Identification and Characterization of MtoA: A Decaheme c-Type Cytochrome of the Neutrophilic Fe(II)-Oxidizing Bacterium Sideroxydans lithotrophicus ES-1

The Gram-negative bacterium Sideroxydans lithotrophicus ES-1 (ES-1) grows on FeCO3 or FeS at oxic–anoxic interfaces at circumneutral pH, and the ES-1-mediated Fe(II) oxidation occurs extracellularly. However, the molecular mechanisms underlying ES-1’s ability to oxidize Fe(II) remain unknown. Survey of the ES-1 genome for candidate genes for microbial extracellular Fe(II) oxidation revealed that it contained a three-gene cluster encoding homologs of Shewanella oneidensis MR-1 (MR-1) MtrA, MtrB, and CymA that are involved in extracellular Fe(III) reduction. Homologs of MtrA and MtrB were also previously shown to be involved in extracellular Fe(II) oxidation by Rhodopseudomonas palustris TIE-1. To distinguish them from those found in MR-1, the identified homologs were named MtoAB and CymAES-1. Cloned mtoA partially complemented an MR-1 mutant without MtrA with regards to ferrihydrite reduction. Characterization of purified MtoA showed that it was a decaheme c-type cytochrome and oxidized soluble Fe(II). Oxidation of Fe(II) by MtoA was pH- and Fe(II)-complexing ligand-dependent. Under conditions tested, MtoA oxidized Fe(II) from pH 7 to pH 9 with the optimal rate at pH 9. MtoA oxidized Fe(II) complexed with different ligands at different rates. The reaction rates followed the order Fe(II)Cl2 > Fe(II)–citrate > Fe(II)–NTA > Fe(II)–EDTA with the second-order rate constants ranging from 6.3 × 10-3 µM-1 s-1 for oxidation of Fe(II)Cl2 to 1.0 × 10-3 µM-1 s-1 for oxidation of Fe(II)–EDTA. Thermodynamic modeling showed that redox reaction rates for the different Fe(II)-complexes correlated with their respective estimated reaction-free energies. Collectively, these results demonstrate that MtoA is a functional Fe(II)-oxidizing protein that, by working in concert with MtoB and CymAES-1, may oxidize Fe(II) at the bacterial surface and transfer released electrons across the bacterial cell envelope to the quinone pool in the inner membrane during extracellular Fe(II) oxidation by ES-1

Identification and characterization of MtoA: a decaheme \u3ci\u3ec\u3c/i\u3e-type cytochrome of the neutrophilic Fe(II)-oxidizing bacterium Sideroxydans lithotrophicus ES-1

The Gram-negative bacterium Sideroxydans lithotrophicus ES-1(ES-1) grows on FeCO3 or FeS at oxic–anoxic interfaces at circumneutral pH, and the ES-1-mediated Fe(II) oxidation occurs extracellularly. However, the molecular mechanisms underlying ES-1’s ability to oxidize Fe(II) remain unknown. Survey of the ES-1 genome for candidate genes formicrobial extracellular Fe(II) oxidation revealed that it contained a three-genecluster encoding homologs of Shewanella oneidensis MR-1(MR-1) MtrA, MtrB, and CymA that are involved in extracellular Fe(III) reduction. Homologs of MtrA and MtrB were also previously shown to be involved in extracellular Fe(II) oxidation by Rhodopseudomonas palustris TIE-1. To distinguish them from those found in MR-1, the identified homologs were named MtoAB andCymAES-1. Cloned mtoA partially complemented an MR-1 mutant without MtrA with regards to ferrihydrite reduction. Characterization of purified MtoA showed that it was a decaheme c-type cytochrome and oxidized soluble Fe(II). Oxidation of Fe(II) by MtoA was pH- and Fe (II) – complexing ligand-dependent.Under conditions tested, MtoA oxidized Fe(II) from pH 7 to pH 9 with the optimal rate at pH 9. MtoA oxidized Fe(II) complexed with different ligands at different rates. The reaction rates followed the order Fe(II)Cl2\u3e Fe(II) –citrate\u3e Fe(II)–NTA\u3eFe(II)–EDTA with the second-order rate constants ranging from 6.3×10−3μM−1s−1 for oxidation of Fe(II) Cl2 to 1.0 × 10−3 μM−1s−1 for oxidation of Fe (II)–EDTA

Defending Our Public Biological Databases as a Global Critical Infrastructure

Progress in modern biology is being driven, in part, by the large amounts of freely available data in public resources such as the International Nucleotide Sequence Database Collaboration (INSDC), the world's primary database of biological sequence (and related) information. INSDC and similar databases have dramatically increased the pace of fundamental biological discovery and enabled a host of innovative therapeutic, diagnostic, and forensic applications. However, as high-value, openly shared resources with a high degree of assumed trust, these repositories share compelling similarities to the early days of the Internet. Consequently, as public biological databases continue to increase in size and importance, we expect that they will face the same threats as undefended cyberspace. There is a unique opportunity, before a significant breach and loss of trust occurs, to ensure they evolve with quality and security as a design philosophy rather than costly “retrofitted” mitigations. This Perspective surveys some potential quality assurance and security weaknesses in existing open genomic and proteomic repositories, describes methods to mitigate the likelihood of both intentional and unintentional errors, and offers recommendations for risk mitigation based on lessons learned from cybersecurity

The Succinated Proteome

The post-translational modifications (PTMs) of cysteine residues include oxidation, S-glutathionylation, S-nitrosylation, and succination, all of which modify protein function or turnover in response to a changing intracellular redox environment. Succination is a chemical modification of cysteine in proteins by the Krebs cycle intermediate, fumarate, yielding S-(2-succino) cysteine (2SC). Intracellular fumarate concentration and succination of proteins are increased by hyperpolarization of the inner mitochondrial membrane, in concert with mitochondrial, endoplasmic reticulum (ER) and oxidative stress in 3T3 adipocytes grown in high glucose medium and in adipose tissue in obesity and diabetes in mice. Increased succination of proteins is also detected in the kidney of a fumarase deficient conditional knock-out mouse which develops renal cysts. A wide range of proteins are subject to succination, including enzymes, adipokines, cytoskeletal proteins, and ER chaperones with functional cysteine residues. There is also some overlap between succinated and glutathionylated proteins, suggesting that the same low pK(a) thiols are targeted by both. Succination of adipocyte proteins in diabetes increases as a result of nutrient excess derived mitochondrial stress and this is inhibited by uncouplers, which discharge the mitochondrial membrane potential (ΔΨ(m)) and relieve the electron transport chain. 2SC therefore serves as a biomarker of mitochondrial stress or dysfunction in chronic diseases, such as obesity, diabetes, and cancer, and recent studies suggest that succination is a mechanistic link between mitochondrial dysfunction, oxidative and ER stress, and cellular progression toward apoptosis. In this article, we review the history of the succinated proteome and the challenges associated with measuring this non-enzymatic PTM of proteins by proteomics approaches

Perspective on Improving Environmental Monitoring of Biothreats

For more than a decade, the United States has performed environmental monitoring by collecting and analyzing air samples for a handful of biological threat agents (BTAs) in order to detect a possible biological attack. This effort has faced numerous technical challenges including timeliness, sampling efficiency, sensitivity, specificity, and robustness. The cost of city-wide environmental monitoring using conventional technology has also been a challenge. A large group of scientists with expertise in bioterrorism defense met to assess the objectives and current efficacy of environmental monitoring and to identify operational and technological changes that could enhance its efficacy and cost-effectiveness, thus enhancing its value. The highest priority operational change that was identified was to abandon the current concept of city-wide environmental monitoring because the operational costs were too high and its value was compromised by low detection sensitivity and other environmental factors. Instead, it was suggested that the focus should primarily be on indoor monitoring and secondarily on special-event monitoring because objectives are tractable and these operational settings are aligned with likelihood and risk assessments. The highest priority technological change identified was the development of a reagent-less, real-time sensor that can identify a potential airborne release and trigger secondary tests of greater sensitivity and specificity for occasional samples of interest. This technological change could be transformative with the potential to greatly reduce operational costs and thereby create the opportunity to expand the scope and effectiveness of environmental monitoring

Detection and Identification of Heme <i>c</i>‑Modified Peptides by Histidine Affinity Chromatography, High-Performance Liquid Chromatography–Mass Spectrometry, and Database Searching

Multiheme c-type cytochromes (proteins with covalently attached heme <i>c</i> moieties) play important roles in extracellular metal respiration in dissimilatory metal-reducing bacteria. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) characterization of c-type cytochromes is hindered by the presence of multiple heme groups, since the heme <i>c</i> modified peptides are typically not observed or, if observed, not identified. Using a recently reported histidine affinity chromatography (HAC) procedure, we enriched heme <i>c</i> tryptic peptides from purified bovine heart cytochrome <i>c</i>, two bacterial decaheme cytochromes, and subjected these samples to LC–MS/MS analysis. Enriched bovine cytochrome <i>c</i> samples yielded 3- to 6-fold more confident peptide–spectrum matches to heme <i>c</i> containing peptides than unenriched digests. In unenriched digests of the decaheme cytochrome MtoA from <i>Sideroxydans lithotrophicus</i> ES-1, heme <i>c</i> peptides for 4 of the 10 expected sites were observed by LC–MS/MS; following HAC fractionation, peptides covering 9 out of 10 sites were obtained. Heme <i>c</i> peptide spiked into <i>E. coli</i> lysates at mass ratios as low as 1 × 10^–4 was detected with good signal-to-noise after HAC and LC–MS/MS analysis. In addition to HAC, we have developed a proteomics database search strategy that takes into account the unique physicochemical properties of heme <i>c</i> peptides. The results suggest that accounting for the double thioether link between heme <i>c</i> and peptide, and the use of the labile heme fragment as a reporter ion, can improve database searching results. The combination of affinity chromatography and heme-specific informatics yielded increases in the number of peptide–spectrum matches of 20–100-fold for bovine cytochrome <i>c</i>

Detection and Identification of Heme <i>c</i>‑Modified Peptides by Histidine Affinity Chromatography, High-Performance Liquid Chromatography–Mass Spectrometry, and Database Searching

Multiheme c-type cytochromes (proteins with covalently attached heme <i>c</i> moieties) play important roles in extracellular metal respiration in dissimilatory metal-reducing bacteria. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) characterization of c-type cytochromes is hindered by the presence of multiple heme groups, since the heme <i>c</i> modified peptides are typically not observed or, if observed, not identified. Using a recently reported histidine affinity chromatography (HAC) procedure, we enriched heme <i>c</i> tryptic peptides from purified bovine heart cytochrome <i>c</i>, two bacterial decaheme cytochromes, and subjected these samples to LC–MS/MS analysis. Enriched bovine cytochrome <i>c</i> samples yielded 3- to 6-fold more confident peptide–spectrum matches to heme <i>c</i> containing peptides than unenriched digests. In unenriched digests of the decaheme cytochrome MtoA from <i>Sideroxydans lithotrophicus</i> ES-1, heme <i>c</i> peptides for 4 of the 10 expected sites were observed by LC–MS/MS; following HAC fractionation, peptides covering 9 out of 10 sites were obtained. Heme <i>c</i> peptide spiked into <i>E. coli</i> lysates at mass ratios as low as 1 × 10^–4 was detected with good signal-to-noise after HAC and LC–MS/MS analysis. In addition to HAC, we have developed a proteomics database search strategy that takes into account the unique physicochemical properties of heme <i>c</i> peptides. The results suggest that accounting for the double thioether link between heme <i>c</i> and peptide, and the use of the labile heme fragment as a reporter ion, can improve database searching results. The combination of affinity chromatography and heme-specific informatics yielded increases in the number of peptide–spectrum matches of 20–100-fold for bovine cytochrome <i>c</i>

Dynameomics:a comprehensive database of protein dynamics

The dynamic behavior of proteins is important for an understanding of their function and folding. We have performed molecular dynamics simulations of the native state and unfolding pathways of over 1000 proteins, representing the majority of folds in globular proteins. These data are stored and organized using an innovative database approach, which can be mined to obtain both general and specific information about the dynamics and folding/unfolding of proteins, relevant subsets thereof, and individual proteins. Here we describe the project in general terms and the type of information contained in the database. Then we provide examples of mining the database for information relevant to protein folding, structure building, the effect of single-nucleotide polymorphisms, and drug design. The native state simulation data and corresponding analyses for the 100 most populated metafolds, together with related resources, are publicly accessible through www.dynameomics.org

Investigation of Yersinia pestis Laboratory Adaptation through a Combined Genomics and Proteomics Approach

The bacterial pathogen Yersinia pestis, the cause of plague in humans and animals, normally has a sylvatic lifestyle, cycling between fleas and mammals. In contrast, laboratory-grown Y. pestis experiences a more constant environment and conditions that it would not normally encounter. The transition from the natural environment to the laboratory results in a vastly different set of selective pressures, and represents what could be considered domestication. Understanding the kinds of adaptations Y. pestis undergoes as it becomes domesticated will contribute to understanding the basic biology of this important pathogen. In this study, we performed a parallel serial passage experiment (PSPE) to explore the mechanisms by which Y. pestis adapts to laboratory conditions, hypothesizing that cells would undergo significant changes in virulence and nutrient acquisition systems. Two wild strains were serially passaged in 12 independent populations each for ~750 generations, after which each population was analyzed using whole-genome sequencing, LC-MS/MS proteomic analysis, and GC/MS metabolomics. We observed considerable parallel evolution in the endpoint populations, detecting multiple independent mutations in ail, pepA, and zwf, suggesting that specific selective pressures are shaping evolutionary responses. Complementary LC-MS/MS proteomic data provide physiological context to the observed mutations, and reveal regulatory changes not necessarily associated with specific mutations, including changes in amino acid metabolism and cell envelope biogenesis. Proteomic data support hypotheses generated by genomic data in addition to suggesting future mechanistic studies, indicating that future whole-genome sequencing studies be designed to leverage proteomics as a critical complement