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Introduction: Shed snake skin (SSS) is commonly used empirically in ethnomedicine to treat psoriasis, acne, warts, eczema, scabies, open wounds, hemorrhoids, and glaucoma. Although a few studies exist, SSS extracts' in vitro immunological effects have yet to be well described. Therefore, we aimed to investigate the immunomodulatory effects of SSS extract on murine lymphocytes and T cells. Methods: Hexane, methanol, and chloroform extractions were conducted in collected SSS samples. Protein concentrations in the SSS extract were measured. The cytotoxic and anticancer activities were measured using L929 Fibroblast and SK MEL 30 Cell Lines via MTT assay as described in TS EN ISO 10993-5. Immunomodulatory activities of SSS extract on total lymphocytes or enriched CD4+ T cell cultures, their cell-specific pro-inflammatory cytokines (IL-6, IL-1β. IL-12p40, IL-23p19, TNF-α, IL-17A, IFN-γ, IL-10, TGFβ1) levels were measured via FACS ARIA III analysis and related gene expression with Real-Time Quantitative Polymerase Chain Reaction (Rt-qPCR). Results: Hexane, methanol, and chloroform-extracted SSS were tested on SKMEL-30 cells via MTT and revealed a superior anti-proliferative effect for hexane extract of SSS at low concentrations. SSS treatment of murine lymphocytes augmented Tnf-α and IFN-γ levels produced by CD3+ T cells when lymphocytes were activated with anti-CD3/CD28 or LPS stimulation. This effect required the presence of non-T cells, possibly antigen-presenting cells, and was not observed on purified CD4+ T cells. Additionally, SSS significantly blocked suppressive cytokine Tgfb gene expression (but not Il10) without altering in vitro Treg generation/or expansion. Discussion: This is the first in vitro study investigating SSS's anti-tumor and immunomodulatory effects. Our data provide evidence for SSS's antiproliferative activity on SK-MEL-30 cells and its pro-inflammatory role on murine lymphocytes, which warrants further investigation of the potential use of SSS extract with in vivo disease models. [ABSTRACT FROM AUTHOR]

Background/aim: Chemical biological radiological nuclear threats are at an important point in the agenda of world health today, as they can cause mass deaths. B. mallei attracts attention as a potential biological warfare agent due to its features such as multidrug resistance, a rapid transmission mechanism via aerosol, the absence of a complete treatment protocol for the infection it causes, and the absence of an approved vaccine for protection against the bacteria. B. mallei suspect samples must be studied by experienced personnel in biosafety level III laboratories. B mallei is a difficult and troublesome pathogen to diagnose and many unknowns about B. mallei today. Therefore, the aim of the study was to determine the molecular differences and potential resistance genes of B mallei strains. Materials and methods: Determination of the molecular differences and potential resistance genes of B mallei strains with new bioinformatics approaches by comparatively examining the data of 29 B mallei strains, 10 of which were isolated from Türkiye, on the genome list of the National Biotechnology Information Center (NCBI). Results: According to the genome annotations of the origins, the origin containing the highest number of CDS which is 5172 was found as the 11th strain obtained in Türkiye in 1949. The origin with the highest number of pseudogenes was determined as 23,344 (China 7) origin. Two hundred and eighty-five pseudogenes found in this strain were obtained from a knee effusion in Myanmar. According to chromosome 2 data, B. mallei strain was determined as the most similar strain to ATCC 23344, line 11 with NCTC 10229 strain, and SAVP1 strain was determined as the least similar strain. When the antimicrobial resistance gene markers of the isolates included in the study were examined, amrA and amrB, qacG ade, Burkholderia pseudomallei Omp38 were found to be carrying. Conclusion: In terms of public health, it was thought that the data obtained as a result of our study about B mallei, which is defined as a biological weapon, is very valuable for creating treatment protocols to be applied to possible epidemics in the future. In addition, the available genetic epidemiological data of these strains belonging to a category that is dangerous to work with in a laboratory environment were reviewed. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) is increasingly recognized as a major pathogen in adults and shows a disease burden comparable to that for influenza (1). No vaccine is currently available. However, [...]

Background: Acute respiratory infections (ARIs) during pregnancy are associated with poor maternal and fetal outcomes. Methods: Using U.S. Flu Vaccine Effectiveness Network data (2011–2016) from Washington and Michigan, we tested for respiratory viruses among pregnant and non‐pregnant outpatients matched on age, site, and season (n = 191). Results: Among all participants, detection of human coronaviruses and rhinovirus was common. We also observed differences in virus detection by pregnancy status; human coronaviruses and respiratory syncytial virus (RSV) were detected more frequently among pregnant and non‐pregnant participants, respectively. Conclusions: The role of respiratory viruses in maternal ARI morbidity should be further characterized to inform implementation of prevention interventions including maternal vaccines. [ABSTRACT FROM AUTHOR]

This study was aimed to synthesise antimicrobial and therapeutic silver nanoparticles (AgNPs) by green synthesis method using Vitex agnus‐castus L. (VAC) seed extract. The synthesised VAC seed extract coated AgNPs were characterised by UV‐Vis spectrometry, Zeta analysis and FT‐IR spectrometry. The antifungal, antimicrobial, cytotoxicity by MTT assay, and anticarcinogenic activities on HeLa cells line were investigated. VAC‐AgNPs exhibited high antimicrobial activity against S. aureus with a zone diameter of 11.3±0.6 mm. In addition, the cytotoxicity of VAC‐AgNPs was evaluated using the L929 fibroblast cell line and the HeLa cells line, with AgNPs applied at six different concentrations. At a concentration of 5.0 mM, viability was found to be 47.6 %±4.2 % in L929 fibroblast cells and 39.6 %±7.9 % in HeLa cells, suggesting a potential antiproliferative effect of VAC‐AgNPs on HeLa cancer cells. The IC50 values of VAC‐AgNPs against the HeLa cell line were calculated as 2.94 μg/ml. [ABSTRACT FROM AUTHOR]

The One Health approach shows that people, animals, plants, and environmental factors can affect each other. Phages are one of the mobile genetic elements. Quinolones are a critical group of antibiotics for both human and animal health and monitoring their antimicrobial resistance is very important. The aim of the study is to determine the frequency of the quinolone resistance gene in bacteriophage DNA fractions obtained from healthy calf stool samples. In our study, 50 samples from 6-9 months old calves, which were found to be healthy and not treated with any group of antibiotics in Sanlıurfa province, were included. DNA isolation was made from phage lysates of stool samples and specific primers were used qnrA, qnrB and qnrS genes. qPCR was performed on LightCycler480. Despite not receiving any antibiotic treatment, qnrB was the most detected gene among the phage DNA fractions detected in 11 calves. While qnrA, qnrB and qnrS quinolone resistance genes were detected together in one sample, qnrB and qnrS resistance genes were found together in two samples. Our data, obtained from the study in Türkiye to search for antimicrobial resistance genes in phage fractions, showed the importance of the One Health approach and determined that it was highly effective in quinolone resistance gene shedding in healthy calves that had never been treated with antibiotics. It has been concluded that in empirical treatment with quinolone, attention should be paid to all living things and unconscious antibiotic use may cause the spread of resistance genes more than expected. [ABSTRACT FROM AUTHOR]

Within the scope of the "National Ruam Eradication Project" carried out between 2000-2001, Glanders was eradicated in our country. Unfortunately, 81 horses were culled in Türkiye in December 2019, following the detection of Epidemic in horses in Büyükada. In 2019, Glanders were reported in horses in Uşak and Bolu. No human cases have been reported. Türkiye is at risk for Glanders because of its geography. Therefore, reliable and fastly detection of Burkholderia mallei by PCR in a short time will prevent the distribution of unwanted infections that may occur in the future throughout the country. In this study; Burkholderia mallei strains isolated from horses were verified and optimized by PCR. The use of PCR for the detection of Burkholderia mallei was performed for the first time in our country. It has been concluded that the PCR as a diagnostic method with high reliability and sensitivity safely used together with diagnosis of Glanders. [ABSTRACT FROM AUTHOR]

Objective: Raw milk is an important source of nutrients. Therefore, today, there is a great demand for raw milk consumption. The positive side of milk consumption on growth and development cannot be ignored, but unfortunately, pathogens in raw milk are always potential public health risks for transmission pathogens. Bacteria such as Enterobacteriaceae in normal flora can cause serious problems due to their extended-spectrum beta-lactamase (ESBL) production. These bacteria and their resistance genes have been reported in raw milk. In this matter, the aim of the study is to determine the status of blaCTX-M-1, blaCTXM-2, blaTEM and blaSHV genes responsible for the production of ESBL enzyme in Escherichia coli and Klebsiella spp. strains to identify risk factors in raw milk consumption and to gain an understanding of the epidemiology of these resistant strains. Materials and methods: A total of different 50 raw milk samples were collected and subjected to phenotypic microbiological analysis and Real-time PCR targeting blaCTX-M-1, blaCTX-M-2, blaTEM and blaSHV genes. In the phenotypic analyses, suspicious isolates were identified by classical microbiological methods and antibiotic resistance profiles were revealed. Results: These results indicated that raw milk is a potential reservoir for ESBL producing E. Coli, Klebsiella spp. strains are obviously significant. And It was determined that CTX-M-basedESBL genes are predominant in ESBL production. Conclusion: The present study revealed that raw milk is epidemiologically involved in the transmission of ESBL genes. Raw milk could be distributed to ESBL genes widely and is consumed in Şanlıurfa. [ABSTRACT FROM AUTHOR]

In recent years, researchers have shown an increased interest in using medicinal plants for the synthesis of silver nanoparticles (AgNPs) having various therapeutic properties. Centella asiatica (CA), a medicinal plant, has been used to treat minor burn wounds, psoriasis, and hypertrophic wounds among many other pathological conditions. The current study aimed to synthesize CA coated AgNPs (CA-AgNPs) with appropriate biocompatibility and various therapeutic properties, including antimicrobial and analgesic activities. The synthesized CA-AgNPs were characterized by ultraviolet-visible (UV-Vis) spectroscopy, zeta potential measurements, and fourier transform infrared (FT-IR) spectroscopy. The formation of spherical CA-AgNPs was confirmed by a single surface plasmon resonance (SPR) peak emerging at 420 nm wavelength by UV-Vis. The average hydrodynamic diameter and zeta potential of the particles were found to be 29.5 nm and -24.5 mV, respectively. The FT-IR analyses showed that the AgNPs were coated and stabilized by bioactive compounds from the CA extract. MTT cytotoxicity assay revealed that CA-AgNPs at ≤1 mM concentrations exhibited biocompatibility for L929 fibroblast cells. The antimicrobial activity of CA-AgNPs was confirmed by significant inhibition of Staphylococcus aureus and Escherichia coli. In addition, the analgesic effect of CA-AgNPs was investigated for the first time in the literature by tail-flick and hot plate methods, and statistically significant results were obtained for both methods. Taken together, these results suggest that CA-AgNPs can be used as an effective antibacterial and analgesic agent in a variety of biomedical applications, including coating wound dressings. [ABSTRACT FROM AUTHOR]

Copyright of Etlik Veteriner Mikrobiyoloji Dergisi is the property of Veteriner Kontrol Merkez Arastirma Enstitusu and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Previously, an unexplained subcellular localization was reported for a functional fluorescent protein fusion to the response regulator OmpR in Escherichia coli. The pronounced regions of increased fluorescence, or foci, are dependent on OmpR phosphorylation and do not occupy fixed, easily identifiable positions, such as the poles or mid-cell. Here we show that the foci are due to OmpR-YFP (yellow fluorescent protein) fusion binding to specific sites in the chromosome. To identify positions of foci and quantify their fluorescence intensity, we used a simple system to tag virtually any chromosomal location with arrays of lacO or tetO. The brightest foci colocalize with the OmpR-regulated gene ompF, which is strongly expressed under our growth conditions. When we increased OmpR-YFP phosphorylation by stimulating the EnvZ/OmpR system with procaine, we observed a small increase in OmpR-YFP fluorescence at ompF and a significant increase at the OmpR-regulated gene ompC. This supports a model of hierarchical binding of OmpR to the ompF and ompC promoters. Our results explain the inhomogeneous distribution of OmpR-YFP fluorescence in cells and further demonstrate that for a transcription factor expressed at wild-type levels, binding to native sites in the chromosome can be imaged and quantified by fluorescence microscopy. doi: 10.1128/JB.00344-10

Copyright of Etlik Veteriner Mikrobiyoloji Dergisi is the property of Veteriner Kontrol Merkez Arastirma Enstitusu and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Technological developments not only increase the number of product and service alternatives offered to the market but also increase the number of alternative channels in delivering products and services to consumers. In particular, multi -channel and omni-channel strategies, one of the most prominent topics in marketing literature in recent years, support the importance of this development. Therefore, today's consumer has to decide from many alternatives. However, in terms of marketing,understanding how customers make choices among alternatives or to what factors shape preferences is critical. The aim of this study is to examine how the increase in the number of distribution channel alternatives, which is a frequently discussed issue in marketing literature in recent years, has an effect on consumer channel preferences, and in particular the effects of this increasing number of channels on procurement decision process. More specifically, questions which search for answer in the context of research are "Why do consumers prefer to buy products from stores after they product research on the internet?" Or vice versa "why would they prefer to buy products on the internet after having tried the products in the store?" This question is considered to have led to various negativities in terms of the retail sector, despite the increasing number of choices in consumer buying behaviors and increasing brand-consumer interaction in terms of brands. Therefore, this study aims to provide a contribution, in part, to this clarification in the literature by attempting to show what are the elements that lead to these fluctuations between the channels and what are the typical characteristics of those who prefer certain behaviors. In the context of research, a survey was conducted on 387 people in July 2017. Research findings indicate that both types of behavior are common but there are significant differences in the factors affecting the choice of the two behavior patterns, in the product groups and in the demographic characteristics of the persons. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Faculty of Veterinary Medicine, Erciyes University / Erciyes Üniversitesi Veteriner Fakültesi Dergisi is the property of Erciyes University, Faculty of Veterinary Medicine and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The fission yeast Schizosaccharomyces pombe has been successfully used as a model to gain fundamental knowledge in understanding how eukaryotic cells acquire copper during vegetative growth. These studies have revealed the existence of a heteromeric Ctr4-Ctr5 plasma membrane complex that mediates uptake of copper within the cells. Furthermore, additional studies have led to the identification of one of the first vacuolar copper transporters, Ctr6, as well as the copper-responsive Cuf1 transcription factor. Recent investigations have extended the use of S. pombe to elucidate new roles for copper metabolism in meiotic differentiation. For example, these studies have led to the discovery of Mfc1, which turned out to be the first example of a meiosis-specific copper transporter. Whereas copper-dependent transcriptional regulation of the Ctr family members is under the control of Cuf1 during mitosis or meiosis, meiosis-specific copper transporter Mfc1 is regulated by the recently discovered transactivator Mca1. It is foreseeable that identification of novel meiotic copper-related proteins will serve as stepping stones to unravel fundamental aspects of copper homoeostasis. [ABSTRACT FROM AUTHOR]

The Rhodobacter capsulatus cbb3-type cytochrome c oxidase (cbb3-Cox) belongs to the heme-copper oxidase superfamily, and its subunits are encoded by the ccoNOQP operon. Biosynthesis of this enzyme is complex and needs dedicated biogenesis genes (ccoGHIS). It also relies on the c-type cytochrome maturation (Ccm) process, which requires the ccmABCDEFGHI genes, because two of the cbb3-Cox subunits (CcoO and CcoP) are c-type cytochromes. Recently, we reported that mutants lacking CcoA, a major facilitator superfamily type transporter, produce very small amounts of cbb3-Cox unless the growth medium is supplemented with copper. In this work, we isolated "Cu-unresponsive" derivatives of a ccoA deletion strain that exhibited no cbb3-Cox activity even upon Cu supplementation. Molecular characterization of these mutants revealed missense mutations in the ccmA or cctnF gene, required for the Ccm process. As expected, Cu-unresponsive mutants lacked the CcoO and CcoP subunits due to Ccm defects, but remarkably, they contained the CcoN subunit of cbb3-Cox. Subsequent construction and examination of single ccm knockout mutants demonstrated that membrane insertion and stability of CcoN occurred in the absence of the Ccm process. Moreover, while the ccm knockout mutants were completely incompetent for photosynthesis, the Cu-unresponsive mutants grew photosynthetically at lower rates and produced smaller amounts of cytochromes c1 and c2 than did a wild-type strain due to their restricted Ccm capabilities. These findings demonstrate that different levels of Ccm efficiency are required for the production of various c-type cytochromes and reveal for the first time that maturation of the heme-Cu-con-taining subunit CcoN of R. capsulatus cbb3-Cox proceeds independently of that of the c-type cytochromes during the biogenesis of this enzyme. [ABSTRACT FROM AUTHOR]

Abstract: The cbb 3-type cytochrome c oxidases (cbb 3-Cox) constitute the second most abundant cytochrome c oxidase (Cox) group after the mitochondrial-like aa 3-type Cox. They are present in bacteria only, and are considered to represent a primordial innovation in the domain of Eubacteria due to their phylogenetic distribution and their similarity to nitric oxide (NO) reductases. They are crucial for the onset of many anaerobic biological processes, such as anoxygenic photosynthesis or nitrogen fixation. In addition, they are prevalent in many pathogenic bacteria, and important for colonizing low oxygen tissues. Studies related to cbb 3-Cox provide a fascinating paradigm for the biogenesis of sophisticated oligomeric membrane proteins. Complex subunit maturation and assembly machineries, producing the c-type cytochromes and the binuclear heme b 3 -CuB center, have to be coordinated precisely both temporally and spatially to yield a functional cbb 3-Cox enzyme. In this review we summarize our current knowledge on the structure, regulation and assembly of cbb 3-Cox, and provide a highly tentative model for cbb 3-Cox assembly and formation of its heme b 3-CuB binuclear center. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes. [Copyright &y& Elsevier]

International Journal of Paediatric Dentistry 2011; 21: 29-34 The aim of this in vitro study was to evaluate the effects of using only phosphoric acid or a self-etch bonding agent under clear and opaque fissure sealants on laser fluorescence (LF) readings and the reproducibility of the laser device. Eighty extracted permanent molars, ranged from sound to carious, were randomly divided into four groups: phosphoric acid + opaque sealant (group I), Clearfil S3 Bond (Kuraray, Kurashiki, Japan) + opaque sealant (group II), phosphoric acid + clear sealant (group III), and Clearfil S3 Bond + clear sealant (group IV). The teeth were measured using an LFpen device, before and after sealing. Data were analysed using the Spearman's correlation, Wilcoxon signed rank, and Mann-Whitney U-test. Except group IV, there was a statistically significant decrease in fluorescence after the application of sealants ( P < 0.05). The decrease of LFpen readings in the opaque sealant groups was more significant than the clear sealant groups ( P < 0.05). But for both sealants, the difference between phosphoric acid and Clearfil S3 Bond groups was nonsignificant ( P > 0.05). There was a statistically significant decrease in fluorescence for both clear and opaque sealant groups. However, clear sealant with Clearfil S3 Bond does not influence the LFpen readings. [ABSTRACT FROM AUTHOR]

Background/aim: Chemical biological radiological nuclear threats are at an important point in the agenda of world health today, as they can cause mass deaths. B. mallei attracts attention as a potential biological warfare agent due to its features such as multidrug resistance, a rapid transmission mechanism via aerosol, the absence of a complete treatment protocol for the infection it causes, and the absence of an approved vaccine for protection against the bacteria. B. mallei suspect samples must be studied by experienced personnel in biosafety level III laboratories. B mallei is a difficult and troublesome pathogen to diagnose and many unknowns about B. mallei today. Therefore, the aim of the study was to determine the molecular differences and potential resistance genes of B mallei strains., Materials and Methods: Determination of the molecular differences and potential resistance genes of B mallei strains with new bioinformatics approaches by comparatively examining the data of 29 B mallei strains, 10 of which were isolated from Türkiye, on the genome list of the National Biotechnology Information Center (NCBI)., Results: According to the genome annotations of the origins, the origin containing the highest number of CDS which is 5172 was found as the 11th strain obtained in Türkiye in 1949. The origin with the highest number of pseudogenes was determined as 23,344 (China 7) origin. Two hundred and eighty-five pseudogenes found in this strain were obtained from a knee effusion in Myanmar. According to chromosome 2 data, B. mallei strain was determined as the most similar strain to ATCC 23344, line 11 with NCTC 10229 strain, and SAVP1 strain was determined as the least similar strain. When the antimicrobial resistance gene markers of the isolates included in the study were examined, amrA and amrB , qacG ade, Burkholderia pseudomallei Omp38 were found to be carrying., Conclusion: In terms of public health, it was thought that the data obtained as a result of our study about B mallei , which is defined as a biological weapon, is very valuable for creating treatment protocols to be applied to possible epidemics in the future. In addition, the available genetic epidemiological data of these strains belonging to a category that is dangerous to work with in a laboratory environment were reviewed., (© TÜBİTAK.)

Unlabelled: Copper is an essential micronutrient used as a metal cofactor by a variety of enzymes, including cytochrome c oxidase (Cox). In all organisms from bacteria to humans, cellular availability and insertion of copper into target proteins are tightly controlled due to its toxicity. The major subunit of Cox contains a copper atom that is required for its catalytic activity. Previously, we identified CcoA (a member of major facilitator superfamily transporters) as a component required for cbb3-type Cox production in the Gram-negative, facultative phototroph Rhodobacter capsulatus. Here, first we demonstrate that CcoA is a cytoplasmic copper importer. Second, we show that bypass suppressors of a ccoA deletion mutant suppress cbb3-Cox deficiency by increasing cellular copper content and sensitivity. Third, we establish that these suppressors are single-base-pair insertion/deletions located in copA, encoding the major P1B-type ATP-dependent copper exporter (CopA) responsible for copper detoxification. A copA deletion alone has no effect on cbb3-Cox biogenesis in an otherwise wild-type background, even though it rescues the cbb3-Cox defect in the absence of CcoA and renders cells sensitive to copper. We conclude that a hitherto unknown functional interplay between the copper importer CcoA and the copper exporter CopA controls intracellular copper homeostasis required for cbb3-Cox production in bacteria like R. capsulatus., Importance: Copper (Cu) is an essential micronutrient required for many processes in the cell. It is found as a cofactor for heme-copper containing cytochrome c oxidase enzymes at the terminus of the respiratory chains of aerobic organisms by catalyzing reduction of dioxygen (O2) to water. Defects in the biogenesis and copper insertion into cytochrome c oxidases lead to mitochondrial diseases in humans. This work shows that a previously identified Cu transporter (CcoA) is a Cu importer and illustrates the link between two Cu transporters, the importer CcoA and the exporter CopA, required for Cu homeostasis and Cu trafficking to cytochrome c oxidase in the cell.

The Rhodobacter capsulatus cbb(3)-type cytochrome c oxidase (cbb(3)-Cox) belongs to the heme-copper oxidase superfamily, and its subunits are encoded by the ccoNOQP operon. Biosynthesis of this enzyme is complex and needs dedicated biogenesis genes (ccoGHIS). It also relies on the c-type cytochrome maturation (Ccm) process, which requires the ccmABCDEFGHI genes, because two of the cbb(3)-Cox subunits (CcoO and CcoP) are c-type cytochromes. Recently, we reported that mutants lacking CcoA, a major facilitator superfamily type transporter, produce very small amounts of cbb(3)-Cox unless the growth medium is supplemented with copper. In this work, we isolated "Cu-unresponsive" derivatives of a ccoA deletion strain that exhibited no cbb(3)-Cox activity even upon Cu supplementation. Molecular characterization of these mutants revealed missense mutations in the ccmA or ccmF gene, required for the Ccm process. As expected, Cu-unresponsive mutants lacked the CcoO and CcoP subunits due to Ccm defects, but remarkably, they contained the CcoN subunit of cbb(3)-Cox. Subsequent construction and examination of single ccm knockout mutants demonstrated that membrane insertion and stability of CcoN occurred in the absence of the Ccm process. Moreover, while the ccm knockout mutants were completely incompetent for photosynthesis, the Cu-unresponsive mutants grew photosynthetically at lower rates and produced smaller amounts of cytochromes c(1) and c(2) than did a wild-type strain due to their restricted Ccm capabilities. These findings demonstrate that different levels of Ccm efficiency are required for the production of various c-type cytochromes and reveal for the first time that maturation of the heme-Cu-containing subunit CcoN of R. capsulatus cbb(3)-Cox proceeds independently of that of the c-type cytochromes during the biogenesis of this enzyme.

The cbb(3)-type cytochrome c oxidases (cbb(3)-Cox) constitute the second most abundant cytochrome c oxidase (Cox) group after the mitochondrial-like aa(3)-type Cox. They are present in bacteria only, and are considered to represent a primordial innovation in the domain of Eubacteria due to their phylogenetic distribution and their similarity to nitric oxide (NO) reductases. They are crucial for the onset of many anaerobic biological processes, such as anoxygenic photosynthesis or nitrogen fixation. In addition, they are prevalent in many pathogenic bacteria, and important for colonizing low oxygen tissues. Studies related to cbb(3)-Cox provide a fascinating paradigm for the biogenesis of sophisticated oligomeric membrane proteins. Complex subunit maturation and assembly machineries, producing the c-type cytochromes and the binuclear heme b(3)-Cu(B) center, have to be coordinated precisely both temporally and spatially to yield a functional cbb(3)-Cox enzyme. In this review we summarize our current knowledge on the structure, regulation and assembly of cbb(3)-Cox, and provide a highly tentative model for cbb(3)-Cox assembly and formation of its heme b(3)-Cu(B) binuclear center. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Unlabelled: The acquisition, delivery, and incorporation of metals into their respective metalloproteins are important cellular processes. These processes are tightly controlled in order to prevent exposure of cells to free-metal concentrations that could yield oxidative damage. Copper (Cu) is one such metal that is required as a cofactor in a variety of proteins. However, when present in excessive amounts, Cu is toxic due to its oxidative capability. Cytochrome c oxidases (Coxs) are among the metalloproteins whose assembly and activity require the presence of Cu in their catalytic subunits. In this study, we focused on the acquisition of Cu for incorporation into the heme-Cu binuclear center of the cbb(3)-type Cox (cbb(3)-Cox) in the facultative phototroph Rhodobacter capsulatus. Genetic screens identified a cbb(3)-Cox defective mutant that requires Cu(2+) supplementation to produce an active cbb(3)-Cox. Complementation of this mutant using wild-type genomic libraries unveiled a novel gene (ccoA) required for cbb(3)-Cox biogenesis. In the absence of CcoA, the cellular Cu content decreases and cbb(3)-Cox assembly and activity become defective. CcoA shows homology to major facilitator superfamily (MFS)-type transporter proteins. Members of this family are known to transport small solutes or drugs, but so far, no MFS protein has been implicated in cbb(3)-Cox biogenesis. These findings provide novel insights into the maturation and assembly of membrane-integral metalloproteins and on a hitherto-unknown function(s) of MFS-type transporters in bacterial Cu acquisition., Importance: Biogenesis of energy-transducing membrane-integral enzymes, like the heme copper-containing cytochrome c oxidases, and the acquisition of transition metals, like copper, as their catalytic cofactors are vital processes for all cells. These widespread and well-controlled processes are poorly understood in all organisms, including bacteria. Defects in these processes lead to severe mitochondrial diseases in humans and poor crop yields in plants. In this study, using the facultative phototroph Rhodobacter capsulatus as a model organism, we report on the discovery of a novel major facilitator superfamily (MFS)-type transporter (CcoA) that affects cellular copper content and cbb(3)-type cytochrome c oxidase production in bacteria.