Mixed heavy metal stress induces global iron starvation response.

Multiple heavy metal contamination is an increasingly common global problem. Heavy metals have the potential to disrupt microbially mediated biogeochemical cycling. However, systems-level studies on the effects of combinations of heavy metals on bacteria are lacking. For this study, we focused on the Oak Ridge Reservation (ORR; Oak Ridge, TN, USA) subsurface which is contaminated with several heavy metals and high concentrations of nitrate. Using a native Bacillus cereus isolate that represents a dominant species at this site, we assessed the combined impact of eight metal contaminants, all at site-relevant concentrations, on cell processes through an integrated multi-omics approach that included discovery proteomics, targeted metabolomics, and targeted gene-expression profiling. The combination of eight metals impacted cell physiology in a manner that could not have been predicted from summing phenotypic responses to the individual metals. Exposure to the metal mixture elicited a global iron starvation response not observed during individual metal exposures. This disruption of iron homeostasis resulted in decreased activity of the iron-cofactor-containing nitrate and nitrite reductases, both of which are important in biological nitrate removal at the site. We propose that the combinatorial effects of simultaneous exposure to multiple heavy metals is an underappreciated yet significant form of cell stress in the environment with the potential to disrupt global nutrient cycles and to impede bioremediation efforts at mixed waste sites. Our work underscores the need to shift from single- to multi-metal studies for assessing and predicting the impacts of complex contaminants on microbial systems.

Iron- and aluminium-induced depletion of molybdenum in acidic environments impedes the nitrogen cycle.

Anthropogenic nitrate contamination is a serious problem in many natural environments. Nitrate removal by microbial action is dependent on the metal molybdenum (Mo), which is required by nitrate reductase for denitrification and dissimilatory nitrate reduction to ammonium. The soluble form of Mo, molybdate (MoO4 2- ), is incorporated into and adsorbed by iron (Fe) and aluminium (Al) (oxy) hydroxide minerals. Herein we used Oak Ridge Reservation (ORR) as a model nitrate-contaminated acidic environment to investigate whether the formation of Fe- and Al-precipitates could impede microbial nitrate removal by depleting Mo. We demonstrate that Fe and Al mineral formation that occurs as the pH of acidic synthetic groundwater is increased, decreases soluble Mo to low picomolar concentrations, a process proposed to mimic environmental diffusion of acidic contaminated groundwater. Analysis of ORR sediments revealed recalcitrant Mo in the contaminated core that co-occurred with Fe and Al, consistent with Mo scavenging by Fe/Al precipitates. Nitrate removal by ORR isolate Pseudomonas fluorescens N2A2 is virtually abolished by Fe/Al precipitate-induced Mo depletion. The depletion of naturally occurring Mo in nitrate- and Fe/Al-contaminated acidic environments like ORR or acid mine drainage sites has the potential to impede microbial-based nitrate reduction thereby extending the duration of nitrate in the environment.

Characterization of membrane-bound sulfane reductase: A missing link in the evolution of modern day respiratory complexes.

Hyperthermophilic archaea contain a hydrogen gas-evolving,respiratory membrane-bound NiFe-hydrogenase (MBH) that is very closely related to the aerobic respiratory complex I. During growth on elemental sulfur (S°), these microorganisms also produce a homologous membrane-bound complex (MBX), which generates H2S. MBX evolutionarily links MBH to complex I, but its catalytic function is unknown. Herein, we show that MBX reduces the sulfane sulfur of polysulfides by using ferredoxin (Fd) as the electron donor, and we rename it membrane-bound sulfane reductase (MBS). Two forms of affinity-tagged MBS were purified from genetically engineered Pyrococcus furiosus (a hyperthermophilic archaea species): the 13-subunit holoenzyme (S-MBS) and a cytoplasmic 4-subunit catalytic subcomplex (C-MBS). S-MBS and C-MBS reduced dimethyl trisulfide (DMTS) with comparable Km (∼490 µm) and Vmax values (12 µmol/min/mg). The MBS catalytic subunit (MbsL), but not that of complex I (NuoD), retains two of four NiFe-coordinating cysteine residues of MBH. However, these cysteine residues were not involved in MBS catalysis because a mutant P. furiosus strain (MbsLC85A/C385A) grew normally with S°. The products of the DMTS reduction and properties of polysulfides indicated that in the physiological reaction, MBS uses Fd (Eo' = -480 mV) to reduce sulfane sulfur (Eo' -260 mV) and cleave organic (RS n R, n ≥ 3) and anionic polysulfides (S n2-, n ≥ 4) but that it does not produce H2S. Based on homology to MBH, MBS also creates an ion gradient for ATP synthesis. This work establishes the electrochemical reaction catalyzed by MBS that is intermediate in the evolution from proton- to quinone-reducing respiratory complexes.

Smartphone Analytics: Mobilizing the Lab into the Cloud for Omic-Scale Analyses.

Active data screening is an integral part of many scientific activities, and mobile technologies have greatly facilitated this process by minimizing the reliance on large hardware instrumentation. In order to meet with the increasingly growing field of metabolomics and heavy workload of data processing, we designed the first remote metabolomic data screening platform for mobile devices. Two mobile applications (apps), XCMS Mobile and METLIN Mobile, facilitate access to XCMS and METLIN, which are the most important components in the computer-based XCMS Online platforms. These mobile apps allow for the visualization and analysis of metabolic data throughout the entire analytical process. Specifically, XCMS Mobile and METLIN Mobile provide the capabilities for remote monitoring of data processing, real time notifications for the data processing, visualization and interactive analysis of processed data (e.g., cloud plots, principle component analysis, box-plots, extracted ion chromatograms, and hierarchical cluster analysis), and database searching for metabolite identification. These apps, available on Apple iOS and Google Android operating systems, allow for the migration of metabolomic research onto mobile devices for better accessibility beyond direct instrument operation. The utility of XCMS Mobile and METLIN Mobile functionalities was developed and is demonstrated here through the metabolomic LC-MS analyses of stem cells, colon cancer, aging, and bacterial metabolism.

Characterization of ten heterotetrameric NDP-dependent acyl-CoA synthetases of the hyperthermophilic archaeon Pyrococcus furiosus.

The hyperthermophilic archaeon Pyrococcus furiosus grows by fermenting peptides and carbohydrates to organic acids. In the terminal step, acyl-CoA synthetase (ACS) isoenzymes convert acyl-CoA derivatives to the corresponding acid and conserve energy in the form of ATP. ACS1 and ACS2 were previously purified from P. furiosus and have α 2 ß 2 structures but the genome contains genes encoding three additional α-subunits. The ten possible combinations of α and ß genes were expressed in E. coli and each resulted in stable and active α 2 ß 2 isoenzymes. The α-subunit of each isoenzyme determined CoA-based substrate specificity and between them they accounted for the CoA derivatives of fourteen amino acids. The ß-subunit determined preference for adenine or guanine nucleotides. The GTP-generating isoenzymes are proposed to play a role in gluconeogenesis by producing GTP for GTP-dependent phosphoenolpyruvate carboxykinase and for other GTP-dependent processes. Transcriptional and proteomic data showed that all ten isoenzymes are constitutively expressed indicating that both ATP and GTP are generated from the metabolism of most of the amino acids. A phylogenetic analysis showed that the ACSs of P. furiosus and other members of the Thermococcales are evolutionarily distinct from those found throughout the rest of biology, including those of other hyperthermophilic archaea.

Correlating the transcriptome, proteome, and metabolome in the environmental adaptation of a hyperthermophile.

We have performed a comprehensive characterization of global molecular changes for a model organism Pyrococcus furiosus using transcriptomic (DNA microarray), proteomic, and metabolomic analysis as it undergoes a cold adaptation response from its optimal 95 to 72 degrees C. Metabolic profiling on the same set of samples shows the down-regulation of many metabolites. However, some metabolites are found to be strongly up-regulated. An approach using accurate mass, isotopic pattern, database searching, and retention time is used to putatively identify several metabolites of interest. Many of the up-regulated metabolites are part of an alternative polyamine biosynthesis pathway previously established in a thermophilic bacterium Thermus thermophilus. Arginine, agmatine, spermidine, and branched polyamines N4-aminopropylspermidine and N4-( N-acetylaminopropyl)spermidine were unambiguously identified based on their accurate mass, isotopic pattern, and matching of MS/MS data acquired under identical conditions for the natural metabolite and a high purity standard. Both DNA microarray and semiquantitative proteomic analysis using a label-free spectral counting approach indicate the down-regulation of a large majority of genes with diverse predicted functions related to growth such as transcription, amino acid biosynthesis, and translation. Some genes are, however, found to be up-regulated through the measurement of their relative mRNA and protein levels. The complimentary information obtained by the various "omics" techniques is used to catalogue and correlate the overall molecular changes.