Shewanella oneidensis MR-1 Sensory Box Protein Involved in Aerobic and Anoxic Growth

ABSTRACTAlthough little is known of potential function for conserved signaling proteins, it is hypothesized that such proteins play important roles to coordinate cellular responses to environmental stimuli. In order to elucidate the function of a putative sensory box protein (PAS domains) inShewanella oneidensisMR-1, the physiological role of SO3389 was characterized. The predicted open reading frame (ORF) encodes a putative sensory box protein that has PAS, GGDEF, and EAL domains, and an in-frame deletion mutant was constructed (ΔSO3389) with approximately 95% of the ORF deleted. Under aerated conditions, wild-type and mutant cultures had similar growth rates, but the mutant culture had a lower growth rate under static, aerobic conditions. Oxygen consumption rates were lower for mutant cultures (1.5-fold), and wild-type cultures also maintained lower dissolved oxygen concentrations under aerated growth conditions. When transferred to anoxic conditions, the mutant did not grow with fumarate, iron(III), or dimethyl sulfoxide (DMSO) as electron acceptors. Biochemical assays demonstrated the expression of differentc-type cytochromes as well as decreased fumarate reductase activity in the mutant transferred to anoxic growth conditions. Transcriptomic studies showed the inability of the mutant to up-express and down-express genes, includingc-type cytochromes (e.g., SO4047/SO4048, SO3285/SO3286), reductases (e.g., SO0768, SO1427), and potential regulators (e.g., SO1329). The complemented strain was able to grow when transferred from aerobic to anoxic growth conditions with the tested electron acceptors. The modeled structure for the SO3389 PAS domains was highly similar to the crystal structures of FAD-binding PAS domains that are known O2/redox sensors. Based on physiological, genomic, and bioinformatic results, we suggest that the sensory box protein, SO3389, is an O2/redox sensor that is involved in optimization of aerobic growth and transitions to anoxia inS. oneidensisMR-1.

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Causal Role of Single Nucleotide Polymorphisms within themprFGene of Staphylococcus aureus in Daptomycin Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01184-13 ◽

2013 ◽

Vol 57 (11) ◽

pp. 5658-5664 ◽

Cited By ~ 48

Author(s):

Soo-Jin Yang ◽

Nagendra N. Mishra ◽

Aileen Rubio ◽

Arnold S. Bayer

Keyword(s):

Staphylococcus Aureus ◽

Single Nucleotide Polymorphisms ◽

Point Mutations ◽

Nucleotide Polymorphisms ◽

Wild Type ◽

Single Nucleotide ◽

Content Type ◽

Reading Frame ◽

A Charge

ABSTRACTSingle nucleotide polymorphisms (SNPs) within themprFopen reading frame (ORF) have been commonly observed in daptomycin-resistant (DAPr)Staphylococcus aureusstrains. Such SNPs are usually associated with a gain-in-function phenotype, in terms of either increased synthesis or enhanced translocation (flipping) of lysyl-phosphatidylglycerol (L-PG). However, it is unclear if suchmprFSNPs are causal in DAPrstrains or are merely a biomarker for this phenotype. In this study, we used an isogenic set ofS. aureusstrains: (i) Newman, (ii) its isogenic ΔmprFmutant, and (iii) several intransplasmid complementation constructs, expressing either a wild-type or point-mutated form of themprFORF cloned from two isogenic DAP-susceptible (DAPs)-DAPrstrain pairs (616-701 and MRSA11/11-REF2145). Complementation of the ΔmprFstrain with singly point-mutatedmprFgenes (mprFS295LormprFT345A) revealed that (i) individual and distinct point mutations within themprFORF can recapitulate phenotypes observed in donor strains (i.e., changes in DAP MICs, positive surface charge, and cell membrane phospholipid profiles) and (ii) these gain-in-function SNPs (i.e., enhanced L-PG synthesis) likely promote reduced DAP binding toS. aureusby a charge repulsion mechanism. Thus, for these two DAPrstrains, the definedmprFSNPs appear to be causally related to this phenotype.

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Identification of a bacterial di-haem cytochrome c peroxidase from Methylomicrobium album BG8

Microbiology ◽

10.1099/mic.0.037119-0 ◽

2010 ◽

Vol 156 (9) ◽

pp. 2682-2690 ◽

Cited By ~ 8

Author(s):

O. A. Karlsen ◽

Ø. Larsen ◽

H. B. Jensen

Keyword(s):

Copper Ions ◽

Sequence Similarity ◽

Physiological Role ◽

Growth Conditions ◽

Inverse Pcr ◽

Restriction Enzyme Digestion ◽

Methylococcus Capsulatus ◽

Gene Encoding ◽

Reading Frame ◽

Methylomicrobium Album

The nucleotide sequence of an open reading frame (corB) downstream of the copper-repressible CorA-encoding gene of the methanotrophic bacterium Methylomicrobium album BG8 was obtained by restriction enzyme digestion and inverse PCR. The amino acid sequence deduced from this gene showed significant sequence similarity to the surface-associated di-haem cytochrome c peroxidase (SACCP) previously isolated from Methylococcus capsulatus (Bath), including both c-type haem-binding motifs. Homology analysis placed this protein, phylogenetically, within the subfamily containing the M. capsulatus SACCP of the bacterial di-haem cytochrome c peroxidase (BCCP) family of proteins. Immunospecific recognition confirmed synthesis of the M. album CorB as a protein non-covalently associated with the outer membrane and exposed to the periplasm. corB expression is regulated by the availability of copper ions during growth and the protein is most abundant in M. album when grown at a low copper-to-biomass ratio, indicating an important physiological role of CorB under these growth conditions. corB was co-transcribed with the gene encoding CorA, constituting a copper-responding operon, which appears to be under the control of a σ 54-dependent promoter. M. album CorB is the second isolated member of the recently described subfamily of the BCCP family of proteins. So far, these proteins have only been described in methanotrophic bacteria.

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Distinct Physiological Roles of the Three Ferredoxins Encoded in the Hyperthermophilic Archaeon Thermococcus kodakarensis

mBio ◽

10.1128/mbio.02807-18 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 7

Author(s):

Brett W. Burkhart ◽

Hallie P. Febvre ◽

Thomas J. Santangelo

Keyword(s):

Electron Flux ◽

Electron Flow ◽

Physiological Role ◽

High Energy ◽

Electron Acceptors ◽

Content Type ◽

Maximal Energy ◽

Thermococcus Kodakarensis ◽

Small Proteins ◽

Energy Gains

ABSTRACT Control of electron flux is critical in both natural and bioengineered systems to maximize energy gains. Both small molecules and proteins shuttle high-energy, low-potential electrons liberated during catabolism through diverse metabolic landscapes. Ferredoxin (Fd) proteins—an abundant class of Fe-S-containing small proteins—are essential in many species for energy conservation and ATP production strategies. It remains difficult to model electron flow through complicated metabolisms and in systems in which multiple Fd proteins are present. The overlap of activity and/or limitations of electron flux through each Fd can limit physiology and metabolic engineering strategies. Here we establish the interplay, reactivity, and physiological role(s) of the three ferredoxin proteins in the model hyperthermophile Thermococcus kodakarensis. We demonstrate that the three loci encoding known Fds are subject to distinct regulatory mechanisms and that specific Fds are utilized to shuttle electrons to separate respiratory and energy production complexes during different physiological states. The results obtained argue that unique physiological roles have been established for each Fd and that continued use of T. kodakarensis and related hydrogen-evolving species as bioengineering platforms must account for the distinct Fd partnerships that limit flux to desired electron acceptors. Extrapolating our results more broadly, the retention of multiple Fd isoforms in most species argues that specialized Fd partnerships are likely to influence electron flux throughout biology. IMPORTANCE High-energy electrons liberated during catabolic processes can be exploited for energy-conserving mechanisms. Maximal energy gains demand these valuable electrons be accurately shuttled from electron donor to appropriate electron acceptor. Proteinaceous electron carriers such as ferredoxins offer opportunities to exploit specific ferredoxin partnerships to ensure that electron flux to critical physiological pathways is aligned with maximal energy gains. Most species encode many ferredoxin isoforms, but very little is known about the role of individual ferredoxins in most systems. Our results detail that ferredoxin isoforms make largely unique and distinct protein interactions in vivo and that flux through one ferredoxin often cannot be recovered by flux through a different ferredoxin isoform. The results obtained more broadly suggest that ferredoxin isoforms throughout biological life have evolved not as generic electron shuttles, but rather serve as selective couriers of valuable low-potential electrons from select electron donors to desirable electron acceptors.

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Pseudomonas aeruginosa Ethanol Oxidation by AdhA in Low-Oxygen Environments

Journal of Bacteriology ◽

10.1128/jb.00393-19 ◽

2019 ◽

Vol 201 (23) ◽

Cited By ~ 3

Author(s):

Alex W. Crocker ◽

Colleen E. Harty ◽

John H. Hammond ◽

Sven D. Willger ◽

Pedro Salazar ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Alcohol Dehydrogenase ◽

Ethanol Oxidation ◽

Physiological Role ◽

Wild Type ◽

Low Oxygen ◽

Content Type ◽

Anoxic Conditions ◽

Oxygen Conditions ◽

Acetate Accumulation

ABSTRACT Pseudomonas aeruginosa has a broad metabolic repertoire that facilitates its coexistence with different microbes. Many microbes secrete products that P. aeruginosa can then catabolize, including ethanol, a common fermentation product. Here, we show that under oxygen-limiting conditions P. aeruginosa utilizes AdhA, an NAD-linked alcohol dehydrogenase, as a previously undescribed means for ethanol catabolism. In a rich medium containing ethanol, AdhA, but not the previously described PQQ-linked alcohol dehydrogenase, ExaA, oxidizes ethanol and leads to the accumulation of acetate in culture supernatants. AdhA-dependent acetate accumulation and the accompanying decrease in pH promote P. aeruginosa survival in LB-grown stationary-phase cultures. The transcription of adhA is elevated by hypoxia and under anoxic conditions, and we show that it is regulated by the Anr transcription factor. We have shown that lasR mutants, which lack an important quorum sensing regulator, have higher levels of Anr-regulated transcripts under low-oxygen conditions than their wild-type counterparts. Here, we show that a lasR mutant, when grown with ethanol, has an even larger decrease in pH than the wild type (WT) that is dependent on both anr and adhA. The large increase in AdhA activity is similar to that of a strain expressing a hyperactive Anr-D149A variant. Ethanol catabolism in P. aeruginosa by AdhA supports growth on ethanol as a sole carbon source and electron donor in oxygen-limited settings and in cells growing by denitrification under anoxic conditions. This is the first demonstration of a physiological role for AdhA in ethanol oxidation in P. aeruginosa. IMPORTANCE Ethanol is a common product of microbial fermentation, and the Pseudomonas aeruginosa response to and utilization of ethanol are relevant to our understanding of its role in microbial communities. Here, we report that the putative alcohol dehydrogenase AdhA is responsible for ethanol catabolism and acetate accumulation under low-oxygen conditions and that it is regulated by Anr.

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Comparative c-type cytochrome expression analysis in Shewanella oneidensis strain MR-1 and Anaeromyxobacter dehalogenans strain 2CP-C grown with soluble and insoluble oxidized metal electron acceptors

Biochemical Society Transactions ◽

10.1042/bst20120182 ◽

2012 ◽

Vol 40 (6) ◽

pp. 1204-1210 ◽

Cited By ~ 14

Author(s):

Silke Nissen ◽

Xiaoxin Liu ◽

Karuna Chourey ◽

Robert L. Hettich ◽

Darlene D. Wagner ◽

...

Keyword(s):

Deletion Mutant ◽

Shewanella Oneidensis ◽

Genetic System ◽

Growth Conditions ◽

Electron Acceptors ◽

Genetic Approach ◽

Gene Deletions ◽

Distinct Phenotype ◽

Biochemical Studies ◽

Metal Electron

The genomes of Shewanella oneidensis strain MR-1 and Anaeromyxobacter dehalogenans strain 2CP-C encode 40 and 69 putative c-type cytochrome genes respectively. Deletion mutant and biochemical studies have assigned specific functions to a few c-type cytochromes involved in electron transfer to oxidized metals in S. oneidensis strain MR-1. Although promising, the genetic approach is limited to gene deletions that produce a distinct phenotype and to an organism for which a genetic system is available. To investigate and compare c-type cytochrome expression in S. oneidensis strain MR-1 and Anaeromyxobacter dehalogenans strain 2CP-C more comprehensively, proteomic measurements were used to characterize lysates of cells grown with soluble Fe(III) (as ferric citrate) and insoluble Mn(IV) (as MnO2) as electron acceptors. Strain MR-1 expressed 19 and 20, and strain 2CP-C expressed 27 and 25, c-type cytochromes when grown with Fe(III) and Mn(IV) respectively. The majority of c-type cytochromes (77% for strain MR-1 and 63% for strain 2CP-C) were expressed under both growth conditions; however, the analysis also revealed unique c-type cytochromes that were specifically expressed in cells grown with soluble Fe(III) or insoluble Mn(IV). Proteomic characterization proved to be a promising approach for determining the c-type cytochrome complement expressed under different growth conditions, and will help to elucidate the specific functions of more c-type cytochromes that are the basis for Shewanella and Anaeromyxobacter respiratory versatility.

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Characterization of BcaA, a Putative Classical Autotransporter Protein in Burkholderia pseudomallei

Infection and Immunity ◽

10.1128/iai.01453-12 ◽

2013 ◽

Vol 81 (4) ◽

pp. 1121-1128 ◽

Cited By ~ 9

Author(s):

Cristine G. Campos ◽

Luke Borst ◽

Peggy A. Cotter

Keyword(s):

Burkholderia Pseudomallei ◽

Efflux Pump ◽

Outer Membrane Proteins ◽

Lung Epithelial Cells ◽

Wild Type ◽

Passenger Domain ◽

Content Type ◽

Reading Frame ◽

Type V

ABSTRACTBurkholderia pseudomalleiis a tier 1 select agent, and the causative agent of melioidosis, a disease with effects ranging from chronic abscesses to fulminant pneumonia and septic shock, which can be rapidly fatal. Autotransporters (ATs) are outer membrane proteins belonging to the type V secretion system family, and many have been shown to play crucial roles in pathogenesis. The open reading frame Bp1026b_II1054 (bcaA) inB. pseudomalleistrain 1026b is predicted to encode a classical autotransporter protein with an approximately 80-kDa passenger domain that contains a subtilisin-related domain. Immediately 3′ tobcaAis Bp11026_II1055 (bcaB), which encodes a putative prolyl 4-hydroxylase. To investigate the role of these genes in pathogenesis, large in-frame deletion mutations ofbcaAandbcaBwere constructed in strain Bp340, an efflux pump mutant derivative of the melioidosis clinical isolate 1026b. Comparison of Bp340ΔbcaAand Bp340ΔbcaBmutants to wild-typeB. pseudomalleiin vitrodemonstrated similar levels of adherence to A549 lung epithelial cells, but the mutant strains were defective in their ability to invade these cells and to form plaques. In a BALB/c mouse model of intranasal infection, similar bacterial burdens were observed after 48 h in the lungs and liver of mice infected with Bp340ΔbcaA, Bp340ΔbcaB, and wild-type bacteria. However, significantly fewer bacteria were recovered from the spleen of Bp340ΔbcaA-infected mice, supporting the idea of a role for this AT in dissemination or in survival in the passage from the site of infection to the spleen.

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Inactivation of Escherichia coli by Polychromatic Simulated Sunlight: Evidence for and Implications of a Fenton Mechanism Involving Iron, Hydrogen Peroxide, and Superoxide

Applied and Environmental Microbiology ◽

10.1128/aem.02419-13 ◽

2013 ◽

Vol 80 (3) ◽

pp. 935-942 ◽

Cited By ~ 17

Author(s):

Michael B. Fisher ◽

Kara L. Nelson

Keyword(s):

Escherichia Coli ◽

Hydrogen Peroxide ◽

Enteric Bacteria ◽

Growth Conditions ◽

Simulated Sunlight ◽

Wild Type ◽

Intracellular Iron ◽

Content Type ◽

E Coli ◽

Degree Of Sensitization

ABSTRACTSunlight inactivation ofEscherichia colihas previously been shown to accelerate in the presence of oxygen, exogenously added hydrogen peroxide, and bioavailable forms of exogenously added iron. In this study, mutants unable to effectively scavenge hydrogen peroxide or superoxide were found to be more sensitive to polychromatic simulated sunlight (without UVB wavelengths) than wild-type cells, while wild-type cells grown under low-iron conditions were less sensitive than cells grown in the presence of abundant iron. Furthermore, prior exposure to simulated sunlight was found to sensitize cells to subsequent hydrogen peroxide exposure in the dark, but this effect was attenuated for cells grown with low iron. Mutants deficient in recombination DNA repair were sensitized to simulated sunlight (without UVB wavelengths), but growth in the presence of iron chelators reduced the degree of sensitization conferred by this mutation. These findings support the hypothesis that hydrogen peroxide, superoxide, and intracellular iron all participate in the photoinactivation ofE. coliand further suggest that the inactivation rate of enteric bacteria in the environment may be strongly dependent on iron availability and growth conditions.

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The genes YNI1 and YNR1, encoding nitrite reductase and nitrate reductase respectively in the yeast Hansenula polymorpha, are clustered and co-ordinately regulated

Biochemical Journal ◽

10.1042/bj3170089 ◽

1996 ◽

Vol 317 (1) ◽

pp. 89-95 ◽

Cited By ~ 30

Author(s):

Nélida BRITO ◽

Julio AVILA ◽

M. Dolores PEREZ ◽

Celedonio GONZALEZ ◽

José M. SIVERIO

Keyword(s):

Nitrate Reductase ◽

Nitrite Reductase ◽

Reductase Activity ◽

Hansenula Polymorpha ◽

Enzymic Activity ◽

Wild Type ◽

Reading Frame ◽

Dna Library ◽

Genomic Dna Library ◽

Nitrate And Nitrite

The nitrite reductase-encoding gene (YNI1) from the yeast Hansenula polymorpha was isolated from a lambda EMBL3 H. polymorpha genomic DNA library, using as a probe a 481 bp DNA fragment from the gene of Aspergillus nidulans encoding nitrite reductase (niiA). An open reading frame of 3132 bp, encoding a putative protein of 1044 amino acids with high similarity with nitrite reductases from fungi, was located by DNA sequencing in the phages λNB5 and λJA13. Genes YNI1 and YNR1 (encoding nitrate reductase) are clustered, separated by 1700 bp. Northern blot analysis showed that expression of YNI1 and YNR1 is co-ordinately regulated; induced by nitrate and nitrite and repressed by sources of reduced nitrogen, even in the presence of nitrate. A mutant lacking nitrite reductase activity was obtained by deletion of the chromosomal copy of YNI1. The mutant does not grow in nitrate or in nitrite; it exhibits a similar level of transcription of YNR1 to the wild type, but the nitrate reductase enzymic activity is only about 50% of the wild type. In the presence of nitrate the Δyni1::URA3 mutant extrudes approx. 24 nmol of nitrite/h per mg of yeast (wet weight), about five times more than the wild type.

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Rapidly Correcting Frameshift Mutations in the Mycobacterium tuberculosis orn Gene Produce Reversible Ethambutol Resistance and Small-Colony-Variant Morphology

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00213-20 ◽

2020 ◽

Vol 64 (9) ◽

Author(s):

Hassan Safi ◽

Subramanya Lingaraju ◽

Shuyi Ma ◽

Seema Husain ◽

Mainul Hoque ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Frameshift Mutation ◽

Relative Increase ◽

Wild Type ◽

Content Type ◽

Reading Frame ◽

Small Colony Variant ◽

Large Colony ◽

Small Colony ◽

High Level

ABSTRACT We have identified a previously unknown mechanism of reversible high-level ethambutol (EMB) resistance in Mycobacterium tuberculosis that is caused by a reversible frameshift mutation in the M. tuberculosis orn gene. A frameshift mutation in orn produces the small-colony-variant (SCV) phenotype, but this mutation does not change the MICs of any drug for wild-type M. tuberculosis. However, the same orn mutation in a low-level EMB-resistant double embB-aftA mutant (MIC = 8 μg/ml) produces an SCV with an EMB MIC of 32 μg/ml. Reversible resistance is indistinguishable from a drug-persistent phenotype, because further culture of these orn-embB-aftA SCV mutants results in rapid reversion of the orn frameshifts, reestablishing the correct orn open reading frame, returning the culture to normal colony size, and reversing the EMB MIC back to that (8 μg/ml) of the parental strain. Transcriptomic analysis of orn-embB-aftA mutants compared to wild-type M. tuberculosis identified a 27-fold relative increase in the expression of embC, which is a cellular target for EMB. Expression of embC in orn-embB-aftA mutants was also increased 5-fold compared to that in the parental embB-aftA mutant, whereas large-colony orn frameshift revertants of the orn-embB-aftA mutant had levels of embC expression similar to that of the parental embB-aftA strain. Reversible frameshift mutants may contribute to a reversible form of microbiological drug resistance in human tuberculosis.

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Enabling Unbalanced Fermentations by Using Engineered Electrode-Interfaced Bacteria

mBio ◽

10.1128/mbio.00190-10 ◽

2010 ◽

Vol 1 (5) ◽

Cited By ~ 95

Author(s):

Jeffrey M. Flynn ◽

Daniel E. Ross ◽

Kristopher A. Hunt ◽

Daniel R. Bond ◽

Jeffrey A. Gralnick

Keyword(s):

Waste Product ◽

Shewanella Oneidensis ◽

Redox Balance ◽

Strain Engineering ◽

Electron Acceptors ◽

Content Type ◽

Whole Cells ◽

Conversion Rates ◽

Substrate Conversion ◽

Current Production

ABSTRACTCellular metabolism is a series of tightly linked oxidations and reductions that must be balanced. Recycling of intracellular electron carriers during fermentation often requires substrate conversion to undesired products, while respiration demands constant addition of electron acceptors. The use of electrode-based electron acceptors to balance biotransformations may overcome these constraints. To test this hypothesis, the metal-reducing bacteriumShewanella oneidensiswas engineered to stoichiometrically convert glycerol into ethanol, a biotransformation that will not occur unless two electrons are removed via an external reaction, such as electrode reduction. Multiple modules were combined into a single plasmid to alterS. oneidensismetabolism: a glycerol module, consisting ofglpF,glpK,glpD, andtpiAfromEscherichia coli, and an ethanol module containingpdcandadhfromZymomonas mobilis. A further increase in product yields was accomplished through knockout ofpta, encoding phosphate acetyltransferase, shifting flux toward ethanol and away from acetate production. In this first-generation demonstration, conversion of glycerol to ethanol required the presence of an electrode to balance the reaction, and electrode-linked rates were on par with volumetric conversion rates observed in engineeredE. coli. Linking microbial biocatalysis to current production can eliminate redox constraints by shifting other unbalanced reactions to yield pure products and serve as a new platform for next-generation bioproduction strategies.IMPORTANCEAll reactions catalyzed by whole cells or enzymes must achieve redox balance. In rare cases, conversion can be achieved via perfectly balanced fermentations, allowing all electron equivalents to be recovered in a single product. In most biotransformations, organisms must produce a mixture of acids, gasses, and/or alcohols, and no amount of enzyme or strain engineering can overcome this fundamental requirement. Stoichiometric conversion of glycerol, a waste product from biodiesel transesterification, into ethanol and CO2with no side products represents such an impossible fermentation, due to the more reduced state of glycerol than of ethanol and CO2. The unbalanced conversion of glycerol to ethanol has been viewed as having only two solutions: fermenting glycerol to ethanol and potentially useful coproducts or “burning off” excess electrons via careful introduction of oxygen. Here, we use the glycerol-to-ethanol example to demonstrate a third strategy, using bacteria directly interfaced to electrodes.

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