C-type Cytochromes Research Articles

Comparing electrochemical and spectroscopic properties of three cytochrome nanowires from Geobacter sulfurreducens.

The model anaerobic bacterium, Geobacter sulfurreducens, produces filaments that mediate extracellular electron transfer as part of its respiratory metabolism in oxygen-free environments. The physical, biological, and chemical principles supporting conductivity in protein filaments such as those produced by Geobacter have considerable importance for our understanding of biogeochemical cycles, impacts of climate change, and advanced biomedical devices. We have recently discovered that G. sulfurreducens produces at least three such filaments, which structural studies revealed to be polymers of multiheme cytochromes, OmcS, OmcE, and OmcZ. All three filaments are composed of these cytochrome subunits with bis-histidine-coordinated hemes forming a chain along the fiber axis. Our work aims to understand the structure-property relationships in these conductive filaments, including those that directly support long-range electron transport. Our structural studies show that the arrangement of heme in OmcS and OmcE are nearly identical, despite lacking sequence homology. These two filaments also exhibit heme coordinated across the subunit-subunit interface by histidines from neighboring subunits. The structure of OmcZ is distinct in that it lacks this interfacial heme. One heme per subunit is offset from the axial chain and largely exposed to solvent. Moreover, all heme pairs in these filaments, and indeed most heme pairs within multiheme c-type cytochrome structures in the Protein Databank, cluster into common rotation angle and separation distance arrangement, suggesting conserved electron transfer motifs across domains of life. Spectroscopic comparison between OmcS, OmcE, and OmcZ filaments, and their disassembled monomers will be discussed. Additionally, an outline of the biophysical and electrochemical characterizations of cytochrome filaments and their monomeric subunits will be addressed. The new concepts that will be concluded from the investigation of conductive cytochrome filaments in Geobacter will lead to a better understanding of biological structures supporting long-range electronic conductivity.

Microbial nanowires: Cytochrome filaments in Geobacter and more

While early genetic and low-resolution structural observations suggested that extracellular conductive filaments on metal-reducing organisms such as Geobacter were composed of type IV pili, it has now been established that bacterial c-type cytochromes can polymerize to form extracellular filaments capable of long-range electron transport. Here we have used cryo-EM and revealed atomic structures of all three such cytochrome filaments, formed from the hexaheme cytochrome OmcS, OmcE, and OmcZ in Geobacter. In contrast to the linear heme chains in OmcS and OmcE from the same organism, the packing of hemes, heme:heme angles, and between-subunit heme coordination is quite different in OmcZ. A branched heme arrangement within OmcZ leads to a highly surface exposed heme in every subunit, which may account for the formation of conductive biofilm networks, and explain the higher measured conductivity of OmcZ filaments. This new structural evidence suggests that conductive cytochrome polymers arose independently on more than one occasion from different ancestral multiheme proteins. Based on those findings we further identified cytochromes in other prokaryotes.

Polyphasic Characterization of Geotalea uranireducens NIT-SL11 Newly Isolated from a Complex of Sewage Sludge and Microbially Reduced Graphene Oxide

Graphene oxide (GO), a chemically oxidized sheet of graphite, has been used as a conductive carbon carrier of microbes to boost various bioelectrochemical reactions. However, the types of microbes that can reduce GO have rarely been investigated. In this study, a strain of GO-reducing bacteria, named NIT-SL11, which was obtained from a hydrogel of microbially reduced GO and anaerobic sludge that converts sewage to electricity, was phylogenically identified as a novel strain of Geotalea uraniireducens. Considering the current lack of information on the electrogenic ability of the bacterium and its physicochemical and chemotaxonomic characteristics, the polyphasic characterization of the Geotalea uraniireducens strain NIT-SL11 was performed. NIT-SL11 utilized various organic acids, such as lactate, benzoate, and formate, as electron donors and exhibited respiration using GO, electrodes, fumarate, and malate. The strain contained C16:1ω7c and C16:0 as the major fatty acids and MK-8 and 9 as the major respiratory quinones. The complete genome of NIT-SL11 was 4.7 Mbp in size with a G+C content of 60.9%, and it encoded 80 putative c-type cytochromes and 23 type IV pili-related proteins. The possible extracellular electron transfer (EET) pathways of the strain were the porin-cytochrome (Pcc) EET pathway and type IV pili-based pathway.

Effect of Mo2C-functionalized electrode interface on enhancing microbial cathode electrocatalysis: Beyond electrochemical hydrogen evolution

Mo2C has recently been introduced into microbial cathode electrocatalysis due to its well-known catalytic activity for hydrogen evolution reaction (HER), but a low electrode potential (i.e., a high energy input) is required usually. However, in the range of potential where HER cannot occur, its effect on microorganism/cathode interfacial kinetics remains unknown. Herein, an active Mo2C interface is constructed on conventional carbon cloth electrode to investigate its impact on electric-driven fumarate reduction by a typical electroactive microorganism (Shewanella oneidensis MR-1) at a potential of −0.36 V (vs. standard hydrogen electrode) that cannot meet the HER requirement. The Mo2C-functionalized electrode achieves a greatly increased current consumption density of ∼0.041 mA cm−2, about three times that of the control, indicating a faster fumarate reduction rate. This substantially proves that the enhancement of microbial electrocatalytic kinetics at Mo2C-functionalized interface is not confined to the induced hydrogen evolution. By analyzing its interaction with extracellular electron transfer mediators (e.g., c-type cytochromes and riboflavin), a robust inward extracellular electron transfer process based on the rapid electrochemical reaction of riboflavin at the Mo2C-functionalized interface is elaborated. This work provides a new understanding of the mechanism by which the nanostructured Mo2C interface enhances microbial cathode electrocatalysis.

A New Electron Shuttling Pathway Mediated by Lipophilic Phenoxazine via the Interaction with Periplasmic and Inner Membrane Proteins of Shewanella oneidensis MR-1.

Although it has been established that electron mediators substantially promote extracellular electron transfer (EET), electron shuttling pathways are not fully understood. Here, a new electron shuttling pathway was found in the EET process by Shewanella oneidensis MR-1 with resazurin, a lipophilic electron mediator. With resazurin, the genes encoding outer-membrane cytochromes (mtrCBA and omcA) were downregulated. Although cytochrome deletion substantially reduced biocurrent generation to 1-12% of that of wild-type (WT) cells, the presence of resazurin restored biocurrent generation to 168 μA·cm-2 (ΔmtrA/omcA/mtrC), nearly equivalent to that of WT cells (194 μA·cm-2), indicating that resazurin-mediated electron transfer was not dependent on the Mtr pathway. Biocurrent generation by resazurin was much lower in ΔcymA and ΔmtrA/omcA/mtrC/fccA/cctA mutants (4 and 6 μA·cm-2) than in WT cells, indicating a key role of FccA, CctA, and CymA in this process. The effectiveness of resazurin in EET of Mtr cytochrome mutants is also supported by cyclic voltammetry, resazurin reduction kinetics, and in situ c-type cytochrome spectroscopy results. The findings demonstrated that low molecular weight, lipophilic electron acceptors, such as phenoxazine and phenazine, may facilitate electron transfer directly from periplasmic and inner membrane proteins, thus providing new insight into the roles of exogenous electron mediators in electron shuttling in natural and engineered biogeochemical systems.

Electroactive bacteria as whole-cell biosensor for sensitive detection of hydrogen peroxide

Hydrogen peroxide (H2O2) is a widely used strong oxidizer and has harmful effects on human health with excessive exposure. Herein, we proposed a simple and effective method for H2O2 detection by utilizing electroactive bacteria (EAB) as a whole-cell biosensor. The proposed method was achieved by utilizing the multiheme c-type cytochromes (central components of microbial extracellular electron transfer) of model EAB Shewanella oneidensis MR-1 to catalyze the oxidation of tetramethylbenzidine in the presence of H2O2. Owing to the excellent peroxidase-like activity of the multiheme c-cyts, this H2O2 detection method has a wide dynamic range between 0.1 and 1000 μM and a low detection limit of 0.17 μM. The proposed method, when coupled with glucose oxidase, displayed a high sensitivity (0.57 μM) and broad linear range (1 μM - 10 mM) for glucose. Moreover, this method has high specificity and good recovery for detecting H2O2-spiked real samples. Overall, this study has developed a novel biosensing platform that provides an alternative approach to H2O2 detection.

Whole genome analyses for c-type cytochromes associated with respiratory chains in the extreme thermophile, Thermus thermophilus.

In thermophilic microorganisms, c-type cytochrome (cyt) proteins mainly function in the respiratory chain as electron carriers. Genome analyses at the beginning of this century revealed a variety of genes harboring the heme c motif. Here, we describe the results of surveying genes with the heme c motif, CxxCH, in a genome database comprising four strains of Thermus thermophilus, including strain HB8, and the confirmation of 19 c-type cytochromes among 27 selected genes. We analyzed the 19 genes, including the expression of four, by a bioinformatics approach to elucidate their individual attributes. One of the approaches included an analysis based on the secondary structure alignment pattern between the heme c motif and the 6th ligand. The predicted structures revealed many cyt c domains with fewer β-strands, such as mitochondrial cyt c, in addition to the β-strand unique to Thermus inserted in cyt c domains, as in T. thermophilus cyt c552 and caa3 cyt c oxidase subunit IIc. The surveyed thermophiles harbor potential proteins with a variety of cyt c folds. The gene analyses led to the development of an index for the classification of cyt c domains. Based on these results, we propose names for T. thermophilus genes harboring the cyt c fold.

Effect of chromium and copper stress on antibiotic resistance genes transfer and associated metabolic mechanism in electrochemical system treating sulfanilamide wastewater

To investigate the electrochemical process and metabolic mechanism of sulfonamide wastewater treatment under chromium and copper stress in bioelectrochemical system, electron transfer and antibiotic resistance genes (ARGs) transfer were investigated. Metagenomics was used to reveal metabolic pathways, antibiotic resistance mechanisms, carbohydrate active enzymes, etc. The results indicated that copper stress not only did not affect the electricity production performance of the electrochemical system, but also promoted the electron transfer efficiency. Copper stress can also effectively control ARGs and mobile genetic elements (MGEs). The metagenomics analysis showed that Cu increased the abundance of coxA and cox1 genes in the electrochemical system and increased the C-type cytochrome, thereby improving the ability of electron transfer. In contrast, chromium stress affected electrochemical system performance and accelerated ARGs transfer, and increasing the risk of resistance gene horizontal transfer. The mechanism of antibiotic resistance was mainly through the efflux function of antibiotics (61.5%), which was mainly related to Proteobacteria (51.5%) and Firmicutes (25.4%). The results also indicated that the glycolysis process was promoted by Cu, while the process was negatively affected by Cr. This study revealed the effect of chromium and copper stress on the metabolic mechanisms of microorganisms in bioelectrochemical system at the genetic level and provided a theoretical basis for risk control of livestock and poultry wastewater.

Global occurrence of the bacteria with capability for extracellular reduction of iodate.

The γ-proteobacterium Shewanella oneidensis MR-1 reduces iodate to iodide extracellularly. Both dmsEFAB and mtrCAB gene clusters are involved in extracellular reduction of iodate by S. oneidensis MR-1. DmsEFAB reduces iodate to hypoiodous acid and hydrogen peroxide (H2O2). Subsequently, H2O2 is reduced by MtrCAB to facilitate DmsEFAB-mediated extracellular reduction of iodate. To investigate the distribution of bacteria with the capability for extracellular reduction of iodate, bacterial genomes were systematically searched for both dmsEFAB and mtrCAB gene clusters. The dmsEFAB and mtrCAB gene clusters were found in three Ferrimonas and 26 Shewanella species. Coexistence of both dmsEFAB and mtrCAB gene clusters in these bacteria suggests their potentials for extracellular reduction of iodate. Further analyses demonstrated that these bacteria were isolated from a variety of ecosystems, including the lakes, rivers, and subsurface rocks in East and Southeast Asia, North Africa, and North America. Importantly, most of the bacteria with both dmsEFAB and mtrCAB gene clusters were found in different marine environments, which ranged from the Arctic Ocean to Antarctic coastal marine environments as well as from the Atlantic Ocean to the Indian and Pacific Oceans. Widespread distribution of the bacteria with capability for extracellular reduction of iodate around the world suggests their significant importance in global biogeochemical cycling of iodine. The genetic organization of dmsEFAB and mtrCAB gene clusters also varied substantially. The identified mtrCAB gene clusters often contained additional genes for multiheme c-type cytochromes. The numbers of dmsEFAB gene cluster detected in a given bacterial genome ranged from one to six. In latter, duplications of dmsEFAB gene clusters occurred. These results suggest different paths for these bacteria to acquire their capability for extracellular reduction of iodate.

Enhancing the extracellular electron transfer ability via Polydopamine@S. oneidensis MR-1 for Cr(VI) reduction

Microbial reduction of hexavalent chromium (Cr (VI)) shows better efficiency and cost-effectiveness. However, immobilization of Cr (III) remains a challenge as there is a limited supply of electron donors. A greener and cleaner option for donating external electrons was using bioelectrochemical systems to perform the microbial reduction of Cr(VI). In this system, we constructed a polydopamine (PDA) decorated Shewanella oneidensis MR-1 (S. oneidensis MR-1) bioelectrode with bidirectional electron transport, abbreviated as PDA@S. oneidensis MR-1. The conjugated PDA distributed on the intracellular and extracellular of individual S. oneidensis MR-1 has been shown to accelerate electron transfer by outer membrane C-type cytochromes and flavin-bound MtrC/OmcA pathway by various electrochemical analyses. As expected, the PDA@S. oneidensis MR-1 biofilm achieved 88.1% Cr (VI) removal efficiency (RE) and 58.1% Cr (III) immobilization efficiency (IE) within 24 h under the autotrophic conditions at the optimal voltage (−150 mV) compared with the control potential (0 mV). The PDA@S. oneidensis MR-1 biofilm showed increased RE activity was attributed to the shortening of the distance between individual bacteria by PDA. This research provides a viable strategy for in situ bioremediation of Cr(VI) polluted aquatic environment.

Cytochrome-mediated direct electron uptake from metallic iron by Methanosarcina acetivorans.

Methane-producing microorganisms accelerate the corrosion of iron-containing metals. Previous studies have inferred that some methanogens might directly accept electrons from Fe(0), but when this possibility was more intensively investigated, H2 was shown to be an intermediary electron carrier between Fe(0) and methanogens. Here, we report that Methanosarcina acetivorans catalyzes direct metal-to-microbe electron transfer to support methane production. Deletion of the gene for the multiheme, outer-surface c-type cytochrome MmcA eliminated methane production from Fe(0), consistent with the key role of MmcA in other forms of extracellular electron exchange. These findings, coupled with the previous demonstration that outer-surface c-type cytochromes are also electrical contacts for electron uptake from Fe(0) by Geobacter and Shewanella species, suggest that the presence of multiheme c-type cytochromes on corrosion surfaces might be diagnostic for direct metal-to-microbe electron transfer and that interfering with cytochrome function might be a strategy to mitigate corrosion.

Lack of Specificity in Geobacter Periplasmic Electron Transfer.

Reduction of extracellular acceptors requires electron transfer across the periplasm. In Geobacter sulfurreducens, three separate cytoplasmic membrane cytochromes are utilized depending on redox potential, and at least five cytochrome conduits span the outer membrane. Because G. sulfurreducens produces 5 structurally similar triheme periplasmic cytochromes (PpcABCDE) that differ in expression level, midpoint potential, and heme biochemistry, many hypotheses propose distinct periplasmic carriers could be used for specific redox potentials, terminal acceptors, or growth conditions. Using a panel of marker-free single, quadruple, and quintuple mutants, little support for these models could be found. Three quadruple mutants containing only one paralog (PpcA, PpcB, and PpcD) reduced Fe(III) citrate and Fe(III) oxide at the same rate and extent, even though PpcB and PpcD were at much lower periplasmic levels than PpcA. Mutants containing only PpcC and PpcE showed defects, but these cytochromes were nearly undetectable in the periplasm. When expressed sufficiently, PpcC and PpcE supported wild-type Fe(III) reduction. PpcA and PpcE from G. metallireducens similarly restored metal respiration in G. sulfurreducens. PgcA, an unrelated extracellular triheme c-type cytochrome, also participated in periplasmic electron transfer. While triheme cytochromes were important for metal reduction, sextuple ΔppcABCDE ΔpgcA mutants grew near wild-type rates with normal cyclic voltammetry profiles when using anodes as electron acceptors. These results reveal broad promiscuity in the periplasmic electron transfer network of metal-reducing Geobacter and suggest that an as-yet-undiscovered periplasmic mechanism supports electron transfer to electrodes. IMPORTANCE Many inner and outer membrane cytochromes used by Geobacter for electron transfer to extracellular acceptors have specific functions. How these are connected by periplasmic carriers remains poorly understood. G. sulfurreducens contains multiple triheme periplasmic cytochromes with unique biochemical properties and expression profiles. It is hypothesized that each could be involved in a different respiratory pathway, depending on redox potential or energy needs. Here, we show that Geobacter periplasmic cytochromes instead show evidence of being highly promiscuous. Any of 6 triheme cytochromes supported similar growth with soluble or insoluble metals, but none were required when cells utilized electrodes. These findings fail to support many models of Geobacter electron transfer, and question why these organisms produce such an array of periplasmic cytochromes.

Spectroscopic identification of the catalytic intermediates of cytochrome c oxidase in respiring heart mitochondria

The catalytic cycle of cytochrome c oxidase (COX) couples the reduction of oxygen to the translocation of protons across the inner mitochondrial membrane and involves several intermediate states of the heme a3-CuB binuclear center with distinct absorbance properties. The absorbance maximum close to 605 nm observed during respiration is commonly assigned to the fully reduced species of hemes a or a3 (R). However, by analyzing the absorbance of isolated enzyme and mitochondria in the Soret (420–450 nm), alpha (560–630 nm) and red (630–700 nm) spectral regions, we demonstrate that the Peroxy (P) and Ferryl (F) intermediates of the binuclear center are observed during respiration, while the R form is only detectable under nearly anoxic conditions in which electrons also accumulate in the higher extinction coefficient low spin a heme. This implies that a large fraction of COX (>50 %) is active, in contrast with assumptions that assign spectral changes only to R and/or reduced heme a. The concentration dependence of the COX chromophores and reduced c-type cytochromes on the transmembrane potential (ΔΨm) was determined in isolated mitochondria during substrate or apyrase titration to hydrolyze ATP. The cytochrome c-type redox levels indicated that soluble cytochrome c is out of equilibrium with respect to both Complex III and COX. Thermodynamic analyses confirmed that reactions involving the chromophores we assign as the P and F species of COX are ΔΨm-dependent, out of equilibrium, and therefore much slower than the ΔΨm-insensitive oxidation of the R intermediate, which is undetectable due to rapid oxygen binding.

The first cultivated representatives of the actinobacterial lineage OPB41 isolated from subsurface environments constitute a novel order Anaerosomatales.

The continental subsurface harbors microbial populations highly enriched in uncultured taxa. OPB41 is an uncultured order-level phylogenetic lineage within the actinobacterial class Coriobacteriia. OPB41 bacteria have a wide geographical distribution, but the physiology and metabolic traits of this cosmopolitan group remain elusive. From two contrasting subsurface environments, a terrestrial mud volcano and a deep subsurface aquifer, located in the central part of Eurasia, within the Caucasus petroleum region, we have isolated two pure cultures of anaerobic actinobacteria belonging to OPB41. The cells of both strains are small non-motile rods forming numerous pili-like appendages. Strain M08DHBT is mesophilic, while strain Es71-Z0120T is a true thermophile having a broad temperature range for growth (25-77°C). Strain M08DHBT anaerobically reduces sulfur compounds and utilizes an aromatic compound 3,4-dihydroxybenzoic acid. Strain Es71-Z0120T is an obligate dissimilatory Fe(III) reducer that is unable to utilize aromatic compounds. Both isolates grow lithotrophically and consume molecular hydrogen or formate using either thiosulfate, elemental sulfur, or Fe(III) as an electron acceptor. Genomes of the strains encode the putative reductive glycine pathway for autotrophic CO2 fixation, Ni-Fe hydrogenases, putative thiosulfate/polysulfide reductases, and multiheme c-type cytochromes presumably involved in dissimilatory Fe(III) reduction. We propose to assign the isolated strains to the novel taxa of the species-order levels and describe strain M08DHBT as Anaerosoma tenue gen. nov., sp. nov., and strain Es71-Z0120T as Parvivirga hydrogeniphila gen. nov., sp. nov., being members of Anaerosomatales ord. nov. This work expands the knowledge of the diversity, metabolic functions, and ecological role of the phylum Actinomycetota.

Boosting bioelectricity generation in microbial fuel cells via biomimetic Fe-N-S-C nanozymes

Anode performance has been regarded as a crucial factor determining long-term stability and electricity generation of microbial fuel cells (MFCs), which restricts by the difficult extracellular electron transfer (EET) on the microbe/anode interface. Herein, inspired by biological enzyme systems, this study synthesized the biomimetic nanozymes with Fe-N-S-C active sites as the anode materials of MFCs, which was similar to the hemes of c-type cytochromes (c-Cyts) for boosting EET process. As excepted, an obviously faster start-up and a much higher power density were achieved by the MFCs equipped with Fe-N-S-C nanozymes (startup time, 3.5 d; power density, 2366 ± 34 mW m−2) than that based on traditional carbon cloth (startup time, 5.6 d; power density, 1009 ± 26 mW m−2). Such unique features of Fe-N-S-C nanozymes anode not only greatly favored the bacterial adhesion and the electroactive bacteria enrichment on the anode surface, but also efficiently facilitated the EET process between the electroactive bacteria and anode surface. This study provided a feasible strategy for designing the novel MFC anode materials from the perspective of bionic enzyme.

Cysteine-Mediated Extracellular Electron Transfer of Lysinibacillus varians GY32.

Microbial extracellular electron transfer (EET) is essential in many natural and engineering processes. Compared with the versatile EET pathways of Gram-negative bacteria, the EET of Gram-positive bacteria has been studied much less and is mainly limited to the flavin-mediated pathway. Here, we investigate the EET pathway of a Gram-positive filamentous bacterium Lysinibacillus varians GY32. Strain GY32 has a wide electron donor spectrum (including lactate, acetate, formate, and some amino acids) in electrode respiration. Transcriptomic, proteomic, and electrochemical analyses show that the electrode respiration of GY32 mainly depends on electron mediators, and c-type cytochromes may be involved in its respiration. Fluorescent sensor and electrochemical analyses demonstrate that strain GY32 can secrete cysteine and flavins. Cysteine added shortly after inoculation into microbial fuel cells accelerated EET, showing cysteine is a new endogenous electron mediator of Gram-positive bacteria, which provides novel information to understand the EET networks in natural environments. IMPORTANCE Extracellular electron transport (EET) is a key driving force in biogeochemical element cycles and microbial chemical-electrical-optical energy conversion on the Earth. Gram-positive bacteria are ubiquitous and even dominant in EET-enriched environments. However, attention and knowledge of their EET pathways are largely lacking. Gram-positive bacterium Lysinibacillus varians GY32 has extremely long cells (>1 mm) and conductive nanowires, promising a unique and enormous role in the microenvironments where it lives. Its capability to secrete cysteine renders it not only an EET pathway to respire and survive, but also an electrochemical strategy to connect and shape the ambient microbial community at a millimeter scale. Moreover, its incapability of using flavins as an electron mediator suggests that the common electron mediator is species-dependent. Therefore, our results are important to understanding the EET networks in natural and engineering processes.

Structures of the CcmABCD heme release complex at multiple states

Cytochromes c use heme as a cofactor to carry electrons in respiration and photosynthesis. The cytochrome c maturation system I, consisting of eight membrane proteins (CcmABCDEFGH), results in the attachment of heme to cysteine residues of cytochrome c proteins. Since all c-type cytochromes are periplasmic, heme is first transported to a periplasmic heme chaperone, CcmE. A large membrane complex, CcmABCD has been proposed to carry out this transport and linkage to CcmE, yet the structural basis and mechanisms underlying the process are unknown. We describe high resolution cryo-EM structures of CcmABCD in an unbound form, in complex with inhibitor AMP-PNP, and in complex with ATP and heme. We locate the ATP-binding site in CcmA and the heme-binding site in CcmC. Based on our structures combined with functional studies, we propose a hypothetic model of heme trafficking, heme transfer to CcmE, and ATP-dependent release of holoCcmE from CcmABCD. CcmABCD represents an ABC transporter complex using the energy of ATP hydrolysis for the transfer of heme from one binding partner (CcmC) to another (CcmE).

Manganese oxidation counteracts the deleterious effect of low temperatures on biofilm formation in Pseudomonas sp. MOB-449.

Mn removal from groundwater by biological sand filter technology is negatively impacted by low temperatures in winter periods. Therefore, the need to study Mn(II)-oxidizing bacteria (MOB) having the potential to oxidize Mn(II) and form biofilms at low temperatures is imperative. These MOB can have potential as inocula for sand filter bioaugmentation strategies to optimize Mn removal during winter periods. We previously showed that a Pseudomonas sp. MOB-449 (MOB-449), isolated from a Mn biofilter, oxidizes Mn(II) in a biofilm-dependent way at low temperatures. In this work, MOB-449 Mn(II) oxidation and growth capacities were evaluated under planktonic and biofilm conditions at different temperatures. At 18°C, MOB-449 showed enhanced biofilm formation due to the addition of Mn(II) to the medium correlating with Mn(II) oxidation, compared to biofilms grown in control medium. Moreover, this enhancement on biofilm formation due to the addition of Mn(II) was only observed at 18°C. At this temperature, Mn(II) oxidation in membrane fractions collected from biofilms was induced by uncoupling oxidative phosphorylation from the electron transport chain with 2,4-Dinitrophenol. In Pseudomonas, a role for c-type cytochrome in Mn(II) oxidation has been demonstrated. Accordingly, transcriptional profiles of all terminal oxidases genes found in MOB-449 showed an induction of cytochrome c terminal oxidases expression mediated by Mn(II) oxidation at 18°C. Finally, heme peroxidase activity assays and MS analysis revealed that PetC, a cytochrome c5, and also CcmE, involved in the cytochrome c biogenesis machinery, are induced at 18°C only in the presence of Mn(II). These results present evidence supporting that cytochromes c and also the cytochrome c terminal oxidases are activated at low temperatures in the presence of Mn(II). Overall, this work demonstrate that in MOB-449 Mn(II) oxidation is activated at low temperatures to gain energy, suggesting that this process is important for survival under adverse environmental conditions and contributing to the understanding of the physiological role of bacterial Mn(II) oxidation.

An improved method for rapid evaluation of enzymatic cis/trans isomerization of C18:1 monounsaturated fatty acids

Cytochrome c-type cis/trans fatty acid isomerase (CTI) is a promising candidate for directly controlling cis/trans fatty acid isomerism in lipids-related food products like partially hydrogenated vegetable oils. In this study, to establish a sophisticated analysis platform for the CTI assay, we constructed the reversed micelle reaction system and improved the processes of methylation and GC-FID analysis of C18:1cis/trans monounsaturated fatty acid (MUFA) isomers. Highly stable AOT/isooctane reversed micelles were formed in the presence of periplasmic fractions of Pseudomonas putida KT2440. Using a mid-content cyanopropyl phase DB-FastFAME column, C18:1cis/trans-MUFAs were analyzed rapidly and resolved with resolution factors over 1.34. Based on the newly established assay, the catalytic activity of the periplasmic fraction was precisely determined, and its kinetic parameters (Vmax and Km) were derived as 0.021 mM·min−1 and 0.68 mM, respectively. The following results can provide practical information for investigating CTIs in the fields of food and lipid chemistry.

Vanadate reducing bacteria and archaea may use different mechanisms to reduce vanadate in vanadium contaminated riverine ecosystems as revealed by the combination of DNA-SIP and metagenomic-binning

Vanadium (V) is a transitional metal that poses health risks to exposed humans. Microorganisms play an important role in remediating V contamination by reducing more toxic and mobile vanadate (V(V)) to less toxic and mobile V(IV). In this study, DNA-stable isotope probing (SIP) coupled with metagenomic-binning was used to identify microorganisms responsible for V(V) reduction and determine potential metabolic mechanisms in cultures inoculated with a V-contaminated river sediment. Anaeromyxobacter and Geobacter spp. were identified as putative V(V)-reducing bacteria, while Methanosarcina spp. were identified as putative V(V)-reducing archaea. The bacteria may use the two nitrate reductases NarG and NapA for respiratory V(V) reduction, as has been demonstrated previously for other species. It is proposed that Methanosarcina spp. may reduce V(V) via anaerobic methane oxidation pathways (AOM-V) rather than via respiratory V(V) reduction performed by their bacterial counterparts, as indicated by the presence of genes associated with anaerobic methane oxidation coupled with metal reduction in the metagenome assembled genome (MAG) of Methanosarcina. Briefly, methane may be oxidized through the "reverse methanogenesis" pathway to produce electrons, which may be further captured by V(V) to promote V(V) reduction. More specially, V(V) reduction by members of Methanosarcina may be driven by electron transport (CoMS-SCoB heterodisulfide reductase (HdrDE), F420H2 dehydrogenases (Fpo), and multi-heme c-type cytochrome (MHC)). The identification of putative V(V)-reducing bacteria and archaea and the prediction of their different pathways for V(V) reduction expand current knowledge regarding the potential fate of V(V) in contaminated sites.