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1.
Zootaxa ; 4617(1): zootaxa.4617.1.1, 2019 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-31716327

RESUMO

The species of the millipede genus Antichiropus Attems, 1911 found in the Pilbara region of Western Australia are reviewed, and 33 new species are described. The new species are: A. anguinus Car, n. sp., A. antius Car, n. sp., A. apricus Car, n. sp., A. cirratus Car, n. sp., A. confragus Car, n. sp., A. cristatus Car, n. sp., A. cucumeraceous Car, n. sp., A. cunicularis Car, n. sp, A. echinus Car, n. sp., A. filiolus Car, n. sp., A. forcipatus Car, n. sp., A. georginae Car, n. sp., A. gibbus Car, n. sp., A. hystricosus Car, n. sp., A. julianneae Car, n. sp., A. literulus Car, n. sp., A. lucyae Car, n. sp., A. nicholasi Car, n. sp., A. nimbus Car, n. sp., A. patriciae Car, n. sp., A. pendiculus Car, n. sp., A. picus Car, n. sp., A. procerus Car, n. sp., A. quaestionis Car, n. sp., A. rupinus Car, n. sp., A. salutus Car, n. sp., A. servulus Car, n. sp., A. simmonsi Car, n. sp., A. sloanae Car, n. sp., A. spathion Car, n. sp., A. uvulus Car, n. sp., A. verutus Car, n. sp. and A. vindicatus Car, n. sp.. The number of described Antichiropus species now stands at 72. Two species (A. julianneae Car, n. sp. and A. pendiculus Car, n. sp.) lack one diagnostic feature of the genus, namely a solenomere process, but are included here because they conform to the genus definition in all other characters. We also obtained sequence data from four mitochondrial genes (cytochrome c oxidase subunit 1 [COI], cytochrome c oxidase subunit 3 [COIII], cytochrome B [CytB], and 12S rRNA [12S]), and one nuclear gene (28S rRNA [28S]) for 19 species. Three main clades were recovered: one in the northern Pilbara, one in the southern Pilbara, and one just outside the south-western margin of the Pilbara.


Assuntos
Artrópodes , Besouros , Animais , Artrópodes/genética , Grupo dos Citocromos c , RNA Ribossômico 28S , Austrália Ocidental
2.
Biochim Biophys Acta Proteins Proteom ; 1867(11): 140265, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31437585

RESUMO

Many c-type cytochromes (cyts) can form domain-swapped oligomers. The positively charged Hydrogenobacter thermophilus (HT) cytochrome (cyt) c552 forms domain-swapped oligomers during expression in the Escherichia coli (E. coli) expression system, but the factors influencing the oligomerization remain unrevealed. Here, we found that the dimer of the negatively charged Shewanella violacea (SV) cyt c5 exhibits a domain-swapped structure, in which the N-terminal helix is exchanged between protomers, similar to the structures of the HT cyt c552 and Pseudomonas aeruginosa (PA) cyt c551 domain-swapped dimers. Positively charged horse cyt c and HT cyt c552 domain swapped during expression in E. coli, whereas negatively charged PA cyt c551 and SV cyt c5 did not. Oligomers were formed during expression in E. coli for HT cyt c552 attached to either a co- or post-translational signal peptide for transportation through the cytoplasm membrane, but not for PA cyt c551 attached to either signal peptide. HT cyt c552 formed oligomers in E. coli in the presence and absence of rare codons. More oligomers were obtained from the in vitro folding of horse cyt c and HT cyt c552 by the addition of negatively charged liposomes during folding, whereas the amount of oligomers for the in vitro folding of PA cyt c551 and SV cyt c5 did not change significantly by the addition. These results indicate that the protein surface charge affects the oligomerization of c-type cyts in cells; positively charged c-type cyts assemble on a negatively charged membrane, inducing formation of domain-swapped oligomers during folding.


Assuntos
Proteínas de Bactérias/química , Grupo dos Citocromos c/química , Multimerização Proteica , Pseudomonas aeruginosa/enzimologia , Shewanella/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Grupo dos Citocromos c/genética , Grupo dos Citocromos c/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Domínios Proteicos , Pseudomonas aeruginosa/genética , Shewanella/genética , Propriedades de Superfície
3.
mBio ; 10(4)2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31431546

RESUMO

Extracellular electron transfer is the key process underpinning the development of bioelectrochemical systems for the production of energy or added-value compounds. Thermincola potens JR is a promising Gram-positive bacterium to be used in these systems because it is thermophilic. In this paper, we describe the structural and functional properties of the nonaheme cytochrome OcwA, which is the terminal reductase of this organism. The structure of OcwA, determined at 2.2-Å resolution, shows that the overall fold and organization of the hemes are not related to other metal reductases and instead are similar to those of multiheme cytochromes involved in the biogeochemical cycles of nitrogen and sulfur. We show that, in addition to solid electron acceptors, OcwA can also reduce soluble electron shuttles and oxyanions. These data reveal that OcwA can work as a multipurpose respiratory enzyme allowing this organism to grow in environments with rapidly changing availability of terminal electron acceptors without the need for transcriptional regulation and protein synthesis.IMPORTANCE Thermophilic Gram-positive organisms were recently shown to be a promising class of organisms to be used in bioelectrochemical systems for the production of electrical energy. These organisms present a thick peptidoglycan layer that was thought to preclude them to perform extracellular electron transfer (i.e., exchange catabolic electrons with solid electron acceptors outside the cell). In this paper, we describe the structure and functional mechanisms of the multiheme cytochrome OcwA, the terminal reductase of the Gram-positive bacterium Thermincola potens JR found at the cell surface of this organism. The results presented here show that this protein can take the role of a respiratory "Swiss Army knife," allowing this organism to grow in environments with soluble and insoluble substrates. Moreover, it is shown that it is unrelated to terminal reductases found at the cell surface of other electroactive organisms. Instead, OcwA is similar to terminal reductases of soluble electron acceptors. Our data reveal that terminal oxidoreductases of soluble and insoluble substrates are evolutionarily related, providing novel insights into the evolutionary pathway of multiheme cytochromes.


Assuntos
Membrana Celular/metabolismo , Grupo dos Citocromos c/metabolismo , Transporte de Elétrons/fisiologia , Bactérias Gram-Positivas/metabolismo , Oxirredutases/metabolismo , Peptococcaceae/metabolismo , Grupo dos Citocromos c/química , Eletricidade , Elétrons , Heme/química , Heme/metabolismo , Cinética , Metais/metabolismo , Oxirredução , Oxirredutases/química , Conformação Proteica
4.
Biochim Biophys Acta Bioenerg ; 1860(10): 148033, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31226315

RESUMO

Respiration in aerobic Actinobacteria involves a cytochrome bc1-aa3 supercomplex with a diheme cytochrome c1, first isolated from Corynebacterium glutamicum. Synthesis of a functional cytochrome c oxidase requires incorporation of CuA, CuB, heme a, and heme a3. In contrast to eukaryotes and α-proteobacteria, this process is poorly understood in Actinobacteria. Here, we analyzed the role of a Surf1 homolog of C. glutamicum in the formation of a functional bc1-aa3 supercomplex. Deletion of the surf1 gene (cg2460) in C. glutamicum caused a growth defect and cytochrome spectra revealed reduced levels of cytochrome c and a and an increased level of cytochrome d. Membranes of the Δsurf1 strain had lost the ability to oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine, suggesting that Surf1 is essential for the formation of a functional cytochrome aa3 oxidase. In contrast to the wild type, a bc1-aa3 supercomplex could not be purified from solubilized membranes of the Δsurf1 mutant. A transcriptome comparison revealed that the genes of the SigC regulon including those for cytochrome bd oxidase were upregulated in the Δsurf1 strain as well as the copper deprivation-inducible gene ctiP. Complementation studies showed that the Surf1 homologs of Corynebacterium diphtheriae, Mycobacterium smegmatis and Mycobacterium tuberculosis could at least partially abolish the growth defect of the C. glutamicum Δsurf1 mutant, suggesting that Surf1 is a conserved assembly factor for actinobacterial cytochrome aa3 oxidase.


Assuntos
Actinobacteria/química , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Proteínas de Membrana/fisiologia , Proteínas Mitocondriais/fisiologia , Proteínas de Bactérias , Corynebacterium glutamicum/química , Grupo dos Citocromos c , Citocromos c1 , Complexo III da Cadeia de Transporte de Elétrons , Oxirredutases/fisiologia
5.
Ann Ital Chir ; 90: 174-181, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31182701

RESUMO

INTRODUCTION: Due to irreversible damage following head trauma, many overlapping pathophysiological events occur including excitotoxicity, acidotoxicity, ionic imbalance, edema, oxidative stress inflammation and apoptosis. MATERIAL AND METHODS: In this this study, after the rats were separated in to groups theserats were fed throughout fourteen days with betaine, omega-3 or betaine+omega-3 combination in physiological limits prior to the trauma. After a closed head trauma, the damaged brain tissues were collected for biochemically and histologically analyses. This examination involved analyses of levels of caspase-3 and cytochrome C and neuron-specific enolase (NSE) levels in brain tissue. RESULTS: These analyses showed that traumatic brain injury (TBI) caused an increase in the levels of caspase-3, cytochrome C and neuron-specific enolase (NED) in the brain tissues examined. DISCUSSION: In this study, apoptotic and/or necrotic cell death via mitochondrial cytochrome C caspase pathway in traumatized cells and neuron-specific enolase (NED) increase indicative of neuronal damage confirmed the research hypothesis. CONCLUSION: Level of the biomarkers induced by brain injury in the groups fed with betaine, omega-3 and betaine+omega-3 combination before the traumatic damage approximated to that of control group values, suggesting that these products may have a neuroprotective role. KEY WORDS: Betain, Caspase-3, Cytochrome C and Neuron-specific enolase, Omega-3, Traumatic brain injury.


Assuntos
Betaína/administração & dosagem , Lesões Encefálicas Traumáticas/prevenção & controle , Suplementos Nutricionais , Ácidos Graxos Ômega-3/administração & dosagem , Animais , Biomarcadores/análise , Química Encefálica , Caspase 3/análise , Grupo dos Citocromos c/análise , Fosfopiruvato Hidratase/análise , Ratos
6.
Adv Microb Physiol ; 74: 1-96, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31126529

RESUMO

The family Geobacteraceae, with its only valid genus Geobacter, comprises deltaproteobacteria ubiquitous in soil, sediments, and subsurface environments where metal reduction is an active process. Research for almost three decades has provided novel insights into environmental processes and biogeochemical reactions not previously known to be carried out by microorganisms. At the heart of the environmental roles played by Geobacter bacteria is their ability to integrate redox pathways and regulatory checkpoints that maximize growth efficiency with electron donors derived from the decomposition of organic matter while respiring metal oxides, particularly the often abundant oxides of ferric iron. This metabolic specialization is complemented by versatile metabolic reactions, respiratory chains, and sensory networks that allow specific members to adaptively respond to environmental cues to integrate organic and inorganic contaminants in their oxidative and reductive metabolism, respectively. Thus, Geobacteraceae are important members of the microbial communities that degrade hydrocarbon contaminants under iron-reducing conditions and that contribute, directly or indirectly, to the reduction of radionuclides, toxic metals, and oxidized species of nitrogen. Their ability to produce conductive pili as nanowires for discharging respiratory electrons to solid-phase electron acceptors and radionuclides, or for wiring cells in current-harvesting biofilms highlights the unique physiological traits that make these organisms attractive biological platforms for bioremediation, bioenergy, and bioelectronics application. Here we review some of the most notable physiological features described in Geobacter species since the first model representatives were recovered in pure culture. We provide a historical account of the environmental research that has set the foundation for numerous physiological studies and the laboratory tools that had provided novel insights into the role of Geobacter in the functioning of microbial communities from pristine and contaminated environments. We pay particular attention to latest research, both basic and applied, that has served to expand the field into new directions and to advance interdisciplinary knowledge. The electrifying physiology of Geobacter, it seems, is alive and well 30 years on.


Assuntos
Condutividade Elétrica , Geobacter/fisiologia , Biodegradação Ambiental , Biofilmes/crescimento & desenvolvimento , Biotecnologia , Grupo dos Citocromos c/genética , Grupo dos Citocromos c/metabolismo , Transporte de Elétrons/fisiologia , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Geobacter/classificação , Metais/metabolismo , Oxirredução
7.
Bioelectrochemistry ; 129: 18-25, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31075535

RESUMO

Periplasmic c-type cytochromes are essential for the electron transport between the cytoplasmic membrane bound menquinol oxidase CymA and the terminal ferric iron reductase MtrABC in the outer membrane of Shewanella oneidensis cells. Either STC or FccA are necessary for periplasmic electron transfer. We followed the hypothesis that the elimination of potential competing reactions in the periplasm and the simultaneous overexpression of STC (cctA) could lead to an accelerated electron transfer to the cell surface. The genes nrfA, ccpA, napB and napA were replaced by cctA. This led to a 1.7-fold increased ferric iron reduction rate and a 23% higher current generation in a bioelectrochemical system. Moreover, the quadruple mutant had a higher periplasmic flavin content. Further deletion of fccA and its replacement by cctA resulted in a strain with ferric iron reduction rates similar to the wild type and a lower concentration of periplasmic flavin compared to the quadruple mutant. A transcriptomic analysis revealed that the quadruple mutant had a 3.7-fold higher cctA expression which could not be further increased by the replacement of fccA. This work indicates that a synthetic adaptation of Shewanella towards extracellular respiration holds potential for increased respiratory rates and consequently higher current densities.


Assuntos
Proteínas de Bactérias/metabolismo , Grupo dos Citocromos c/metabolismo , Proteínas Periplásmicas/metabolismo , Shewanella/metabolismo , Proteínas de Bactérias/genética , Grupo dos Citocromos c/genética , Transporte de Elétrons , Desenho de Equipamento , Fumaratos/metabolismo , Deleção de Genes , Dosagem de Genes , Engenharia Genética/métodos , Lactatos/metabolismo , Proteínas Periplásmicas/genética , Shewanella/genética , Transcriptoma , Regulação para Cima
8.
Proc Natl Acad Sci U S A ; 116(9): 3425-3430, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30755526

RESUMO

The bacterium Shewanella oneidensis has evolved a sophisticated electron transfer (ET) machinery to export electrons from the cytosol to extracellular space during extracellular respiration. At the heart of this process are decaheme proteins of the Mtr pathway, MtrC and MtrF, located at the external face of the outer bacterial membrane. Crystal structures have revealed that these proteins bind 10 c-type hemes arranged in the peculiar shape of a staggered cross that trifurcates the electron flow, presumably to reduce extracellular substrates while directing electrons to neighboring multiheme cytochromes at either side along the membrane. Especially intriguing is the design of the heme junctions trifurcating the electron flow: they are made of coplanar and T-shaped heme pair motifs with relatively large and seemingly unfavorable tunneling distances. Here, we use electronic structure calculations and molecular simulations to show that the side chains of the heme rings, in particular the cysteine linkages inserting in the space between coplanar and T-shaped heme pairs, strongly enhance electronic coupling in these two motifs. This results in an [Formula: see text]-fold speedup of ET steps at heme junctions that would otherwise be rate limiting. The predicted maximum electron flux through the solvated proteins is remarkably similar for all possible flow directions, suggesting that MtrC and MtrF shuttle electrons with similar efficiency and reversibly in directions parallel and orthogonal to the outer membrane. No major differences in the ET properties of MtrC and MtrF are found, implying that the different expression levels of the two proteins during extracellular respiration are not related to redox function.


Assuntos
Grupo dos Citocromos c/genética , Transporte de Elétrons/genética , Modelos Moleculares , Shewanella/genética , Sequência de Aminoácidos/genética , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Grupo dos Citocromos c/química , Citocromos/química , Citocromos/genética , Elétrons , Heme/química , Heme/genética , Oxirredução , Shewanella/química , Shewanella/patogenicidade
9.
Extremophiles ; 23(2): 239-248, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30689055

RESUMO

The stability of dimeric cytochrome c' from a thermophile, as compared with that of a homologous mesophilic counterpart, is attributed to strengthened interactions around the heme and at the subunit-subunit interface, both of which are molecular interior regions. Here, we showed that interactions in the equivalent interior regions of homologous cytochromes c' from two psychrophiles, Shewanella benthica and Shewanella violacea (SBCP and SVCP, respectively) were similarly weakened as compared with those of the counterparts of psychrophilic Shewanella livingstonensis and mesophilic Shewanella amazonensis (SLCP and SACP, respectively), and consistently the stability of SVCP, SLCP, and SACP increased in that order. Therefore, the stability of cytochromes c' from the psychrophile, mesophile, and thermophile is systematically regulated in their molecular interior regions. Unexpectedly, however, the stability of SBCP was significantly higher than that of SVCP, and the former had additional molecular surface interactions. Collectively, SBCP had weakened interior interactions like SVCP did, but the former was stabilized at the molecular surface as compared with the latter, implying complex multiple adaptation of the proteins because the psychrophilic sources of SBCP and SVCP are also piezophilic, thriving in deep-sea extreme environments of low temperature and high hydrostatic pressure.


Assuntos
Adaptação Fisiológica , Proteínas de Bactérias/metabolismo , Grupo dos Citocromos c/metabolismo , Shewanella/metabolismo , Proteínas de Bactérias/química , Temperatura Baixa , Grupo dos Citocromos c/química , Estabilidade Enzimática , Pressão Hidrostática , Shewanella/genética
10.
Appl Environ Microbiol ; 85(3)2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30446562

RESUMO

The metal-reducing gammaproteobacterium Shewanella oneidensis reduces iodate (IO3 -) as an anaerobic terminal electron acceptor. Microbial IO3 - electron transport pathways are postulated to terminate with nitrate (NO3 -) reductase, which reduces IO3 - as an alternative electron acceptor. Recent studies with S. oneidensis, however, have demonstrated that NO3 - reductase is not involved in IO3 - reduction. The main objective of the present study was to determine the metal reduction and protein secretion genes required for IO3 - reduction by Shewanella oneidensis with lactate, formate, or H2 as the electron donor. With all electron donors, the type I and type V protein secretion mutants retained wild-type IO3 - reduction activity, while the type II protein secretion mutant lacking the outer membrane secretin GspD was impaired in IO3 - reduction. Deletion mutants lacking the cyclic AMP receptor protein (CRP), cytochrome maturation permease CcmB, and inner membrane-tethered c-type cytochrome CymA were impaired in IO3 - reduction with all electron donors, while deletion mutants lacking c-type cytochrome MtrA and outer membrane ß-barrel protein MtrB of the outer membrane MtrAB module were impaired in IO3 - reduction with only lactate as an electron donor. With all electron donors, mutants lacking the c-type cytochromes OmcA and MtrC of the metal-reducing extracellular electron conduit MtrCAB retained wild-type IO3 - reduction activity. These findings indicate that IO3 - reduction by S. oneidensis involves electron donor-dependent metal reduction and protein secretion pathway components, including the outer membrane MtrAB module and type II protein secretion of an unidentified IO3 - reductase to the S. oneidensis outer membrane.IMPORTANCE Microbial iodate (IO3 -) reduction is a major component in the biogeochemical cycling of iodine and the bioremediation of iodine-contaminated environments; however, the molecular mechanism of microbial IO3 - reduction is poorly understood. Results of the present study indicate that outer membrane (type II) protein secretion and metal reduction genes encoding the outer membrane MtrAB module of the extracellular electron conduit MtrCAB are required for IO3 - reduction by S. oneidensis On the other hand, the metal-reducing c-type cytochrome MtrC of the extracellular electron conduit is not required for IO3 - reduction by S. oneidensis These findings indicate that the IO3 - electron transport pathway terminates with an as yet unidentified IO3 - reductase that associates with the outer membrane MtrAB module to deliver electrons extracellularly to IO3.


Assuntos
Proteínas de Bactérias/metabolismo , Iodatos/metabolismo , Metais/metabolismo , Shewanella/metabolismo , Proteínas de Bactérias/genética , Biodegradação Ambiental , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/metabolismo , Grupo dos Citocromos c/metabolismo , Formiatos/metabolismo , Ácido Láctico/metabolismo , Nitrato Redutase/genética , Nitrato Redutase/metabolismo , Oxirredução , Shewanella/genética
11.
Physiol Plant ; 166(1): 199-210, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30499233

RESUMO

Cytochrome c550 is an extrinsic component in the luminal side of photosystem II (PSII) in cyanobacteria, as well as in eukaryotic algae from the red photosynthetic lineage including, among others, diatoms. We have established that cytochrome c550 from the diatom Phaeodactylum tricornutum can be obtained as a complete protein from the membrane fraction of the alga, although a C-terminal truncated form is purified from the soluble fractions of this diatom as well as from other eukaryotic algae. Eukaryotic cytochromes c550 show distinctive electrostatic features as compared with cyanobacterial cytochrome c550 . In addition, co-immunoseparation and mass spectrometry experiments, as well as immunoelectron microscopy analyses, indicate that although cytochrome c550 from P. tricornutum is mainly located in the thylakoid domain of the chloroplast - where it interacts with PSII - , it can also be found in the chloroplast pyrenoid, related with proteins linked to the CO2 concentrating mechanism and assimilation. These results thus suggest new alternative functions of this heme protein in eukaryotes.


Assuntos
Grupo dos Citocromos c/metabolismo , Diatomáceas/metabolismo , Cloroplastos/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo
12.
Curr Genet ; 65(1): 65-70, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29947971

RESUMO

Phenotypic diversity helps populations persist in changing and often unpredictable environments. One diversity-generating strategy is for individuals to switch randomly between phenotypic states such that one subpopulation has high fitness in the present environment, and another subpopulation has high fitness in an environment that might be encountered in the future. This sort of biological bet hedging can be found in all domains of life. Here, we discuss a recently described example from the bacterium Escherichia coli. When exposed to both oxygen and trimethylamine oxide (TMAO), E. coli hedges its bets on the possibility of oxygen loss by generating high cell-to-cell variability in the expression of the TMAO respiratory system. If oxygen is rapidly depleted from the environment, only those cells that had been expressing the TMAO respiratory system at high levels can continue to grow. This particular bet-hedging scheme possesses some unusual characteristics, most notably the decoupling of gene expression noise from the mean expression level. This decoupling allows bacteria to sense oxygen and regulate the amount of variability in TMAO reductase expression (that is, to turn bet hedging on or off) without having to adjust the mean TMAO reductase expression level. In this review, we discuss the features of the TMAO signaling pathway that permit the decoupling of gene expression noise from the mean and the regulation of bet hedging. We also highlight some open questions regarding the TMAO respiratory system and its regulatory architecture that may be relevant to many signaling systems.


Assuntos
Escherichia coli/genética , Metilaminas/farmacologia , Oxigênio/farmacologia , Transdução de Sinais/efeitos dos fármacos , Grupo dos Citocromos c/genética , Grupo dos Citocromos c/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Óperon/genética , Oxidantes/farmacologia , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Transdução de Sinais/genética
13.
J Appl Microbiol ; 126(3): 826-841, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30585693

RESUMO

AIMS: Cyanobacteria are prokaryotes performing oxygenic photosynthesis, and they can be engineered to harness solar energy for production of commodity and high-value chemicals by means of synthetic biology. The Cu2+ -regulated petJ promoter (PpetJ ), which controls the expression of the endogenous cytochrome c553, can be used for expression of foreign products in Synechocystis 6803. We aimed to disclose potential bottlenecks in application of the PpetJ in synthetic biology approaches. METHODS AND RESULTS: Quantitative label-free mass spectrometry revealed global proteome changes which occurred during nutrient conditions which repress or activate of PpetJ in Synechocystis 6803. CONCLUSIONS: Some irreversible proteome alterations were discovered due to the copper stress, including downregulation of the ribosomal proteins, significant changes in protein amounts of the cell surface layer and the outer and inner membranes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed limitations in the use of PpetJ for biotechnological applications.


Assuntos
Proteínas de Bactérias , Cobre/farmacologia , Grupo dos Citocromos c , Regiões Promotoras Genéticas/genética , Proteoma , Synechocystis , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Grupo dos Citocromos c/genética , Grupo dos Citocromos c/metabolismo , Proteoma/efeitos dos fármacos , Proteoma/genética , Synechocystis/química , Synechocystis/efeitos dos fármacos , Synechocystis/genética , Synechocystis/metabolismo
14.
Photosynth Res ; 139(1-3): 281-293, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29691716

RESUMO

The thermophilic purple sulfur bacterium Thermochromatium tepidum possesses four main water-soluble redox proteins involved in the electron transfer behavior. Crystal structures have been reported for three of them: a high potential iron-sulfur protein, cytochrome c', and one of two low-potential cytochrome c552 (which is a flavocytochrome c) have been determined. In this study, we purified another low-potential cytochrome c552 (LPC), determined its N-terminal amino acid sequence and the whole gene sequence, characterized it with absorption and electron paramagnetic spectroscopy, and solved its high-resolution crystal structure. This novel cytochrome was found to contain five c-type hemes. The overall fold of LPC consists of two distinct domains, one is the five heme-containing domain and the other one is an Ig-like domain. This provides a representative example for the structures of multiheme cytochromes containing an odd number of hemes, although the structures of multiheme cytochromes with an even number of hemes are frequently seen in the PDB database. Comparison of the sequence and structure of LPC with other proteins in the databases revealed several characteristic features which may be important for its functioning. Based on the results obtained, we discuss the possible intracellular function of this LPC in Tch. tepidum.


Assuntos
Chromatiaceae/metabolismo , Grupo dos Citocromos c/química , Grupo dos Citocromos c/metabolismo , Heme/química , Heme/metabolismo , Cristalografia por Raios X , Citocromos c/química , Citocromos c/metabolismo , Transporte de Elétrons/genética , Transporte de Elétrons/fisiologia
15.
Biosci Biotechnol Biochem ; 83(2): 221-232, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30304991

RESUMO

Aquaculture suffers from a number of diseases caused by Aeromonas hydrophila. Biofilm can protect bacteria from antibiotic therapy. To identify the genes those play crucial roles in A. hydrophila biofilm formation, a library of mini-Tn10 transposon insertion mutants of A. hydrophila B11 has been constructed, and 10 mutants were subjected to biofilm formation assay. The biofilm formation ability of mutant (B188) was significantly decreased compared with B11. The DNA sequence flanking the mini-Tn10 transposon inserted showed that an ORF of approximately 576 bp of the mutant strain B188 was inserted. This ORF putatively displays the highest identity (92%) with the cytochrome c4 gene (cyt-c4) of A. hydrophila subsp. hydrophila ATCC 7966. Silencing cyt-c4 led to deficiencies in biofilm formation, adhesion, drug resistance and pathogenicity of A. hydrophila, which suggests that cyt-c4 plays crucial role in the biofilm formation and virulence mechanisms of A. hydrophila. ABBREVIATIONS: GEN: gentamycin; SDZ: sulfadiazine; AK: amikacin; P: penicillin; CFP: cefoperazone; LEV: levofloxacin; MH: minocycline; FFC: florfenicol; TE: tetracycline; AMP: ampicillin; KAN: kanamycin; STR: streptomycin; SXT: sulfamethoxazole/trimethoprim; DO: doxycycline; OT: Oxytetracycline.


Assuntos
Aeromonas hydrophila/genética , Aeromonas hydrophila/metabolismo , Biofilmes , Grupo dos Citocromos c/genética , Inativação Gênica , Genes Bacterianos , Aeromonas hydrophila/patogenicidade , Sequência de Aminoácidos , Aquicultura , Aderência Bacteriana/genética , Sequência de Bases , Elementos de DNA Transponíveis , Farmacorresistência Bacteriana/genética , Mutagênese Insercional , Fases de Leitura Aberta , Interferência de RNA , Virulência
16.
J Biol Inorg Chem ; 24(1): 21-29, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30390140

RESUMO

Microperoxidase-11 (MP11) is an undecapeptide derived from horse heart cytochrome c, which is considered as a heme-protein model. Here, the reductive nitrosylation of ferric MP11 (MP11(III)) under anaerobic conditions has been investigated between pH 7.4 and 9.2, at T = 20.0 °C. At pH ≤ 7.7, NO binds reversibly to MP11(III) leading to the formation of the MP11(III)-NO complex. However, between pH 8.2 and 9.2, the addition of NO to MP11(III) leads to the formation of ferrous nitrosylated MP11(II) (MP11(II)-NO). In fact, the transient MP11{FeNO}6 species is converted to ferrous deoxygenated MP11 (MP11(II)) by OH-- and H2O-based catalysis, which represents the rate-limiting step of the whole reaction. Then, MP11(II) binds NO very rapidly leading to MP11(II)-NO formation. Over the whole pH range explored, the apparent values of kon, koff, and K (= koff/kon) for MP11(III)(-NO) (de)nitrosylation are essentially pH independent, ranging between 5.8 × 105 M-1 s-1 and 1.6 × 106 M-1 s-1, between 1.9 s-1 and 3.7 s-1, and between 1.4 × 10-6 M and 4.6 × 10-6 M, respectively. Values of the apparent pseudo-first-order rate constant for the MP11{FeNO}6 conversion to MP11(II) (i.e., h) increase linearly with pH; the apparent values [Formula: see text] and [Formula: see text] are 7.2 × 102 M-1 s-1 and 2.5 × 10-4 s-1, respectively. Present data confirm that MP11 is a useful molecular model to highlight the role of the protein matrix on the heme-based reactivity.


Assuntos
Grupo dos Citocromos c/metabolismo , Peroxidases/metabolismo , Animais , Grupo dos Citocromos c/química , Cavalos , Compostos de Ferro/química , Compostos de Ferro/metabolismo , Miocárdio/enzimologia , Compostos Nitrosos/química , Compostos Nitrosos/metabolismo , Oxirredução , Peroxidases/química
17.
ACS Synth Biol ; 7(12): 2726-2736, 2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30396267

RESUMO

The relative scarcity of well-defined genetic and metabolic linkages to material properties impedes biological production of inorganic materials. The physiology of electroactive bacteria is intimately tied to inorganic transformations, which makes genetically tractable and well-studied electrogens, such as Shewanella oneidensis, attractive hosts for material synthesis. Notably, this species is capable of reducing a variety of transition-metal ions into functional nanoparticles, but exact mechanisms of nanoparticle biosynthesis remain ill-defined. We report two key factors of extracellular electron transfer by S. oneidensis, the outer membrane cytochrome, MtrC, and soluble redox shuttles (flavins), that affect Pd nanoparticle formation. Changes in the expression and availability of these electron transfer components drastically modulated particle synthesis rate and phenotype, including their structure and cellular localization. These relationships may serve as the basis for biologically tailoring Pd nanoparticle catalysts and could potentially be used to direct the biogenesis of other metal nanomaterials.


Assuntos
Nanopartículas Metálicas/química , Paládio/química , Shewanella/metabolismo , Grupo dos Citocromos c/deficiência , Grupo dos Citocromos c/genética , Grupo dos Citocromos c/metabolismo , Transporte de Elétrons , Elétrons , Expressão Gênica , Nanopartículas Metálicas/toxicidade , Oxirredução , Tamanho da Partícula , Fenótipo , Shewanella/efeitos dos fármacos
18.
Phys Chem Chem Phys ; 20(40): 25648-25656, 2018 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-30289415

RESUMO

Electrochemical surface plasmon resonance (ESPR) monitors faradaic processes optically by the change in refractive index that occurs with a change in redox state at the electrode surface. Here we apply ESPR to investigate the anode-grown Geobacter sulfurreducens biofilm (GSB), a model system used to study electroactive microbial biofilms (EABFs) which perform electrochemical reactions using electrodes as metabolic electron acceptors or donors. A substantial body of evidence indicates that electron transfer reactions among hemes of c-type cytochromes (c-Cyt) play major roles in the extracellular electron transfer (EET) pathways that connect intracellular metabolic processes of cells in an EABF to the electrode surface. The results reported here reveal that when the potential of the electrode is changed from relatively oxidizing (0.40 V vs. SHE) to reducing (-0.55 V vs. SHE) and then back to oxidizing, 70% of c-Cyt residing closest to the biofilm/electrode (within hundreds of nm from the electrode surface) appear to remain trapped in the reduced state, requiring as long as 12 hours to be re-oxidized. c-Cyt storing electrons cannot contribute to EET, yet turnover current resulting from cellular oxidation of acetate coupled with EET to the electrode surface is unaffected. This suggests that a relatively small fraction of c-Cyt residing closest to the biofilm/electrode interface is involved in EET while the majority store electrons. The results also reveal that biomass density at the biofilm/electrode interface increases rapidly during lag phase, reaching its maximum value at the onset of exponential biofilm growth when turnover current begins to rapidly increase.


Assuntos
Biofilmes , Fenômenos Eletromagnéticos , Geobacter/fisiologia , Grupo dos Citocromos c/metabolismo , Eletrodos , Elétrons , Heme/metabolismo , Oxirredução , Ressonância de Plasmônio de Superfície
19.
J Biol Inorg Chem ; 23(7): 1073-1083, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30143872

RESUMO

Heme c is characterized by its covalent attachment to a polypeptide. The attachment is typically to a CXXCH motif in which the two Cys form thioether bonds with the heme, "X" can be any amino acid other than Cys, and the His serves as a heme axial ligand. Some cytochromes c, however, contain heme attachment motifs with three or four intervening residues in a CX3CH or CX4CH motif. Here, the impacts of these variations in the heme attachment motif on heme ruffling and electronic structure are investigated by spectroscopically characterizing CX3CH and CX4CH variants of Hydrogenobacter thermophilus cytochrome c552. In addition, a novel CXCH variant is studied. 1H and 13C NMR, EPR, and resonance Raman spectra of the protein variants are analyzed to deduce the extent of ruffling using previously reported relationships between these spectral data and heme ruffling. In addition, the reduction potentials of these protein variants are measured using protein film voltammetry. The CXCH and CX4CH variants are found to have enhanced heme ruffling and lower reduction potentials. Implications of these results for the use of these noncanonical motifs in nature, and for the engineering of novel heme peptide structures, are discussed.


Assuntos
Grupo dos Citocromos c/química , Heme/química , Bactérias/enzimologia , Grupo dos Citocromos c/metabolismo , Heme/análogos & derivados , Heme/genética , Mutação , Conformação Proteica
20.
Acta Crystallogr D Struct Biol ; 74(Pt 7): 632-642, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29968673

RESUMO

Flavocytochrome c sulfide dehydrogenase from Thioalkalivibrio paradoxus (TpFCC) is a heterodimeric protein consisting of flavin- and monohaem c-binding subunits. TpFCC was co-purified and co-crystallized with the dimeric copper-binding protein TpCopC. The structure of the TpFCC-(TpCopC)2 complex was determined by X-ray diffraction at 2.6 Šresolution. The flavin-binding subunit of TpFCC is structurally similar to those determined previously, and the structure of the haem-binding subunit is similar to that of the N-terminal domain of dihaem FCCs. According to classification based on amino-acid sequence, TpCopC belongs to a high-affinity CopC subfamily characterized by the presence of a conserved His1-Xxx-His3 motif at the N-terminus. Apparently, a unique α-helix which is present in each monomer of TpCopC at the interface with TpFCC plays a key role in complex formation. The structure of the copper-binding site in TpCopC is similar to those in other known CopC structures. His3 is not involved in binding to the copper ion and is 6-7 Šaway from this ion. Therefore, the His1-Xxx-His3 motif cannot be considered to be a key factor in the high affinity of CopC for copper(II) ions. It is suggested that the TpFCC-(TpCopC)2 heterotetramer may be a component of a large periplasmic complex that is responsible for thiocyanate metabolism.


Assuntos
Proteínas de Bactérias/química , Grupo dos Citocromos c/química , Oxirredutases/química , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/química , Conformação Proteica , Thiobacillus/química , Difração de Raios X
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