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1.
Animals (Basel) ; 12(9)2022 Apr 26.
Article in English | MEDLINE | ID: mdl-35565543

ABSTRACT

(1) Background: The Fst statistic is widely used to characterize between-breed relationships. Fst = 0.1 has frequently been taken as indicating genetic distinctiveness between breeds. This study investigates whether this is justified. (2) Methods: A database was created of 35,080 breed pairs and their corresponding Fst values, deduced from microsatellite and SNP studies covering cattle, sheep, goats, pigs, horses, and chickens. Overall, 6560 (19%) of breed pairs were between breeds located in the same country, 7395 (21%) between breeds of different countries within the same region, 20,563 (59%) between breeds located far apart, and 562 (1%) between a breed and the supposed wild ancestor of the species. (3) Results: General values for between-breed Fst were as follows, cattle: microsatellite 0.06-0.12, SNP 0.08-0.15; sheep: microsatellite 0.06-0.10, SNP 0.06-0.17; horses: microsatellite 0.04-0.11, SNP 0.08-0.12; goats: microsatellite 0.04-0.14, SNP 0.08-0.16; pigs: microsatellite 0.06-0.27, SNP 0.15-0.22; chickens: microsatellite 0.05-0.28, SNP 0.08-0.26. (4) Conclusions: (1) Large amounts of Fst data are available for a substantial proportion of the world's livestock breeds, (2) the value for between-breed Fst of 0.1 is not appropriate owing to its considerable variability, and (3) accumulated Fst data may have value for interdisciplinary research.

2.
Ecol Evol ; 11(21): 14873-14887, 2021 Nov.
Article in English | MEDLINE | ID: mdl-34765147

ABSTRACT

Factors influencing grazing behavior in species-rich grasslands have been little studied. Methodologies have mostly had a primary focus on grasslands with lower floristic diversity.We test the hypothesis that grazing behavior is influenced by both animal and plant factors and investigate the relative importance of these factors, using a novel combination of video technology and vegetation classification to analyze bite and step rates.In a semi-natural, partially wooded grassland in northern Estonia, images of the vegetation being grazed and records of steps and bites were obtained from four video cameras, each mounted on the sternum of a sheep, during 41 animal-hours of observation over five days. Plant species lists for the immediate field of view were compiled. Images were partnered by direct observation of the nearest-neighbor relationships of the sheep. TWINSPAN, a standard vegetation classification technique allocating species lists to objectively defined classes by a principal components procedure, was applied to the species lists and 25 vegetation classes (15 open pasture and 10 woodland) were identified from the images.Taking bite and step rates as dependent variables, relative importance of animal factors (sheep identity), relative importance of day, and relative importance of plant factors (vegetation class) were investigated. The strongest effect on bite rates was of vegetation class. Sheep identity was less influential. When the data from woodland were excluded, sheep identity was more important than vegetation class as a source of variability in bite rate on open pasture.The original hypothesis is therefore supported, and we further propose that, at least with sheep in species-rich open pastures, animal factors will be more important in determining grazing behavior than plant factors. We predict quantifiable within-breed and between-breed differences, which could be exploited to optimize conservation grazing practices and contribute to the sustainability of extensive grazing systems.

3.
Sci Rep ; 11(1): 21363, 2021 11 01.
Article in English | MEDLINE | ID: mdl-34725398

ABSTRACT

Small ruminants are suited to a wide variety of habitats and thus represent promising study models for identifying genes underlying adaptations. Here, we considered local Mediterranean breeds of goats (n = 17) and sheep (n = 25) from Italy, France and Spain. Based on historical archives, we selected the breeds potentially most linked to a territory and defined their original cradle (i.e., the geographical area in which the breed has emerged), including transhumant pastoral areas. We then used the programs PCAdapt and LFMM to identify signatures of artificial and environmental selection. Considering cradles instead of current GPS coordinates resulted in a greater number of signatures identified by the LFMM analysis. The results, combined with a systematic literature review, revealed a set of genes with potentially key adaptive roles in relation to the gradient of aridity and altitude. Some of these genes have been previously implicated in lipid metabolism (SUCLG2, BMP2), hypoxia stress/lung function (BMPR2), seasonal patterns (SOX2, DPH6) or neuronal function (TRPC4, TRPC6). Selection signatures involving the PCDH9 and KLH1 genes, as well as NBEA/NBEAL1, were identified in both species and thus could play an important adaptive role.


Subject(s)
Goats/physiology , Sheep/physiology , Acclimatization , Adaptation, Physiological , Altitude , Animals , Breeding , Ecosystem , France , Italy , Mediterranean Region , Spain
4.
Animals (Basel) ; 10(9)2020 Aug 21.
Article in English | MEDLINE | ID: mdl-32825696

ABSTRACT

Foraging behavior of livestock in species-rich, less intensively managed grassland communities will require different methodologies from those appropriate in floristically simple environments. In this pilot study on sheep in species-rich grassland in northern Estonia, foraging behavior and the plant species of the immediate area grazed by the sheep were registered by continually-recording Go-Pro cameras. From three days of observation of five sheep (706 animal-minutes), foraging behavior was documented. Five hundred and thirty-six still images were sampled, and a plant species list was compiled for each. Each plant species was assigned a score indicating its location, in the ecophysiological sense, on the main environmental gradient. The scores of the plant species present were averaged for each image. Thus, the fine structure of foraging behavior could be studied in parallel with the vegetation of the precise area being grazed. As expected, there was considerable individual variation, and we characterized foraging behavior by quantifying the patterns of interspersion of grazing and non-grazing behaviors. This combination of behavior recording and vegetation classification could enable a numerical analysis of the responses of grazing livestock to vegetation conditions.

5.
Genet Sel Evol ; 52(1): 25, 2020 May 14.
Article in English | MEDLINE | ID: mdl-32408891

ABSTRACT

BACKGROUND: In the Neolithic, domestic sheep migrated into Europe and subsequently spread in westerly and northwesterly directions. Reconstruction of these migrations and subsequent genetic events requires a more detailed characterization of the current phylogeographic differentiation. RESULTS: We collected 50 K single nucleotide polymorphism (SNP) profiles of Balkan sheep that are currently found near the major Neolithic point of entry into Europe, and combined these data with published genotypes from southwest-Asian, Mediterranean, central-European and north-European sheep and from Asian and European mouflons. We detected clines, ancestral components and admixture by using variants of common analysis tools: geography-informative supervised principal component analysis (PCA), breed-specific admixture analysis, across-breed [Formula: see text] profiles and phylogenetic analysis of regional pools of breeds. The regional Balkan sheep populations exhibit considerable genetic overlap, but are clearly distinct from the breeds in surrounding regions. The Asian mouflon did not influence the differentiation of the European domestic sheep and is only distantly related to present-day sheep, including those from Iran where the mouflons were sampled. We demonstrate the occurrence, from southeast to northwest Europe, of a continuously increasing ancestral component of up to 20% contributed by the European mouflon, which is assumed to descend from the original Neolithic domesticates. The overall patterns indicate that the Balkan region and Italy served as post-domestication migration hubs, from which wool sheep reached Spain and north Italy with subsequent migrations northwards. The documented dispersal of Tarentine wool sheep during the Roman period may have been part of this process. Our results also reproduce the documented 18th century admixture of Spanish Merino sheep into several central-European breeds. CONCLUSIONS: Our results contribute to a better understanding of the events that have created the present diversity pattern, which is relevant for the management of the genetic resources represented by the European sheep population.


Subject(s)
Genetics, Population/methods , Polymorphism, Single Nucleotide/genetics , Sheep/genetics , Animals , Balkan Peninsula , Breeding/methods , Domestication , Genetic Testing/methods , Genetic Variation/genetics , Genotype , Phylogeny , Phylogeography/methods
6.
Nat Commun ; 10(1): 3109, 2019 07 23.
Article in English | MEDLINE | ID: mdl-31337752

ABSTRACT

Biological responses to climate change have been widely documented across taxa and regions, but it remains unclear whether species are maintaining a good match between phenotype and environment, i.e. whether observed trait changes are adaptive. Here we reviewed 10,090 abstracts and extracted data from 71 studies reported in 58 relevant publications, to assess quantitatively whether phenotypic trait changes associated with climate change are adaptive in animals. A meta-analysis focussing on birds, the taxon best represented in our dataset, suggests that global warming has not systematically affected morphological traits, but has advanced phenological traits. We demonstrate that these advances are adaptive for some species, but imperfect as evidenced by the observed consistent selection for earlier timing. Application of a theoretical model indicates that the evolutionary load imposed by incomplete adaptive responses to ongoing climate change may already be threatening the persistence of species.


Subject(s)
Acclimatization/physiology , Birds/physiology , Climate Change , Phenotype , Animals , Selection, Genetic/physiology , Time Factors
7.
Ecol Evol ; 9(10): 5859-5869, 2019 May.
Article in English | MEDLINE | ID: mdl-31161004

ABSTRACT

Cattle Bos taurus can perform valuable ecological functions in the maintenance of high nature value (HNV) pastoral systems. They have also attracted attention as potentially filling the ecological niches of megaherbivores, notably the extinct aurochs Bos primigenius, in rewilding initiatives. Native cattle breeds are recognized under the 1992 Rio Convention as components of biodiversity. They are used in HNV settings, but their conservation as breeds has rarely been an important consideration for their management in these contexts.The Chillingham herd has been kept under minimal management in Chillingham Park (northern England) for several centuries. Chillingham Park is not a rewilding scenario, but the long-term study of the cattle can be informative for the design of rewilding schemes that involve cattle as megaherbivores. The pastures of the park are species-rich seminatural grasslands.To 2004, pasture management was influenced by the need to provide herbage for a flock of sheep that was under separate ownership, as well as for the cattle. Surveys of the vegetation conducted in 1979 and 2006-2008 showed a decline of plant species richness (species per 100 m2 quadrat) from 33.8 in 1979 to 22.6 in 2006-2008. This was acceptable as the conservation priority has always been the cattle herd. With removal of the sheep from 2004, it became possible to include recovery of plant diversity as a management goal.In 2017, the cattle numbered 111 (64 in 1979). Plant species richness in 2017 had increased to 26.3 species per quadrat. It has therefore been possible at Chillingham both to conserve the cattle herd and to improve plant diversity. While providing basic information of relevance to the management of cattle in free-ranging situations, this study also suggests a general principle, that the management of pastoral landscapes by native breeds of cattle, can deliver multiple conservation benefits.

8.
Microbiology (Reading) ; 164(2): 122-132, 2018 02.
Article in English | MEDLINE | ID: mdl-29111967

ABSTRACT

'Ene'-reductases have attracted significant attention for the preparation of chemical intermediates and biologically active products. To date, research has been focussed primarily on Old Yellow Enzyme-like proteins, due to their ease of handling, whereas 2-enoate reductases from clostridia have received much less attention, because of their oxygen sensitivity and a lack of suitable expression systems. A hypothetical 2-enoate reductase gene, fldZ, was identified in Clostridium sporogenes DSM 795. The encoded protein shares a high degree of homology to clostridial FMN- and FAD-dependent 2-enoate reductases, including the cinnamic acid reductase proposed to be involved in amino acid metabolism in proteolytic clostridia. The gene was cloned and overexpressed in Escherichia coli. Successful expression depended on the use of strictly anaerobic conditions for both growth and enzyme preparation, since FldZ was oxygen-sensitive. The enzyme reduced aromatic enoates, such as cinnamic acid or p-coumaric acid, but not short chain unsaturated aliphatic acids. The ß,ß-disubstituted nitroalkene, (E)-1-nitro-2-phenylpropene, was reduced to enantiopure (R)-1-nitro-2-phenylpropane with a yield of 90 %. By contrast, the α,ß-disubstituted nitroalkene, (E)-2-nitro-1-phenylpropene, was reduced with a moderate yield of 56 % and poor enantioselectivity (16 % ee for (S)-2-nitro-1-phenylpropane). The availability of an expression system for this recombinant clostridial 2-enoate reductase will facilitate future characterisation of this unusual class of 'ene'-reductases, and expand the biocatalytic toolbox available for enantioselective hydrogenation of carbon-carbon double bonds.


Subject(s)
Anaerobiosis , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Clostridium/enzymology , Oxidoreductases Acting on CH-CH Group Donors/genetics , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Bacterial Proteins/biosynthesis , Biocatalysis , Cloning, Molecular , Clostridium/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Gene Expression , Genes, Bacterial/genetics , Oxidation-Reduction , Oxidoreductases Acting on CH-CH Group Donors/biosynthesis , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
9.
Microbiology (Reading) ; 164(2): 133-141, 2018 02.
Article in English | MEDLINE | ID: mdl-29231156

ABSTRACT

Citramalic acid is a central intermediate in a combined biocatalytic and chemocatalytic route to produce bio-based methylmethacrylate, the monomer used to manufacture Perspex and other high performance materials. We developed an engineered E. coli strain and a fed-batch bioprocess to produce citramalate at concentrations in excess of 80 g l-1 in only 65 h. This exceptional efficiency was achieved by designing the production strain and the fermentation system to operate synergistically. Thus, a single gene encoding a mesophilic variant of citramalate synthase from Methanococcus jannaschii, CimA3.7, was expressed in E. coli to convert acetyl-CoA and pyruvate to citramalate, and the ldhA and pflB genes were deleted. By using a bioprocess with a continuous, growth-limiting feed of glucose, these simple interventions diverted substrate flux directly from central metabolism towards formation of citramalate, without problematic accumulation of acetate. Furthermore, the nutritional requirements of the production strain could be satisfied through the use of a mineral salts medium supplemented only with glucose (172 g l-1 in total) and 1.4 g l-1 yeast extract. Using this system, citramalate accumulated to 82±1.5 g l-1, with a productivity of 1.85 g l-1 h-1 and a conversion efficiency of 0.48 gcitramalate g-1glucose. The new bioprocess forms a practical first step for integrated bio- and chemocatalytic production of methylmethacrylate.


Subject(s)
Escherichia coli/genetics , Escherichia coli/metabolism , Malates/metabolism , Metabolic Engineering , Acetyl Coenzyme A/metabolism , Acetyltransferases/genetics , Acetyltransferases/metabolism , Batch Cell Culture Techniques , Escherichia coli/enzymology , Escherichia coli/growth & development , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Fermentation , Genes, Bacterial/genetics , Methanocaldococcus/enzymology , Methanocaldococcus/genetics , Pyruvic Acid/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
10.
Nature ; 535(7611): 241-5, 2016 07 14.
Article in English | MEDLINE | ID: mdl-27362222

ABSTRACT

Differences in phenological responses to climate change among species can desynchronise ecological interactions and thereby threaten ecosystem function. To assess these threats, we must quantify the relative impact of climate change on species at different trophic levels. Here, we apply a Climate Sensitivity Profile approach to 10,003 terrestrial and aquatic phenological data sets, spatially matched to temperature and precipitation data, to quantify variation in climate sensitivity. The direction, magnitude and timing of climate sensitivity varied markedly among organisms within taxonomic and trophic groups. Despite this variability, we detected systematic variation in the direction and magnitude of phenological climate sensitivity. Secondary consumers showed consistently lower climate sensitivity than other groups. We used mid-century climate change projections to estimate that the timing of phenological events could change more for primary consumers than for species in other trophic levels (6.2 versus 2.5-2.9 days earlier on average), with substantial taxonomic variation (1.1-14.8 days earlier on average).


Subject(s)
Climate Change/statistics & numerical data , Ecosystem , Animals , Aquatic Organisms , Climate , Datasets as Topic , Forecasting , Rain , Seasons , Species Specificity , Temperature , Time Factors , United Kingdom
11.
Front Genet ; 6: 314, 2015.
Article in English | MEDLINE | ID: mdl-26539210

ABSTRACT

Livestock conservation practice is changing rapidly in light of policy developments, climate change and diversifying market demands. The last decade has seen a step change in technology and analytical approaches available to define, manage and conserve Farm Animal Genomic Resources (FAnGR). However, these rapid changes pose challenges for FAnGR conservation in terms of technological continuity, analytical capacity and integrative methodologies needed to fully exploit new, multidimensional data. The final conference of the ESF Genomic Resources program aimed to address these interdisciplinary problems in an attempt to contribute to the agenda for research and policy development directions during the coming decade. By 2020, according to the Convention on Biodiversity's Aichi Target 13, signatories should ensure that "…the genetic diversity of …farmed and domesticated animals and of wild relatives …is maintained, and strategies have been developed and implemented for minimizing genetic erosion and safeguarding their genetic diversity." However, the real extent of genetic erosion is very difficult to measure using current data. Therefore, this challenging target demands better coverage, understanding and utilization of genomic and environmental data, the development of optimized ways to integrate these data with social and other sciences and policy analysis to enable more flexible, evidence-based models to underpin FAnGR conservation. At the conference, we attempted to identify the most important problems for effective livestock genomic resource conservation during the next decade. Twenty priority questions were identified that could be broadly categorized into challenges related to methodology, analytical approaches, data management and conservation. It should be acknowledged here that while the focus of our meeting was predominantly around genetics, genomics and animal science, many of the practical challenges facing conservation of genomic resources are societal in origin and are predicated on the value (e.g., socio-economic and cultural) of these resources to farmers, rural communities and society as a whole. The overall conclusion is that despite the fact that the livestock sector has been relatively well-organized in the application of genetic methodologies to date, there is still a large gap between the current state-of-the-art in the use of tools to characterize genomic resources and its application to many non-commercial and local breeds, hampering the consistent utilization of genetic and genomic data as indicators of genetic erosion and diversity. The livestock genomic sector therefore needs to make a concerted effort in the coming decade to enable to the democratization of the powerful tools that are now at its disposal, and to ensure that they are applied in the context of breed conservation as well as development.

12.
Appl Environ Microbiol ; 81(7): 2625-34, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25636853

ABSTRACT

Mevalonate diphosphate decarboxylase (MVD) is an ATP-dependent enzyme that catalyzes the phosphorylation/decarboxylation of (R)-mevalonate-5-diphosphate to isopentenyl pyrophosphate in the mevalonate (MVA) pathway. MVD is a key enzyme in engineered metabolic pathways for bioproduction of isobutene, since it catalyzes the conversion of 3-hydroxyisovalerate (3-HIV) to isobutene, an important platform chemical. The putative homologue from Picrophilus torridus has been identified as a highly efficient variant in a number of patents, but its detailed characterization has not been reported. In this study, we have successfully purified and characterized the putative MVD from P. torridus. We discovered that it is not a decarboxylase per se but an ATP-dependent enzyme, mevalonate-3-kinase (M3K), which catalyzes the phosphorylation of MVA to mevalonate-3-phosphate. The enzyme's potential in isobutene formation is due to the conversion of 3-HIV to an unstable 3-phosphate intermediate that undergoes consequent spontaneous decarboxylation to form isobutene. Isobutene production rates were as high as 507 pmol min(-1) g cells(-1) using Escherichia coli cells expressing the enzyme and 2,880 pmol min(-1) mg protein(-1) with the purified histidine-tagged enzyme, significantly higher than reported previously. M3K is a key enzyme of the novel MVA pathway discovered very recently in Thermoplasma acidophilum. We suggest that P. torridus metabolizes MVA by the same pathway.


Subject(s)
Alkenes/metabolism , Carboxy-Lyases/metabolism , Mevalonic Acid/analogs & derivatives , Mevalonic Acid/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Thermoplasmales/enzymology , Adenosine Triphosphate/metabolism , Carboxy-Lyases/genetics , Carboxy-Lyases/isolation & purification , Cloning, Molecular , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/isolation & purification , Thermoplasmales/genetics , Valerates/metabolism
13.
Proc Natl Acad Sci U S A ; 110(21): 8393-8, 2013 May 21.
Article in English | MEDLINE | ID: mdl-23671089

ABSTRACT

This article examines two strands of discourse on wild capture fisheries; one that focuses on resource sustainability and environmental impacts, another related to food and nutrition security and human well-being. Available data and research show that, for countries most dependent on fish to meet the nutritional requirements of their population, wild capture fisheries remain the dominant supplier. Although, contrary to popular narratives, the sustainability of these fisheries is not always and everywhere in crisis, securing their sustainability is essential and requires considerable effort across a broad spectrum of fishery systems. An impediment to achieving this is that the current research and policy discourses on environmental sustainability of fisheries and food security remain only loosely and superficially linked. Overcoming this requires adoption of a broader sustainability science paradigm to help harness synergies and negotiate tradeoffs between food security, resource conservation, and macroeconomic development goals. The way society chooses to govern fisheries is, however, an ethical choice, not just a technical one, and we recommend adding an ethical dimension to sustainability science as applied to fisheries.


Subject(s)
Conservation of Natural Resources , Developing Countries/economics , Fisheries , Food Supply , Animals , Conservation of Natural Resources/economics , Conservation of Natural Resources/methods , Fisheries/economics , Fisheries/methods , Food Supply/economics , Food Supply/methods , Humans
14.
Mitochondrion ; 12(4): 438-40, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22609322

ABSTRACT

The Chillingham herd of wild Northumbrian cattle remains viable despite over 300 years of in-breeding and a near-homozygous nuclear genome. Here we report the complete mitochondrial DNA sequence using ultra-deep next generation sequencing. Random population sampling of ~10% of the extant herd identified a single mtDNA haplotype harbouring a unique bovine variant present in all other higher mammals (m.11789C/Y421H) which may contribute to their survival.


Subject(s)
Animals, Inbred Strains/genetics , DNA, Mitochondrial/genetics , Animals , Cattle , Cluster Analysis , Genome, Mitochondrial , Haplotypes , High-Throughput Nucleotide Sequencing , Phylogeny
15.
J Anim Ecol ; 80(6): 1134-44, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21668894

ABSTRACT

1. Studies examining changes in the scheduling of breeding in response to climate change have focused on species with well-defined breeding seasons. Species exhibiting year-round breeding have received little attention and the magnitudes of any responses are unknown. 2. We investigated phenological data for an enclosed feral population of cattle (Bos taurus L.) in northern England exhibiting year-round breeding. This population is relatively free of human interference. 3. We assessed whether the timing of births had changed over the last 60 years, in response to increasing winter and spring temperatures, changes in herd density, and a regime of lime fertilisation. 4. Median birth date became earlier by 1·0 days per year. Analyses of the seasonal distribution of calving dates showed that significantly fewer calves were born in summer (decline from 44% of total births to 20%) and significantly more in winter (increase from 12% to 30%) over the study period. The most pronounced changes occurred in winter, with significant increases in both the proportion and number of births. Winter births arise from conceptions in the previous spring, and we considered models that investigated climate and weather variables associated with the winter preceding and the spring of conceptions. 5. The proportion of winter births was higher when the onset of the plant growing season was earlier during the spring of conceptions. This relationship was much weaker during years when the site had been fertilised with lime, suggesting that increased forage biomass was over-riding the impacts of changing plant phenology. When the onset of the growing season was late, winter births increased with female density. 6. Recruitment estimates from a stage-structured state-space population model were significantly negatively correlated with the proportion of births in the preceding winter, suggesting that calves born in winter are less likely to survive than those born in other seasons. 7. This is one of the first studies to document changes in the phenology of a year-round breeder, suggesting that the impact of climate on the scheduling of biological events may be more extensive than previously thought and that impacts may be negative, even for species with relatively flexible breeding strategies.


Subject(s)
Cattle/physiology , Climate Change , Reproduction , Animals , England , Environment , Female , Male , Models, Biological , Parturition , Population Dynamics , Seasons
16.
Microbiology (Reading) ; 156(Pt 10): 2994-3010, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20688826

ABSTRACT

The zoonotic pathogen Campylobacter jejuni NCTC 11168 uses a complex set of electron transport chains to ensure growth with a variety of electron donors and alternative electron acceptors, some of which are known to be important for host colonization. Many of the key redox proteins essential for electron transfer in this bacterium have N-terminal twin-arginine translocase (TAT) signal sequences that ensure their transport across the cytoplasmic membrane in a folded state. By comparisons of 2D gels of periplasmic extracts, gene fusions and specific enzyme assays in wild-type, tatC mutant and complemented strains, we experimentally verified the TAT dependence of 10 proteins with an N-terminal twin-arginine motif. NrfH, which has a TAT-like motif (LRRKILK), was functional in nitrite reduction in a tatC mutant, and was correctly rejected as a TAT substrate by the tatfind and TatP prediction programs. However, the hydrogenase subunit HydA is also rejected by tatfind, but was shown to be TAT-dependent experimentally. The YedY homologue Cj0379 is the only TAT translocated molybdoenzyme of unknown function in C. jejuni; we show that a cj0379c mutant is deficient in chicken colonization and has a nitrosative stress phenotype, suggestive of a possible role for Cj0379 in the reduction of reactive nitrogen species in the periplasm. Only two potential TAT chaperones, NapD and Cj1514, are encoded in the genome. Surprisingly, despite homology to TorD, Cj1514 was shown to be specifically required for the activity of formate dehydrogenase, not trimethylamine N-oxide reductase, and was designated FdhM.


Subject(s)
Bacterial Proteins/metabolism , Campylobacter jejuni/enzymology , Membrane Transport Proteins/metabolism , Molecular Chaperones/metabolism , Animals , Bacterial Proteins/genetics , Campylobacter jejuni/genetics , Chickens , DNA, Bacterial/genetics , Electron Transport , Electron Transport Chain Complex Proteins/metabolism , Genetic Complementation Test , Membrane Transport Proteins/genetics , Molecular Chaperones/genetics , Mutation , Nitric Oxide/metabolism , Nitrites/metabolism , Proteome/metabolism
18.
Environ Microbiol ; 12(3): 576-91, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19919540

ABSTRACT

Methylmenaquinol : fumarate reductase (Mfr) is a newly recognized type of fumarate reductase present in some epsilon-proteobacteria, where the active site subunit (MfrA) is localized in the periplasm, but for which a physiological role has not been identified. We show that the Campylobacter jejuni mfrABE operon is transcribed from a single promoter, with the mfrA gene preceded by a small open reading-frame (mfrX) encoding a C. jejuni-specific polypeptide of unknown function. The growth characteristics and enzyme activities of mutants in the mfrA and menaquinol : fumarate reductase A (frdA) genes show that the cytoplasmic facing Frd enzyme is the major fumarate reductase under oxygen limitation. The Mfr enzyme is shown to be necessary for maximal rates of growth by fumarate respiration and rates of fumarate reduction in intact cells measured by both viologen assays and 1H-NMR were slower in an mfrA mutant. As periplasmic fumarate reduction does not require fumarate/succinate antiport, Mfr may allow more efficient adaptation to fumarate-dependent growth. However, a further rationale for the periplasmic location of Mfr is suggested by the observation that the enzyme also reduces the fumarate analogues mesaconate and crotonate; fermentation products of anaerobes with which C. jejuni shares its gut environment, that are unable to be transported into the cell. Both MfrA and MfrB subunits were localized in the periplasm by immunoblotting and 2D-gel electrophoresis, but an mfrE mutant accumulated unprocessed MfrA in the cytoplasm, suggesting a preassembled MfrABE holoenzyme has to be recognized by the TAT system for translocation to occur. Gene expression studies in chemostat cultures following an aerobic-anaerobic shift showed that mfrA is highly upregulated by oxygen limitation, as would be experienced in vivo. Our results indicate that in addition to a role in fumarate respiration, Mfr allows C. jejuni to reduce analogous substrates specifically present in the host gut environment.


Subject(s)
Bacterial Proteins/metabolism , Campylobacter jejuni/enzymology , Crotonates/metabolism , Fumarates/metabolism , Maleates/metabolism , Periplasm/enzymology , Succinate Dehydrogenase/metabolism , Amino Acid Sequence , Animals , Bacterial Proteins/genetics , Base Sequence , Campylobacter jejuni/growth & development , Gene Expression Regulation, Bacterial , Humans , Molecular Sequence Data , Operon , Oxidation-Reduction , Oxygen/metabolism , Protein Subunits/genetics , Protein Subunits/metabolism , Succinate Dehydrogenase/genetics
19.
J Bacteriol ; 190(24): 8075-85, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18931123

ABSTRACT

Metal ion homeostasis mechanisms in the food-borne human pathogen Campylobacter jejuni are poorly understood. The Cj1516 gene product is homologous to the multicopper oxidase CueO, which is known to contribute to copper tolerance in Escherichia coli. Here we show, by optical absorbance and electron paramagnetic resonance spectroscopy, that purified recombinant Cj1516 contains both T1 and trinuclear copper centers, which are characteristic of multicopper oxidases. Inductively coupled plasma mass spectrometry revealed that the protein contained approximately six copper atoms per polypeptide. The presence of an N-terminal "twin arginine" signal sequence suggested a periplasmic location for Cj1516, which was confirmed by the presence of p-phenylenediamine (p-PD) oxidase activity in periplasmic fractions of wild-type but not Cj1516 mutant cells. Kinetic studies showed that the pure protein exhibited p-PD, ferroxidase, and cuprous oxidase activities and was able to oxidize an analogue of the bacterial siderophore anthrachelin (3,4-dihydroxybenzoate), although no iron uptake impairment was observed in a Cj1516 mutant. However, this mutant was very sensitive to increased copper levels in minimal media, suggesting a role in copper tolerance. This was supported by increased expression of the Cj1516 gene in copper-rich media. A mutation in a second gene, the Cj1161c gene, encoding a putative CopA homologue, was also found to result in copper hypersensitivity, and a Cj1516 Cj1161c double mutant was found to be more copper sensitive than either single mutant. These observations and the apparent lack of alternative copper tolerance systems suggest that Cj1516 (CueO) and Cj1161 (CopA) are major proteins involved in copper homeostasis in C. jejuni.


Subject(s)
Bacterial Proteins/metabolism , Campylobacter jejuni/enzymology , Copper/metabolism , Oxidoreductases/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Campylobacter jejuni/genetics , Ceruloplasmin/genetics , Ceruloplasmin/metabolism , DNA, Bacterial/genetics , Electron Spin Resonance Spectroscopy , Genes, Bacterial , Molecular Sequence Data , Monophenol Monooxygenase/genetics , Monophenol Monooxygenase/metabolism , Mutagenesis, Insertional , Mutation , Oxidation-Reduction , Oxidoreductases/genetics , Oxygen Consumption , Periplasm/metabolism , Plasmids , Sequence Alignment , Siderophores/metabolism
20.
Mol Microbiol ; 68(2): 474-91, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18284594

ABSTRACT

Campylobacter jejuni is a gastrointestinal pathogen of humans but can asymptomatically colonize the avian gut. C. jejuni therefore grows at both 37 degrees C and 42 degrees C, the internal temperatures of humans and birds respectively. Microarray and proteomic studies on temperature regulation in C. jejuni strain 81-176 revealed the upregulation at 42 degrees C of two proteins, Cj0414 and Cj0415, orthologous to gluconate dehydrogenase (GADH) from Pectobacterium cypripedii. 81-176 demonstrated GADH activity, converting d-gluconate to 2-keto-d-gluconate, that was higher at 42 degrees C than at 37 degrees C. In contrast, cj0414 and cj0415 mutants lacked GADH activity. Wild-type but not cj0415 mutant bacteria exhibited gluconate-dependent respiration. Neither strain grew in defined media with d-gluconate or 2-keto-d-gluconate as a sole carbon source, revealing that gluconate was used as an electron donor rather than as a carbon source. When administered to chicks individually or in competition with wild-type, the cj0415 mutant was impaired in establishing colonization. In contrast, there were few significant differences in colonization of BALB/c-ByJ mice in single or mixed infections. These results suggest that the ability of C. jejuni to use gluconate as an electron donor via GADH activity is an important metabolic characteristic that is required for full colonization of avian but not mammalian hosts.


Subject(s)
Bacterial Proteins/metabolism , Campylobacter jejuni/enzymology , Gluconates/metabolism , Oxidoreductases/metabolism , Animals , Bacterial Proteins/genetics , Campylobacter Infections/microbiology , Campylobacter jejuni/chemistry , Campylobacter jejuni/growth & development , Campylobacter jejuni/metabolism , Cecum/microbiology , Chickens , Colony Count, Microbial , Electrophoresis, Gel, Two-Dimensional , Gene Deletion , Gene Expression Profiling , Mice , Mice, Inbred BALB C , Oligonucleotide Array Sequence Analysis , Oxidoreductases/genetics , Oxygen/metabolism , Pectobacterium/enzymology , Pectobacterium/genetics , Proteome/analysis , Sequence Homology, Amino Acid , Temperature , Virulence Factors/genetics , Virulence Factors/metabolism
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