sat, encoding sulfate adenylyl transferase, showed increased levels in the D. vulgaris Hildenborough Rex (RexDvH) mutant relative to the parental strain. The RexDvH-binding site upstream of sat was identified, confirming RexDvH to be a repressor of sat. We established in vitro that the presence of elevated NADH disrupted the interaction between RexDvH and DNA. Examination of the 5' transcriptional start site for the sat mRNA revealed two unique start sites, one for respiring cells that correlated with the RexDvH-binding site and a second for fermenting cells. Collectively, these data support the role of RexDvH as a transcription repressor for sat that senses the redox status of the cell.

Generalized schemes for high-throughput manipulation of the Desulfovibrio vulgaris genome.

The ability to conduct advanced functional genomic studies of the thousands of sequenced bacteria has been hampered by the lack of available tools for making high-throughput chromosomal manipulations in a systematic manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA "parts" to facilitate chromosomal modification at designated loci. These constructs enable an array of downstream applications, including gene replacement and the creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chromosomal modifications in a bacterium that has previously proven difficult to manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation.

Temporal transcriptomic analysis as Desulfovibrio vulgaris Hildenborough transitions into stationary phase during electron donor depletion.

Desulfovibrio vulgaris was cultivated in a defined medium, and biomass was sampled for approximately 70 h to characterize the shifts in gene expression as cells transitioned from the exponential to the stationary phase during electron donor depletion. In addition to temporal transcriptomics, total protein, carbohydrate, lactate, acetate, and sulfate levels were measured. The microarray data were examined for statistically significant expression changes, hierarchical cluster analysis, and promoter element prediction and were validated by quantitative PCR. As the cells transitioned from the exponential phase to the stationary phase, a majority of the down-expressed genes were involved in translation and transcription, and this trend continued at the remaining times. There were general increases in relative expression for intracellular trafficking and secretion, ion transport, and coenzyme metabolism as the cells entered the stationary phase. As expected, the DNA replication machinery was down-expressed, and the expression of genes involved in DNA repair increased during the stationary phase. Genes involved in amino acid acquisition, carbohydrate metabolism, energy production, and cell envelope biogenesis did not exhibit uniform transcriptional responses. Interestingly, most phage-related genes were up-expressed at the onset of the stationary phase. This result suggested that nutrient depletion may affect community dynamics and DNA transfer mechanisms of sulfate-reducing bacteria via the phage cycle. The putative feoAB system (in addition to other presumptive iron metabolism genes) was significantly up-expressed, and this suggested the possible importance of Fe2+ acquisition under metal-reducing conditions. The expression of a large subset of carbohydrate-related genes was altered, and the total cellular carbohydrate levels declined during the growth phase transition. Interestingly, the D. vulgaris genome does not contain a putative rpoS gene, a common attribute of the delta-Proteobacteria genomes sequenced to date, and the transcription profiles of other putative rpo genes were not significantly altered. Our results indicated that in addition to expected changes (e.g., energy conversion, protein turnover, translation, transcription, and DNA replication and repair), genes related to phage, stress response, carbohydrate flux, the outer envelope, and iron homeostasis played important roles as D. vulgaris cells experienced electron donor depletion.

Global analysis of heat shock response in Desulfovibrio vulgaris Hildenborough.

Desulfovibrio vulgaris Hildenborough belongs to a class of sulfate-reducing bacteria (SRB) and is found ubiquitously in nature. Given the importance of SRB-mediated reduction for bioremediation of metal ion contaminants, ongoing research on D. vulgaris has been in the direction of elucidating regulatory mechanisms for this organism under a variety of stress conditions. This work presents a global view of this organism's response to elevated growth temperature using whole-cell transcriptomics and proteomics tools. Transcriptional response (1.7-fold change or greater; Z >/= 1.5) ranged from 1,135 genes at 15 min to 1,463 genes at 120 min for a temperature up-shift of 13 degrees C from a growth temperature of 37 degrees C for this organism and suggested both direct and indirect modes of heat sensing. Clusters of orthologous group categories that were significantly affected included posttranslational modifications; protein turnover and chaperones (up-regulated); energy production and conversion (down-regulated), nucleotide transport, metabolism (down-regulated), and translation; ribosomal structure; and biogenesis (down-regulated). Analysis of the genome sequence revealed the presence of features of both negative and positive regulation which included the CIRCE element and promoter sequences corresponding to the alternate sigma factors sigma(32) and sigma(54). While mechanisms of heat shock control for some genes appeared to coincide with those established for Escherichia coli and Bacillus subtilis, the presence of unique control schemes for several other genes was also evident. Analysis of protein expression levels using differential in-gel electrophoresis suggested good agreement with transcriptional profiles of several heat shock proteins, including DnaK (DVU0811), HtpG (DVU2643), HtrA (DVU1468), and AhpC (DVU2247). The proteomics study also suggested the possibility of posttranslational modifications in the chaperones DnaK, AhpC, GroES (DVU1977), and GroEL (DVU1976) and also several periplasmic ABC transporters.

The systems biology markup language (SBML): a medium for representation and exchange of biochemical network models.

MOTIVATION: Molecular biotechnology now makes it possible to build elaborate systems models, but the systems biology community needs information standards if models are to be shared, evaluated and developed cooperatively. RESULTS: We summarize the Systems Biology Markup Language (SBML) Level 1, a free, open, XML-based format for representing biochemical reaction networks. SBML is a software-independent language for describing models common to research in many areas of computational biology, including cell signaling pathways, metabolic pathways, gene regulation, and others. AVAILABILITY: The specification of SBML Level 1 is freely available from http://www.sbml.org/

Control motifs for intracellular regulatory networks.

A number of technological innovations are yielding unprecedented data on the networks of biochemical, genetic, and biophysical reactions that underlie cellular behavior and failure. These networks are composed of hundreds to thousands of chemical species and structures, interacting via nonlinear and possibly stochastic physical processes. A central goal of modern biology is to optimally use the data on these networks to understand how their design leads to the observed cellular behaviors and failures. Ultimately, this knowledge should enable cellular engineers to redesign cellular processes to meet industrial needs (such as optimal natural product synthesis), aid in choosing the most effective targets for pharmaceuticals, and tailor treatment for individual genotypes. The size and complexity of these networks and the inevitable lack of complete data, however, makes reaching these goals extremely difficult. If it proves possible to modularize these networks into functional subnetworks, then these smaller networks may be amenable to direct analysis and might serve as regulatory motifs. These motifs, recurring elements of control, may help to deduce the structure and function of partially known networks and form the basis for fulfilling the goals described above. A number of approaches to identifying and analyzing control motifs in intracellular networks are reviewed.

Synthetic cell biology.

Synthesis of data into formal models of cellular function is rapidly becoming a necessary industry. The complexity of the interactions among cellular constituents and the quantity of data about these interactions hinders the ability to predict how cells will respond to perturbation and how they can be engineered for industrial or medical purposes. Models provide a systematic framework to describe and analyze these complex systems. In the past few years, models have begun to have an impact on mainstream biology by creating deeper insight into the design rules of cellular signal processing, providing a basis for rational engineering of cells, and for resolving debates about the root causes of certain cellular behaviors. This review covers some of the recent work and challenges in developing these "synthetic cell" models and their growing practical applications.

An endogenous calcium oscillator may control early embryonic division.

Transient elevations in the concentration of free cytosolic calcium ion ([Ca2+]i) promote cell phase transitions in early embryonic division and persist even if these transitions are blocked. These observations suggest that a [Ca2+]i oscillator is an essential timing element of the early embryonic "master clock." We explore this possibility by coupling a [Ca2+]i oscillator model to an early embryonic cell cycle model based on the protein interactions that govern the activity of the M-phase-promoting factor (MPF). We hypothesize three dynamical states of the MPF system and choose parameter sets to represent each. We then investigate how [Ca2+]i dynamics may control early embryonic division in both sea urchin and Xenopus embryos. To investigate both systems, distinct [Ca2+]i profiles matching those observed in sea urchin embryos (in which [Ca2+]i exhibits sharp transients) and Xenopus embryos (in which [Ca2+]i is elevated and oscillates sinusoidally) are imposed on each of the hypothesized dynamical states of MPF. In the first hypothesis, [Ca2+]i oscillations entrain the autonomous MPF oscillator. In the second and third hypotheses, where the MPF system rests in excitatory and bistable states, respectively, [Ca2+]i oscillations drive MPF activation cycles. Simulation results show that hypotheses two and three, in which a [Ca2+]i oscillator is a fundamental timing element of the master clock, best account for key experimental observations and the questions that they raise. Finally, we propose experiments to elucidate further [Ca2+]i regulation and the fundamental components of the early embryonic master clock.

An algorithm for protein engineering: simulations of recursive ensemble mutagenesis.

An algorithm for protein engineering, termed recursive ensemble mutagenesis, has been developed to produce diverse populations of phenotypically related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis. Starting from partially randomized "wild-type" DNA sequences, a highly parallel search of sequence space for peptides fitting an experimenter's criteria is performed. Each iteration uses information gained from the previous rounds to search the space more efficiently. Simulations of the technique indicate that, under a variety of conditions, the algorithm can rapidly produce a diverse population of proteins fitting specific criteria. In the experimental analog, genetic selection or screening applied during recursive ensemble mutagenesis should force the evolution of an ensemble of mutants to a targeted cluster of related phenotypes.

Optimizing nucleotide mixtures to encode specific subsets of amino acids for semi-random mutagenesis.

In random mutagenesis, synthesis of an NNN triplet (i.e. equiprobable A, C, G, and T at each of the three positions in the codon) could be considered an optimal nucleotide mixture because all 20 amino acids are encoded. NN(G,C) might be considered a slightly more intelligent "dope" because the entire set of amino acids is still encoded using only half as many codons. Using a general algorithm described herein, it is possible to formulate more complex doping schemes which encode specific subsets of the twenty amino acids, excluding others from the mix. Maximizing the equiprobability of amino acid residues contributing to such a subset is suggested as an optimal basis for performing semi-random mutagenesis. This is important for reducing the nucleotide complexity of combinatorial cassettes so that "sequence space" can be searched more efficiently. Computer programs have been developed to provide tables of optimized dopes compatible with automated DNA synthesizers.

Applications of imaging spectroscopy in molecular biology. II. Colony screening based on absorption spectra.

Digital imaging spectroscopy has been used to obtain the grayscale spectrum of colored bacterial colonies directly from petri dishes. Up to 500 individual colony spectra can be simultaneously recorded and processed from a single plate. Spectra can be obtained in the visible to near infrared region (400nm-900nm) with 10nm resolution. Instrument response is normalized through run-time radiometric calibration such that each grayscale spectrum can be converted to the ground-state absorption spectrum of the colony. In this study, mutants of the photosynthetic bacterium Rhodobacter capsulatus have been differentiated by the absorption spectra of their pigment-protein complexes. This imaging technique is applicable to chromogenic systems in which colony and/or media color (e.g. indicator plates) provides a quantitative indicator of gene expression.