Validating regulatory predictions from diverse bacteria with mutant fitness data
Not Available
59 BASIC BIOLOGICAL SCIENCES↗
Engineering topics
Arkin, Adam P.
Publications and source records attributed to Arkin, Adam P..
Programming mRNA decay to modulate synthetic circuit resource allocation
Synthetic circuits embedded in host cells compete with cellular processes for limited intracellular resources. Here we show how funnelling of cellular resources, after global transcriptome degradation by the sequence-dependent endoribonuclease MazF, to a synthetic circuit can increase production. Target genes are protected from MazF activity by recoding the gene sequence to eliminate recognition sites, while preserving the amino acid sequence. The expression of a protected fluorescent reporter and flux of a high-value metabolite are significantly enhanced using this genome-scale control strategy. Proteomics measurements discover a host factor in need of protection to improve resource redistribution activity. A computational model demonstrates that the MazF mRNA-decay feedback loop enables proportional control of MazF in an optimal operating regime. Transcriptional profiling of MazF-induced cells elucidates the dynamic shifts in transcript abundance and discovers regulatory design elements. Altogether, our results suggest that manipulation of cellular resource allocation is a key control parameter for synthetic circuit design.
59 BASIC BIOLOGICAL SCIENCES↗
Erratum to: The JBEI quantitative metabolic modeling library (jQMM): A python library for modeling microbial metabolism
Following publication of this article [1], it has come to our attention that an incomplete version of Fig. 7 was included in this article. The complete figure is shown below with the missing text included to the left of the chart.
59 BASIC BIOLOGICAL SCIENCES↗
Dynamic Succession of Groundwater Sulfate-Reducing Communities during Prolonged Reduction of Uranium in a Contaminated Aquifer
To further understand the diversity and dynamics of SRB in response to substrate amendment, we sequenced genes coding for the dissimilatory sulfite reductase (dsrA) in groundwater samples collected after an emulsified vegetable oil (EVO) amendment, which sustained U(VI)-reducing conditions for one year in a fast-flowing aquifer. EVO amendment significantly altered the composition of groundwater SRB communities. Sequences having no closely related-described species dominated (80%) the indigenous SRB communities in nonamended wells. After EVO amendment, Desulfococcus , Desulfobacterium , and Desulfovibrio , known for long-chain-fatty-acid, short-chain-fatty-acid and H 2 oxidation and U(VI) reduction, became dominant accounting for 7 ± 2%, 21 ± 8%, and 55 ± 8% of the SRB communities, respectively. Succession of these SRB at different bioactivity stages based on redox substrates/products (acetate, SO 4 –2 , U(VI), NO 3 – , Fe(II), and Mn(II)) was observed. Desulfovibrio and Desulfococcus dominated SRB communities at 4–31 days, whereas Desulfobacterium became dominant at 80–140 days. By the end of the experiment (day 269), the abundance of these SRB decreased but the overall diversity of groundwater SRB was still higher than non-EVO controls. Up to 62% of the SRB community changes could be explained by groundwater geochemical variables, including those redox substrates/products. A significant ( P < 0.001) correlation was observed between groundwater U(VI) concentrations and Desulfovibrio abundance. Lastly, our results showed that the members of SRB and their dynamics were correlated significantly with slow EVO biodegradation, electron donor production and maintenance of U(VI)-reducing conditions in the aquifer.
54 ENVIRONMENTAL SCIENCES↗
Massive Associative K-biclustering (MAK) v1.0
Massive Associative K-biclustering (MAK) is a method to address the key limitations in other biclustering approaches. In a formal evaluation against new simulated data sets with more realistic properties MAK improves recovery of biclusters exhibiting common behavior across conditions, with the highest F1 scores, a measure of a test's accuracy, and lowest false positive rates, the probability of falsely rejecting the null hypothesis, compared to established methods. The developers show how MAK can be applied to a popular data set from Saccharomyces cerevisiae to uncover new properties not captured by other methods. MAK biclusters showed increased statistical enrichments for known regulatory associations and gene functions, including for nearly 30% of the novel MAK biclusters not found by other methods. Compared to other methods, MAK results had at least two-fold higher coverage of differential expression in the overall dataset, an overlapping bicluster network with distinct modules for protein expression and energy metabolism, and biologically relevant overlapping biclusters including cases of combinatorial regulation. MAK is suited for large datasets with many potentially noisy and overlapping patterns of varying sizes.
JOACHIMIAK, MARCIN↗
Complete genome sequence of Pseudomonas stutzeri strain RCH2 isolated from a Hexavalent Chromium [Cr(VI)] contaminated site
Abstract Hexavalent Chromium [Cr(VI)] is a widespread contaminant found in soil, sediment, and ground water in several DOE sites, including Hanford 100 H area. In order to stimulate microbially mediated reduction of Cr(VI) at this site, a poly-lactate hydrogen release compound was injected into the chromium contaminated aquifer. Targeted enrichment of dominant nitrate-reducing bacteria post injection resulted in the isolation of Pseudomonas stutzeri strain RCH2. P. stutzeri strain RCH2 was isolated using acetate as the electron donor and is a complete denitrifier. Experiments with anaerobic washed cell suspension of strain RCH2 revealed it could reduce Cr(VI) and Fe(III). The genome of strain RCH2 was sequenced using a combination of Illumina and 454 sequencing technologies and contained a circular chromosome of 4.6 Mb and three plasmids. Global genome comparisons of strain RCH2 with six other fully sequenced P. stutzeri strains revealed most genomic regions are conserved, however strain RCH2 has an additional 244 genes, some of which are involved in chemotaxis, Flp pilus biogenesis and pyruvate/2-oxogluturate complex formation.
59 BASIC BIOLOGICAL SCIENCES↗
Temporal Dynamics of In-Field Bioreactor Populations Reflect the Groundwater System and Respond Predictably to Perturbation
Temporal variability complicates testing the influences of environmental variability on microbial community structure and thus function. An in-field bioreactor system was developed to assess oxic versus anoxic manipulations on in-situ groundwater communities. Each sample was sequenced (16S SSU rRNA genes, average 10,000 reads) and biogeochemical parameters monitored by quantifying 53 metals, 12 organic acids, 14 anions and 3 sugars. Changes in dissolved oxygen (DO), pH, and other variables were similar across bioreactors. Sequencing revealed a complex community that fluctuated in-step with the groundwater community, and responded to DO. This also directly influenced the pH and so the biotic impacts of DO and pH shifts are correlated. A null model demonstrated that bioreactor communities were driven in part by experimental conditions but also by stochastic variability and did not accurately capture alterations in diversity during perturbations. We identified two groups of abundant OTUs important to this system; one was abundant in high DO and pH and contained heterotrophs and oxidizers of iron, nitrite, and ammonium, whereas the other was abundant in low DO with the capability to reduce nitrate. In-field bioreactors are a powerful tool for capturing natural microbial community responses to alterations in geochemical factors beyond the bulk phase.
54 ENVIRONMENTAL SCIENCES↗
System-Wide Adaptations of Desulfovibrio alaskensis G20 to Phosphate-Limited Conditions
Not Available
54 ENVIRONMENTAL SCIENCES↗
A Highly Expressed High-Molecular-Weight S-Layer Complex of Pelosinus sp. Strain UFO1 Binds Uranium
Cell suspensions of Pelosinus sp. strain UFO1 were previously shown, using spectroscopic analysis, to sequester uranium as U(IV) complexed with carboxyl and phosphoryl group ligands on proteins. The goal of our present study was to characterize the proteins involved in uranium binding. Virtually all of the uranium in UFO1 cells was associated with a heterodimeric protein, which was termed the uranium-binding complex (UBC). The UBC was composed of two S-layer domain proteins encoded by UFO1_4202 and UFO1_4203. Samples of UBC purified from the membrane fraction contained 3.3 U atoms/heterodimer, but significant amounts of phosphate were not detected. The UBC had an estimated molecular mass by gel filtration chromatography of 15 MDa, and it was proposed to contain 150 heterodimers (UFO1_4203 and UFO1_4202) and about 500 uranium atoms. The UBC was also the dominant extracellular protein, but when purified from the growth medium, it contained only 0.3 U atoms/heterodimer. The two genes encoding the UBC were among the most highly expressed genes within the UFO1 genome, and their expressions were unchanged by the presence or absence of uranium. Therefore, the UBC appears to be constitutively expressed and is the first line of defense against uranium, including by secretion into the extracellular medium. Although S-layer proteins were previously shown to bind U(VI), here we showed that U(IV) binds to S-layer proteins, we identified the proteins involved, and we quantitated the amount of uranium bound. Widespread uranium contamination from industrial sources poses hazards to human health and to the environment. Here in this paper, we identified a highly abundant uranium-binding complex (UBC) from Pelosinus sp. strain UFO1. The complex makes up the primary protein component of the S-layer of strain UFO1 and binds 3.3 atoms of U(IV) per heterodimer. Finally, while other bacteria have been shown to bind U(VI) on their S-layer, we demonstrate here an example of U(IV) bound by an S-layer complex. The UBC provides a potential tool for the microbiological sequestration of uranium for the cleaning of contaminated environments.
59 BASIC BIOLOGICAL SCIENCES↗
A Comparison of the Costs and Benefits of Bacterial Gene Expression
Not Available
59 BASIC BIOLOGICAL SCIENCES↗
Novel Metal Cation Resistance Systems from Mutant Fitness Analysis of Denitrifying Pseudomonas stutzeri
Metal ion transport systems have been studied extensively, but the specificity of a given transporter is often unclear from amino acid sequence data alone. In this study, predicted Cu 2+ and Zn 2+ resistance systems in Pseudomonas stutzeri strain RCH2 are compared with those experimentally implicated in Cu 2+ and Zn 2+ resistance, as determined by using a DNA-barcoded transposon mutant library. Mutant fitness data obtained under denitrifying conditions are combined with regulon predictions to yield a much more comprehensive picture of Cu 2+ and Zn 2+ resistance in strain RCH2. The results not only considerably expand what is known about well-established metal ion exporters (CzcCBA, CzcD, and CusCBA) and their accessory proteins (CzcI and CusF), they also reveal that isolates with mutations in some predicted Cu 2+ resistance systems do not show decreased fitness relative to the wild type when exposed to Cu 2+ . In addition, new genes are identified that have no known connection to Zn 2+ ( corB , corC , Psest_3226, Psest_3322, and Psest_0618) or Cu 2+ resistance (Mrp antiporter subunit gene, Psest_2850, and Psest_0584) but are crucial for resistance to these metal cations. Growth of individual deletion mutants lacking corB , corC , Psest_3226, or Psest_3322 confirmed the observed Zn-dependent phenotypes. Notably, to our knowledge, this is the first time a bacterial homolog of TMEM165, a human gene responsible for a congenital glycosylation disorder, has been deleted and the resulting strain characterized. Finally, the fitness values indicate Cu 2+ - and Zn 2+ -based inhibition of nitrite reductase and interference with molybdenum cofactor biosynthesis for nitrate reductase. These results extend the current understanding of Cu 2+ and Zn 2+ efflux and resistance and their effects on denitrifying metabolism. Here in this study, genome-wide mutant fitness data in P. stutzeri RCH2 combined with regulon predictions identify several proteins of unknown function that are involved in resisting zinc and copper toxicity. For zinc, these include a member of the UPF0016 protein family that was previously implicated in Ca 2+ /H + antiport and a human congenital glycosylation disorder, CorB and CorC, which were previously linked to Mg 2+ transport, and Psest_3322 and Psest_0618, two proteins with no characterized homologs. Experiments using mutants lacking Psest_3226, Psest_3322, corB , corC , or czcI verified their proposed functions, which will enable future studies of these little-characterized zinc resistance determinants. Likewise, Psest_2850, annotated as an ion antiporter subunit, and the conserved hypothetical protein Psest_0584 are implicated in copper resistance. Physiological connections between previous studies and phenotypes presented here are discussed. Functional and mechanistic understanding of transport proteins improves the understanding of systems in which members of the same protein family, including those in humans, can have different functions.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
A Theoretical Lower Bound for Selection on the Expression Levels of Proteins
We use simple models of the costs and benefits of microbial gene expression to show that changing a protein's expression away from its optimum by 2-fold should reduce fitness by at least [Formula: see text], where P is the fraction the cell's protein that the gene accounts for. As microbial genes are usually expressed at above 5 parts per million, and effective population sizes are likely to be above 10(6), this implies that 2-fold changes to gene expression levels are under strong selection, as [Formula: see text], where Ne is the effective population size and s is the selection coefficient. Thus, most gene duplications should be selected against. On the other hand, we predict that for most genes, small changes in the expression will be effectively neutral.
59 BASIC BIOLOGICAL SCIENCES↗
Lateral Gene Transfer in a Heavy Metal-Contaminated-Groundwater Microbial Community
ABSTRACT Unraveling the drivers controlling the response and adaptation of biological communities to environmental change, especially anthropogenic activities, is a central but poorly understood issue in ecology and evolution. Comparative genomics studies suggest that lateral gene transfer (LGT) is a major force driving microbial genome evolution, but its role in the evolution of microbial communities remains elusive. To delineate the importance of LGT in mediating the response of a groundwater microbial community to heavy metal contamination, representative Rhodanobacter reference genomes were sequenced and compared to shotgun metagenome sequences. 16S rRNA gene-based amplicon sequence analysis indicated that Rhodanobacter populations were highly abundant in contaminated wells with low pHs and high levels of nitrate and heavy metals but remained rare in the uncontaminated wells. Sequence comparisons revealed that multiple geochemically important genes, including genes encoding Fe 2+ /Pb 2+ permeases, most denitrification enzymes, and cytochrome c 553 , were native to Rhodanobacter and not subjected to LGT. In contrast, the Rhodanobacter pangenome contained a recombinational hot spot in which numerous metal resistance genes were subjected to LGT and/or duplication. In particular, Co 2+ /Zn 2+ /Cd 2+ efflux and mercuric resistance operon genes appeared to be highly mobile within Rhodanobacter populations. Evidence of multiple duplications of a mercuric resistance operon common to most Rhodanobacter strains was also observed. Collectively, our analyses indicated the importance of LGT during the evolution of groundwater microbial communities in response to heavy metal contamination, and a conceptual model was developed to display such adaptive evolutionary processes for explaining the extreme dominance of Rhodanobacter populations in the contaminated groundwater microbiome. IMPORTANCE Lateral gene transfer (LGT), along with positive selection and gene duplication, are the three main mechanisms that drive adaptive evolution of microbial genomes and communities, but their relative importance is unclear. Some recent studies suggested that LGT is a major adaptive mechanism for microbial populations in response to changing environments, and hence, it could also be critical in shaping microbial community structure. However, direct evidence of LGT and its rates in extant natural microbial communities in response to changing environments is still lacking. Our results presented in this study provide explicit evidence that LGT played a crucial role in driving the evolution of a groundwater microbial community in response to extreme heavy metal contamination. It appears that acquisition of genes critical for survival, growth, and reproduction via LGT is the most rapid and effective way to enable microorganisms and associated microbial communities to quickly adapt to abrupt harsh environmental stresses.
59 BASIC BIOLOGICAL SCIENCES↗
Measurement and modeling of intrinsic transcription terminators
Corrigendum
59 BASIC BIOLOGICAL SCIENCES↗
Exometabolomics Assisted Design and Validation of Synthetic Obligate Mutualism
Synthetic microbial ecology has the potential to enhance the productivity and resiliency of biotechnology processes compared to approaches using single isolates. Engineering microbial consortia is challenging; however, one approach that has attracted significant attention is the creation of synthetic obligate mutualism using auxotrophic mutants that depend on each other for exchange or cross-feeding of metabolites. In this paper, we describe the integration of mutant library fitness profiling with mass spectrometry based exometabolomics as a method for constructing synthetic mutualism based on cross-feeding. Two industrially important species lacking known ecological interactions, Zymomonas mobilis and Escherichia coli, were selected as the test species. Amino acid exometabolites identified in the spent medium of Z. mobilis were used to select three corresponding E. coli auxotrophs (proA, pheA and IlvA), as potential E. coli counterparts for the coculture. A pooled mutant fitness assay with a Z. mobilis transposon mutant library was used to identify mutants with improved growth in the presence of E. coli. An auxotroph mutant in a gene (ZMO0748) with sequence similarity to cysteine synthase A (cysK), was selected as the Z. mobilis counterpart for the coculture. Exometabolomic analysis of spent E. coli medium identified glutathione related metabolites as potentially available for rescue of the Z. mobilis cysteine synthase mutant. Three sets of cocultures between the Z. mobilis auxotroph and each of the three E. coli auxotrophs were monitored by optical density for growth and analyzed by flow cytometry to confirm high cell counts for each species. Taken together, our methods provide a technological framework for creating synthetic mutualisms combining existing screening based methods and exometabolomics for both the selection of obligate mutualism partners and elucidation of metabolites involved in auxotroph rescue.
59 BASIC BIOLOGICAL SCIENCES↗