scholarly journals Genes Linking Copper Trafficking and Homeostasis to the Biogenesis and Activity of the cbb3-Type Cytochrome c Oxidase in the Enteric Pathogen Campylobacter jejuni

2021 ◽  
Vol 12 ◽  
Author(s):  
Nitanshu Garg ◽  
Aidan J. Taylor ◽  
Federica Pastorelli ◽  
Sarah E. Flannery ◽  
Phillip J. Jackson ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Polyacrylamide Gel Electrophoresis ◽  
Structural Similarity ◽  
Copper Chaperone ◽  
Oxygen Binding ◽  
Gene Deletions ◽  
Copper Trafficking ◽  
Food Borne ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Bacterial Gastroenteritis

Bacterial C-type haem-copper oxidases in the cbb3 family are widespread in microaerophiles, which exploit their high oxygen-binding affinity for growth in microoxic niches. In microaerophilic pathogens, C-type oxidases can be essential for infection, yet little is known about their biogenesis compared to model bacteria. Here, we have identified genes involved in cbb3-oxidase (Cco) assembly and activity in the Gram-negative pathogen Campylobacter jejuni, the commonest cause of human food-borne bacterial gastroenteritis. Several genes of unknown function downstream of the oxidase structural genes ccoNOQP were shown to be essential (cj1483c and cj1486c) or important (cj1484c and cj1485c) for Cco activity; Cj1483 is a CcoH homologue, but Cj1484 (designated CcoZ) has structural similarity to MSMEG_4692, involved in Qcr-oxidase supercomplex formation in Mycobacterium smegmatis. Blue-native polyacrylamide gel electrophoresis of detergent solubilised membranes revealed three major bands, one of which contained CcoZ along with Qcr and oxidase subunits. Deletion of putative copper trafficking genes ccoI (cj1155c) and ccoS (cj1154c) abolished Cco activity, which was partially restored by addition of copper during growth, while inactivation of cj0369c encoding a CcoG homologue led to a partial reduction in Cco activity. Deletion of an operon encoding PCuAC (Cj0909) and Sco (Cj0911) periplasmic copper chaperone homologues reduced Cco activity, which was partially restored in the cj0911 mutant by exogenous copper. Phenotypic analyses of gene deletions in the cj1161c–1166c cluster, encoding several genes involved in intracellular metal homeostasis, showed that inactivation of copA (cj1161c), or copZ (cj1162c) led to both elevated intracellular Cu and reduced Cco activity, effects exacerbated at high external Cu. Our work has therefore identified (i) additional Cco subunits, (ii) a previously uncharacterized set of genes linking copper trafficking and Cco activity, and (iii) connections with Cu homeostasis in this important pathogen.

scholarly journals Biofilm Formation by Campylobacter jejuni Is Increased under Aerobic Conditions

2010 ◽  
Vol 76 (7) ◽  
pp. 2122-2128 ◽  
Author(s):  
Mark Reuter ◽  
Arthur Mallett ◽  
Bruce M. Pearson ◽  
Arnoud H. M. van Vliet
Keyword(s):  
Campylobacter Jejuni ◽  
Food Chain ◽  
Biofilm Formation ◽  
Planktonic Cells ◽  
Aerobic Conditions ◽  
Food Borne ◽  
Passive Release ◽  
Bacterial Gastroenteritis ◽  
Microaerobic Conditions ◽  
Developed World

ABSTRACT The microaerophilic human pathogen Campylobacter jejuni is the leading cause of food-borne bacterial gastroenteritis in the developed world. During transmission through the food chain and the environment, the organism must survive stressful environmental conditions, particularly high oxygen levels. Biofilm formation has been suggested to play a role in the environmental survival of this organism. In this work we show that C. jejuni NCTC 11168 biofilms developed more rapidly under environmental and food-chain-relevant aerobic conditions (20% O2) than under microaerobic conditions (5% O2, 10% CO2), although final levels of biofilms were comparable after 3 days. Staining of biofilms with Congo red gave results similar to those obtained with the commonly used crystal violet staining. The level of biofilm formation by nonmotile aflagellate strains was lower than that observed for the motile flagellated strain but nonetheless increased under aerobic conditions, suggesting the presence of flagellum-dependent and flagellum-independent mechanisms of biofilm formation in C. jejuni. Moreover, preformed biofilms shed high numbers of viable C. jejuni cells into the culture supernatant independently of the oxygen concentration, suggesting a continuous passive release of cells into the medium rather than a condition-specific active mechanism of dispersal. We conclude that under aerobic or stressful conditions, C. jejuni adapts to a biofilm lifestyle, allowing survival under detrimental conditions, and that such a biofilm can function as a reservoir of viable planktonic cells. The increased level of biofilm formation under aerobic conditions is likely to be an adaptation contributing to the zoonotic lifestyle of C. jejuni.

scholarly journals Campylobacter jejuni persistently colonizes gnotobiotic altered Schaedler flora C3H/HeN mice and induces mild colitis

2020 ◽  
Vol 367 (20) ◽  
Author(s):  
Meghan Wymore Brand ◽  
Orhan Sahin ◽  
Jesse M Hostetter ◽  
Julian Trachsel ◽  
Qijing Zhang ◽  
...  
Keyword(s):  
Animal Models ◽  
Campylobacter Jejuni ◽  
Gastrointestinal Disease ◽  
Proximal Colon ◽  
Rodent Model ◽  
Mucosal Inflammation ◽  
Colonization Resistance ◽  
Food Borne ◽  
Bacterial Gastroenteritis

ABSTRACT Campylobacter jejuni is a major cause of food-borne human bacterial gastroenteritis but animal models for C. jejuni mediated disease remain limited because C. jejuni poorly colonizes immunocompetent, conventionally-reared (Conv-R) mice. Thus, a reliable rodent model (i.e. persistent colonization) is desirable in order to evaluate C. jejuni-mediated gastrointestinal disease and mechanisms of pathogenicity. As the nature and complexity of the microbiota likely impacts colonization resistance for C. jejuni, Conv-R and gnotobiotic C3H/HeN mice were used to evaluate the persistence of C. jejuni colonization and development of disease. A total of four C. jejuni isolates readily and persistently colonized ASF mice and induced mild mucosal inflammation in the proximal colon, but C. jejuni did not stably colonize nor induce lesions in Conv-R mice. This suggests that the pathogenesis of C. jejuni is influenced by the microbiota, and that ASF mice offer a reproducible model to study the influence of the microbiota on the ability of C. jejuni to colonize the gut and to mediate gastroenteritis.

scholarly journals The HtrA Protease of Campylobacter jejuni Is Required for Heat and Oxygen Tolerance and for Optimal Interaction with Human Epithelial Cells

2005 ◽  
Vol 71 (6) ◽  
pp. 3205-3212 ◽  
Author(s):  
Lone Brøndsted ◽  
Marianne Thorup Andersen ◽  
Mary Parker ◽  
Kirsten Jørgensen ◽  
Hanne Ingmer
Keyword(s):  
Epithelial Cells ◽  
Campylobacter Jejuni ◽  
Wild Type Strain ◽  
Cumene Hydroperoxide ◽  
Misfolded Protein ◽  
Oxygen Tolerance ◽  
Human Epithelial Cells ◽  
Food Borne ◽  
Bacterial Gastroenteritis ◽  
Developed World

ABSTRACT Campylobacter jejuni is a predominant cause of food-borne bacterial gastroenteritis in the developed world. We have investigated the importance of a homologue of the periplasmic HtrA protease in C. jejuni stress tolerance. A C. jejuni htrA mutant was constructed and compared to the parental strain, and we found that growth of the mutant was severely impaired both at 44°C and in the presence of the tRNA analogue puromycin. Under both conditions, the level of misfolded protein is known to increase, and we propose that the heat-sensitive phenotype of the htrA mutant is caused by an accumulation of misfolded protein in the periplasm. Interestingly, we observed that the level of the molecular chaperones DnaK and ClpB was increased in the htrA mutant, suggesting that accumulation of nonnative proteins in the periplasm induces the expression of cytoplasmic chaperones. While lack of HtrA reduces the oxygen tolerance of C. jejuni, the htrA mutant was not sensitive to compounds that increase the formation of oxygen radicals, such as paraquat, cumene hydroperoxide, and H2O2. Using tissue cultures of human epithelial cells (INT407), we found that the htrA mutant adhered to and invaded human epithelial cells with a decreased frequency compared to the wild-type strain. This defect may be a consequence of the observed altered morphology of the htrA mutant. Thus, our results suggest that in C. jejuni, HtrA is important for growth during stressful conditions and has an impact on virulence.

scholarly journals The Native Monomer of Bacillus Pumilus Ribonuclease Does Not Exist Extracellularly

2018 ◽  
Vol 2018 ◽  
pp. 1-7 ◽  
Author(s):  
Olga Ilinskaya ◽  
Vera Ulyanova ◽  
Irina Lisevich ◽  
Elena Dudkina ◽  
Nataliya Zakharchenko ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Protein Concentration ◽  
Bacillus Pumilus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Culture Fluid ◽  
Size Exclusion ◽  
High Protein Concentration ◽  
Exclusion Chromatography ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Hypochromic Effect

Supported by crystallography studies, secreted ribonuclease of Bacillus pumilus (binase) has long been considered to be monomeric in form. Recent evidence obtained using native polyacrylamide gel electrophoresis and size-exclusion chromatography suggests that binase is in fact dimeric. To eliminate ambiguity and contradictions in the data we have measured conformational changes, hypochromic effect, and hydrodynamic radius of binase. The immutability of binase secondary structure upon transition from low to high protein concentration was registered, suggesting the binase dimerization immediately after translocation through the cell membrane and leading to detection of binase dimers only in the culture fluid regardless of ribonuclease concentration. Our results made it necessary to take a fresh look at the binase stability and cytotoxicity towards virus-infected or tumor cells.

scholarly journals Identity and Substrate Specificity of Reductive Dehalogenases Expressed in Dehalococcoides-Containing Enrichment Cultures Maintained on Different Chlorinated Ethenes

2015 ◽  
Vol 81 (14) ◽  
pp. 4626-4633 ◽  
Author(s):  
Xiaoming Liang ◽  
Olivia Molenda ◽  
Shuiquan Tang ◽  
Elizabeth A. Edwards
Keyword(s):  
Specific Activity ◽  
Enrichment Culture ◽  
Polyacrylamide Gel Electrophoresis ◽  
Peptide Sequencing ◽  
Enzyme Assays ◽  
Content Type ◽  
Substrate Preferences ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
History Of ◽  
Different Strains

ABSTRACTMany reductive dehalogenases (RDases) have been identified in organohalide-respiring microorganisms, and yet their substrates, specific activities, and conditions for expression are not well understood. We tested whether RDase expression varied depending on the substrate-exposure history of reductive dechlorinating communities. For this purpose, we used the enrichment culture KB-1 maintained on trichloroethene (TCE), as well as subcultures maintained on the intermediatescis-dichloroethene (cDCE) and vinyl chloride (VC). KB-1 contains a TCE-to-cDCE dechlorinatingGeobacterand severalDehalococcoidesstrains that together harbor many of the known chloroethene reductases. Expressed RDases were identified using blue native polyacrylamide gel electrophoresis, enzyme assays in gel slices, and peptide sequencing. As anticipated but never previously quantified, the RDase fromGeobacterwas only detected transiently at the beginning of TCE dechlorination. TheDehalococcoidesRDase VcrA and smaller amounts of TceA were expressed in the parent KB-1 culture during complete dechlorination of TCE to ethene regardless of time point or amended substrate. TheDehalococcoidesRDase BvcA was only detected in enrichments maintained on cDCE as growth substrates, in roughly equal abundance to VcrA. Only VcrA was detected in subcultures enriched on VC. Enzyme assays revealed that 1,1-DCE, a substrate not used for culture enrichment, afforded the highest specific activity.trans-DCE was substantially dechlorinated only by extracts from cDCE enrichments expressing BvcA. RDase gene distribution indicated enrichment of different strains ofDehalococcoidesas a function of electron acceptor TCE, cDCE, or VC. Each chloroethene reductase has distinct substrate preferences leading to strain selection in mixed communities.

scholarly journals Role of Campylobacter jejuni Respiratory Oxidases and Reductases in Host Colonization

2008 ◽  
Vol 74 (5) ◽  
pp. 1367-1375 ◽  
Author(s):  
Rebecca A. Weingarten ◽  
Jesse L. Grimes ◽  
Jonathan W. Olson
Keyword(s):  
Campylobacter Jejuni ◽  
Anaerobic Respiration ◽  
Wild Type ◽  
Respiratory Enzymes ◽  
Host Colonization ◽  
Enzymatic Function ◽  
Food Borne ◽  
Transport Chain ◽  
Respiratory Oxidases

ABSTRACT Campylobacter jejuni is the leading cause of human food-borne bacterial gastroenteritis. The C. jejuni genome sequence predicts a branched electron transport chain capable of utilizing multiple electron acceptors. Mutants were constructed by disrupting the coding regions of the respiratory enzymes nitrate reductase (napA::Cm), nitrite reductase (nrfA::Cm), dimethyl sulfoxide, and trimethylamine N-oxide reductase (termed Cj0264::Cm) and the two terminal oxidases, a cyanide-insensitive oxidase (cydA::Cm) and cbb3-type oxidase (ccoN::Cm). Each strain was characterized for the loss of the associated enzymatic function in vitro. The strains were then inoculated into 1-week-old chicks, and the cecal contents were assayed for the presence of C. jejuni 2 weeks postinoculation. cydA::Cm and Cj0264c::Cm strains colonized as well as the wild type; napA::Cm and nrfA::Cm strains colonized at levels significantly lower than the wild type. The ccoN::Cm strain was unable to colonize the chicken; no colonies were recovered at the end of the experiment. While there appears to be a role for anaerobic respiration in host colonization, oxygen is the most important respiratory acceptor for C. jejuni in the chicken cecum.

scholarly journals Draft Genome Sequence of the Enteropathogenic Bacterium Campylobacter jejuni Strain cj255

2015 ◽  
Vol 3 (5) ◽  
Author(s):  
Fariha Masood Siddiqui ◽  
Muhammad Ibrahim ◽  
Nighat Noureen ◽  
Zobia Noreen ◽  
Richard W. Titball ◽  
...  
Keyword(s):  
Global Health ◽  
Host Range ◽  
Campylobacter Jejuni ◽  
Genome Sequence ◽  
Draft Genome ◽  
Epidemiological Studies ◽  
Draft Genome Sequence ◽  
Broad Host Range ◽  
Bacterial Gastroenteritis

The enteropathogen Campylobacter jejuni is a global health disaster, being one of the leading causes of bacterial gastroenteritis. Here, we present the draft genome sequence of C. jejuni strain cj255, isolated from a chicken source in Islamabad, Pakistan. The draft genome sequence will aid in epidemiological studies and quarantine of this broad-host-range pathogen.

Partial purification and characterisation of α-amylases from the digestive tract of the Indian major carp Labeo rohita (Hamilton, 1822)

2020 ◽  
Vol 67 (2) ◽  
Author(s):  
Umalatha . ◽  
N. Sridhar ◽  
Jairam Prasad Kushwaha ◽  
Vadlapudi Kumar
Keyword(s):  
Digestive Tract ◽  
Incubation Temperature ◽  
Amylase Activity ◽  
Labeo Rohita ◽  
Polyacrylamide Gel Electrophoresis ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Indian Major Carp ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Acetone Fractionation

Partial purification of α-amylases from the digestive tract of the Indian major carp Labeo rohita (Hamilton, 1822) through acetone fractionation and ion exchange chromatography (DEAE-SephadexA-50) resulted in 8-fold purification with 86% recovery. Characterisation of amylase activity revealed two pH optima at 4.5 and 6.5. Activity was stable over wide pH ranges of 3.5 to 4.5 and 7 to 12. Optimum incubation temperature was 35°C. The enzyme lost 91% activity at 60°C within 15 min and was inhibited by Amylase inhibitor Type-1 (wheat); 1, 10 Phenanthroline, Ethylene diamine tetra-acetate (EDTA) and Phenyl methyl sulphonyl fluoride (PMSF). Heavy metal ions Hg++ and Cu++ strongly inhibited the enzyme activity, while Zn++ and Bi++ inhibited to a lesser extent. Native polyacrylamide gel electrophoresis of the purified amylase fractions revealed four bands, with corresponding molecular weights of 43.59, 52.36, 55.42 and 54.01 kDa. α-Amylase activity from L. rohita exhibited linear hydrolysis of starch upto 7% concentration in 60 min.

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