Reconstruction of Extracellular Respiratory Pathways for Iron(III) Reduction in Shewanella Oneidensis Strain MR-1

Shewanella oneidensis strain MR-1 is a facultative anaerobic bacterium capable of respiring a multitude of electron acceptors, many of which require the Mtr respiratory pathway. The core Mtr respiratory pathway includes a periplasmic c-type cytochrome (MtrA), an integral outer-membrane β-barrel protein (MtrB), and an outer-membrane-anchored c-type cytochrome (MtrC). Together, these components facilitate transfer of electrons from the c-type cytochrome CymA in the cytoplasmic membrane to electron acceptors at and beyond the outer-membrane. The genes encoding these core proteins have paralogs in the S. oneidensis genome (mtrB and mtrA each have four while mtrC has three) and some of the paralogs of mtrC and mtrA are able to form functional Mtr complexes. We demonstrate that of the additional three mtrB paralogs found in the S. oneidensis genome, only MtrE can replace MtrB to form a functional respiratory pathway to soluble iron(III) citrate. We also evaluate which mtrC/mtrA paralog pairs (a total of 12 combinations) are able to form functional complexes with endogenous levels of mtrB paralog expression. Finally, we reconstruct all possible functional Mtr complexes and test them in a S. oneidensis mutant strain where all paralogs have been eliminated from the genome. We find that each combination tested with the exception of MtrA/MtrE/OmcA is able to reduce iron(III) citrate at a level significantly above background. The results presented here have implications toward the evolution of anaerobic extracellular respiration in Shewanella and for future studies looking to increase the rates of substrate reduction for water treatment, bioremediation, or electricity production

What Genetics Offers Geobiology

For over 50 years, the Parker Brothers’ board game “Clue” has maintained its position as the classic family detective game. A murder has been committed in the mansion, but we don’t know where, by whom, or how. Was it Professor Plum in the study with a knife, or Miss Scarlett in the ballroom with a candlestick? Through rolls of the dice, fragments of information patiently accumulated piece-by-piece, and the application of logic, players construct a case to figure out “whodunit”. Because there are several potential solutions to the problem, the key challenge is to figure out what happened by understanding how it happened

Investigating the Possible Role of a Glycosyl Transferase Protein in the Biosynthesis of Long-Chain Hydrocarbons in Shewanella oneidensis

Abstract In the search for alternative sources of energy, new organisms are being looked at as potential biofuel producers. It has been shown that the long-chain hydrocarbons produced by certain bacteria can be broken down into usable fuel. Shewanella oneidensis, a gram-negative bacterium, may be an ideal organism for producing these long-chain hydrocarbons. The hydrocarbons are formed as a result of a head-to-head fatty acid condensation via the enzyme OleA. Because previous attempts to overproduce these long-chain hydrocarbons in recombinant S. oneidensis strains containing the oleA gene from Stenotrophomonas maltophilia have not yielded a significant increase in production over the wild type strain, the question has been raised as to whether or not other proteins might play a role, either directly or indirectly, in the production process. In my thesis work, I deleted the gene SO_3174, which was interrupted in a transposon mutagenesis screen for increased hydrocarbon production, from S. oneidensis. SO_3174 encodes a putative glycosyl transferase protein. I then tried to show that deleting SO_3174 resulted in an increase in hydrocarbon production just as the interruption of the gene had. The deletion strain showed an increased fluorescence in the presence of Nile Red dye, a hydrophobic dye that can be used to indirectly detect hydrocarbon levels. However, the deletion strain did not exhibit increased hydrocarbons during direct analysis of nonpolar extractions. These same results were obtained from a strain containing the SO_3174 deletion and expressing OleA from S. maltophilia. The SO_3174 deletion strain was shown to have lower levels of extracellular polysaccharides than wild type S. oneidensis based on a Congo Red binding assay. From these results, I hypothesized that the lower levels of extracellular sugars resulting from the absence of the glycosyl transferase may have made the membrane of the deletion strain more permeable to the Nile Red dye. Overall, I found that the protein encoded by SO_3174 most likely does not play a role in hydrocarbon biosynthesis in S. oneidensis. S. oneidensis and Hydrocarbons One of the sources of biofuel being examined by researchers is bacteria. Certain strains of bacteria, including S. oneidensis have been found to produce longchain hydrocarbons and ketones that can be broken down for fuel. These hydrocarbons are produced as a result of a head-to-head condensation of fatty acids by the enzyme OleA. From Sukovich et al. S. Oneidensis cells exposed to Nile Red dye From Pinzon et al. The goal of my study was to find a protein that is involved either directly or indirectly in the biosynthesis of long-chain ketones and hydrocarbons. The other goal was to validate the theory of using Nile Red fluorescence to detect changes in hydrocarbon production. My hypothesis was that deleting the gene SO_3174, interrupted in a transposon mutagenesis screen for increased hydrocarbon production, from S. oneidensis and adding Stenotrophomonas maltophilia oleA would result in increased production of hydrocarbons. Screening for Hydrocarbons Nile Red screen results for ΔSO_3174 deletion strain and wild type S. oneidensis with and without S. maltophilia (S.M.) OleA in terms of RFU/OD 600 . For both comparisons, the deletion resulted in a higher Nile Red signal. Hydrocarbon extraction results for ΔSO_3174 deletion strain and wild type S. oneidensis in terms of FID/OD 600 . The deletion did not result in significantly higher hydrocarbons. Hydrocarbon extraction results for ΔSO_3174 deletion strain and wild type S. oneidensis with S.M. OleA in terms of FID/OD 600 . The deletion did not result in significantly higher hydrocarbons. Conclusions In summary, it was found that the deletion of SO_3174 resulted in an increase in Nile Red signal but no significant difference in hydrocarbon production. One possible hypothesis for this is that the reduction in extracellular polysaccharides resulting from the absence of the glycosyl transferase made the membrane more permeable to the Nile Red dye. This claim can be backed up by the fact that a Congo Red binding assay showed the deletion strain had less extracellular sugars than wild type. The next step from here would be to repeat this experiment with another one of the genes found interrupted in the transposon mutagenesis screen and see if deleting it has any effect on hydrocarbon production

Extracellular respiration

Although it has long been known that microbes can generate energy using diverse strategies, only recently has it become clear that a growing number involve electron transfer to or from extracellular substrates. The best-known example of what we will term ‘extracellular respiration’ is electron transfer between microbes and minerals, such as iron and manganese (hydr)oxides. This makes sense, given that these minerals are sparingly soluble. What is perhaps surprising, however, is that a number of substrates that might typically be classified as ‘soluble’ are also respired at the cell surface. There are several reasons why this might be the case: the substrate, in its ecological context, might be associated with a solid surface and thus effectively insoluble; the substrate, while soluble, might simply be too large to transport inside the cell; or the substrate, while benign in one redox state, might become toxic after it is metabolized. In this review, we discuss various examples of extracellular respiration, paying particular attention to what is known about the molecular mechanisms underlying these processes. As will become clear, much remains to be learned about the biochemistry, cell biology and regulation of extracellular respiration, making it a rich field of study for molecular microbiologists

The genetics of geochemistry

Bacteria are remarkable in their metabolic diversity due to their ability to harvest energy from myriad oxidation and reduction reactions. In some cases, their metabolisms involve redox transformations of metal(loid)s, which lead to the precipitation, transformation, or dissolution of minerals. Microorganism/mineral interactions not only affect the geochemistry of modern environments, but may also have contributed to shaping the near-surface environment of the early Earth. For example, bacterial anaerobic respiration of ferric iron or the toxic metalloid arsenic is well known to affect water quality in many parts of the world today, whereas the utilization of ferrous iron as an electron donor in anoxygenic photosynthesis may help explain the origin of Banded Iron Formations, a class of ancient sedimentary deposits. Bacterial genetics holds the key to understanding how these metabolisms work. Once the genes and gene products that catalyze geochemically relevant reactions are understood, as well as the conditions that trigger their expression, we may begin to predict when and to what extent these metabolisms influence modern geochemical cycles, as well as develop a basis for deciphering their origins and how organisms that utilized them may have altered the chemical and physical features of our planet

Fnr (EtrA) acts as a fine-tuning regulator of anaerobic metabolism in \u3cem\u3eShewanella oneidensis\u3c/em\u3e MR-1

Background EtrA in Shewanella oneidensis MR-1, a model organism for study of adaptation to varied redox niches, shares 73.6% and 50.8% amino acid sequence identity with the oxygen-sensing regulators Fnr in E. coli and Anr in Pseudomonas aeruginosa, respectively; however, its regulatory role of anaerobic metabolism in Shewanella spp. is complex and not well understood. Results The expression of the nap genes, nrfA, cymA and hcp was significantly reduced in etrA deletion mutant EtrA7-1; however, limited anaerobic growth and nitrate reduction occurred, suggesting that multiple regulators control nitrate reduction in this strain. Dimethyl sulfoxide (DMSO) and fumarate reductase gene expression was down-regulated at least 2-fold in the mutant, which, showed lower or no reduction of these electron acceptors when compared to the wild type, suggesting both respiratory pathways are under EtrA control. Transcript analysis further suggested a role of EtrA in prophage activation and down-regulation of genes implicated in aerobic metabolism. Conclusion In contrast to previous studies that attributed a minor regulatory role to EtrA in Shewanella spp., this study demonstrates that EtrA acts as a global transcriptional regulator and, in conjunction with other regulators, fine-tunes the expression of genes involved in anaerobic metabolism in S. oneidensis strain MR-1. Transcriptomic and sequence analyses of the genes differentially expressed showed that those mostly affected by the mutation belonged to the Energy metabolism category, while stress-related genes were indirectly regulated in the mutant possibly as a result of a secondary perturbation (e.g. oxidative stress, starvation). We also conclude based on sequence, physiological and expression analyses that this regulator is more appropriately termed Fnr and recommend this descriptor be used in future publications

Fnr (EtrA) acts as a fine-tuning regulator of anaerobic metabolism in Shewanella oneidensis MR-1

BackgroundEtrA in Shewanella oneidensis MR-1, a model organism for study of adaptation to varied redox niches, shares 73.6% and 50.8% amino acid sequence identity with the oxygen-sensing regulators Fnr in E. coli and Anr in Pseudomonas aeruginosa, respectively; however, its regulatory role of anaerobic metabolism in Shewanella spp. is complex and not well understood.ResultsThe expression of the nap genes, nrfA, cymA and hcp was significantly reduced in etrA deletion mutant EtrA7-1; however, limited anaerobic growth and nitrate reduction occurred, suggesting that multiple regulators control nitrate reduction in this strain. Dimethyl sulfoxide (DMSO) and fumarate reductase gene expression was down-regulated at least 2-fold in the mutant, which, showed lower or no reduction of these electron acceptors when compared to the wild type, suggesting both respiratory pathways are under EtrA control. Transcript analysis further suggested a role of EtrA in prophage activation and down-regulation of genes implicated in aerobic metabolism.ConclusionIn contrast to previous studies that attributed a minor regulatory role to EtrA in Shewanella spp., this study demonstrates that EtrA acts as a global transcriptional regulator and, in conjunction with other regulators, fine-tunes the expression of genes involved in anaerobic metabolism in S. oneidensis strain MR-1. Transcriptomic and sequence analyses of the genes differentially expressed showed that those mostly affected by the mutation belonged to the "Energy metabolism" category, while stress-related genes were indirectly regulated in the mutant possibly as a result of a secondary perturbation (e.g. oxidative stress, starvation). We also conclude based on sequence, physiological and expression analyses that this regulator is more appropriately termed Fnr and recommend this descriptor be used in future publications

Stable sub-complexes observed in situ suggest a modular assembly pathway of the bacterial flagellar motor

The self-assembly of cellular macromolecular machines such as the bacterial flagellar motor requires the spatio- temporal synchronization of gene expression, protein localization and association of a dozen or more unique components. In Salmonella and Escherichia coli, a sequential, outward assembly mechanism has been proposed for the flagellar motor starting from the inner membrane, with each subsequent component stabilizing the last. Here, using electron cryo-tomography of intact Legionella pneumophila, Pseudomonas aeruginosa and Shewanella oneidensis cells, we observe stable outer-membrane-embedded sub-complexes of the flagellar motor. These sub- complexes consist of the periplasmic embellished P- and L-rings, in the absence of other flagellar components, and bend the membrane inward dramatically. Additionally, we also observe independent inner-membrane sub- complexes consisting of the C- and MS-rings and export apparatus. These results suggest an alternate model for flagellar motor assembly in which outer- and inner-membrane-associated sub-complexes form independently and subsequently join, enabling later steps of flagellar production to proceed