scholarly journals Identification of a Third Mn(II) Oxidase Enzyme in Pseudomonas putida GB-1

2016 ◽  
Vol 82 (13) ◽  
pp. 3774-3782 ◽  
Author(s):  
Kati Geszvain ◽  
Logan Smesrud ◽  
Bradley M. Tebo
Keyword(s):  
Pseudomonas Putida ◽  
Oxidase Activity ◽  
Sequence Similarity ◽  
Preliminary Investigation ◽  
Model Organism ◽  
Multicopper Oxidase ◽  
Content Type ◽  
Regulatory Pathways ◽  
Heme Peroxidase ◽  
Oxidase Enzyme

ABSTRACTThe oxidation of soluble Mn(II) to insoluble Mn(IV) is a widespread bacterial activity found in a diverse array of microbes. In the Mn(II)-oxidizing bacteriumPseudomonas putidaGB-1, two Mn(II) oxidase genes, namedmnxGandmcoA, were previously identified; each encodes a multicopper oxidase (MCO)-type enzyme. Expression of these two genes is positively regulated by the response regulator MnxR. Preliminary investigation into putative additional regulatory pathways suggested that the flagellar regulators FleN and FleQ also regulate Mn(II) oxidase activity; however, it also revealed the presence of a third, previously uncharacterized Mn(II) oxidase activity inP. putidaGB-1. A strain from which both of the Mn(II) oxidase genes andfleQwere deleted exhibited low levels of Mn(II) oxidase activity. The enzyme responsible was genetically and biochemically identified as an animal heme peroxidase (AHP) with domain and sequence similarity to the previously identified Mn(II) oxidase MopA. In the ΔfleQstrain,P. putidaGB-1 MopA is overexpressed and secreted from the cell, where it actively oxidizes Mn. Thus, deletion offleQunmasked a third Mn(II) oxidase activity in this strain. These results provide an example of an Mn(II)-oxidizing bacterium utilizing both MCO and AHP enzymes.IMPORTANCEThe identity of the Mn(II) oxidase enzyme inPseudomonas putidaGB-1 has been a long-standing question in the field of bacterial Mn(II) oxidation. In the current work, we demonstrate thatP. putidaGB-1 employs both the multicopper oxidase- and animal heme peroxidase-mediated pathways for the oxidation of Mn(II), rendering this model organism relevant to the study of both types of Mn(II) oxidase enzymes. The presence of three oxidase enzymes inP. putidaGB-1 deepens the mystery of why microorganisms oxidize Mn(II) while providing the field with the tools necessary to address this question. The initial identification of MopA as a Mn(II) oxidase in this strain required the deletion of FleQ, a regulator involved in both flagellum synthesis and biofilm synthesis inPseudomonas aeruginosa. Therefore, these results are also an important step toward understanding the regulation of Mn(II) oxidation.

scholarly journals Elimination of Manganese(II,III) Oxidation in Pseudomonas putida GB-1 by a Double Knockout of Two Putative Multicopper Oxidase Genes

2012 ◽  
Vol 79 (1) ◽  
pp. 357-366 ◽  
Author(s):  
Kati Geszvain ◽  
James K. McCarthy ◽  
Bradley M. Tebo
Keyword(s):  
Pseudomonas Putida ◽  
Multicopper Oxidase ◽  
Genome Comparison ◽  
Double Knockout ◽  
Oxidase Gene ◽  
Content Type ◽  
Heme Peroxidase ◽  
A Genome ◽  
Oxidation Phenotype ◽  
Encoding Genes

ABSTRACTBacterial manganese(II) oxidation impacts the redox cycling of Mn, other elements, and compounds in the environment; therefore, it is important to understand the mechanisms of and enzymes responsible for Mn(II) oxidation. In several Mn(II)-oxidizing organisms, the identified Mn(II) oxidase belongs to either the multicopper oxidase (MCO) or the heme peroxidase family of proteins. However, the identity of the oxidase inPseudomonas putidaGB-1 has long remained unknown. To identify theP. putidaGB-1 oxidase, we searched its genome and found several homologues of known or suspected Mn(II) oxidase-encoding genes (mnxG,mofA,moxA, andmopA). To narrow this list, we assumed that the Mn(II) oxidase gene would be conserved among Mn(II)-oxidizing pseudomonads but not in nonoxidizers and performed a genome comparison to 11Pseudomonasspecies. We further assumed that the oxidase gene would be regulated by MnxR, a transcription factor required for Mn(II) oxidation. Two loci met all these criteria: PputGB1_2447, which encodes an MCO homologous to MnxG, and PputGB1_2665, which encodes an MCO with very low homology to MofA. In-frame deletions of each locus resulted in strains that retained some ability to oxidize Mn(II) or Mn(III); loss of oxidation was attained only upon deletion of both genes. These results suggest that PputGB1_2447 and PputGB1_2665 encode two MCOs that are independently capable of oxidizing both Mn(II) and Mn(III). The purpose of this redundancy is unclear; however, differences in oxidation phenotype for the single mutants suggest specialization in function for the two enzymes.

scholarly journals Identification of a PRC2 Accessory Subunit Required for Subtelomeric H3K27 Methylation in Neurospora crassa

2020 ◽  
Vol 40 (11) ◽  
Author(s):  
Kevin J. McNaught ◽  
Elizabeth T. Wiles ◽  
Eric U. Selker
Keyword(s):  
Neurospora Crassa ◽  
Transcriptional Repression ◽  
Sequence Similarity ◽  
Model Organism ◽  
Content Type ◽  
Polycomb Repressive Complex 2 ◽  
H3k27 Methylation ◽  
Accessory Subunit ◽  
Genomic Regions ◽  
Similarity Searches

ABSTRACT Polycomb repressive complex 2 (PRC2) catalyzes methylation of histone H3 at lysine 27 (H3K27) in genomic regions of most eukaryotes and is critical for maintenance of the associated transcriptional repression. However, the mechanisms that shape the distribution of H3K27 methylation, such as recruitment of PRC2 to chromatin and/or stimulation of PRC2 activity, are unclear. Here, using a forward genetic approach in the model organism Neurospora crassa, we identified two alleles of a gene, NCU04278, encoding an unknown PRC2 accessory subunit (PAS). Loss of PAS resulted in losses of H3K27 methylation concentrated near the chromosome ends and derepression of a subset of associated subtelomeric genes. Immunoprecipitation followed by mass spectrometry confirmed reciprocal interactions between PAS and known PRC2 subunits, and sequence similarity searches demonstrated that PAS is not unique to N. crassa. PAS homologs likely influence the distribution of H3K27 methylation and underlying gene repression in a variety of fungal lineages.

scholarly journals Identification and Characterization of a NaCl-Responsive Genetic Locus Involved in Survival during Desiccation in Sinorhizobium meliloti

2013 ◽  
Vol 79 (18) ◽  
pp. 5693-5700 ◽  
Author(s):  
Jan A. C. Vriezen ◽  
Frans J. de Bruijn ◽  
Klaus Nüsslein
Keyword(s):  
Sinorhizobium Meliloti ◽  
Agricultural Soils ◽  
Sequence Similarity ◽  
Genetic Locus ◽  
Model Organism ◽  
Normal Growth ◽  
Growth Conditions ◽  
Amino Acid Sequence Similarity ◽  
Content Type ◽  
Genetic Mechanisms

ABSTRACTTheRhizobiaceaeare a bacterial family of enormous agricultural importance due to the ability of its members to fix atmospheric nitrogen in an intimate relationship with plants. Their survival as naturally occurring soil bacteria in agricultural soils as well as popular seed inocula is affected directly by drought and salinity. Survival after desiccation in the presence of NaCl is enabled by underlying genetic mechanisms in the model organismSinorhizobium meliloti1021. Since salt stress parallels a loss in water activity, the identification of NaCl-responsive loci may identify loci involved in survival during desiccation. This approach enabled identification of the lociasnOandnggby their reduced ability to grow on increased NaCl concentrations, likely due to their inability to produce the osmoprotectant N-acetylglutaminylglutamine (NAGGN). In addition, the mutant harboringngg::Tn5luxABwas affected in its ability to survive desiccation and responded to osmotic stress. The desiccation sensitivity may have been due to secondary functions of Ngg (N-acetylglutaminylglutamine synthetase)-like cell wall metabolism as suggested by the presence of ad-alanine-d-alanine ligase (dAla-dAla) domain and by sensitivity of the mutant to β-lactam antibiotics.asnO::Tn5luxABis expressed during the stationary phase under normal growth conditions. Amino acid sequence similarity to enzymes producing β-lactam inhibitors and increased resistance to β-lactam antibiotics may indicate thatasnOis involved in the production of a β-lactam inhibitor.

scholarly journals Heterologous Expression and Characterization of the Manganese-Oxidizing Protein from Erythrobacter sp. Strain SD21

2014 ◽  
Vol 80 (21) ◽  
pp. 6837-6842 ◽  
Author(s):  
Katherine Nakama ◽  
Michael Medina ◽  
Ahn Lien ◽  
Jordan Ruggieri ◽  
Krystle Collins ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Sequence Similarity ◽  
Cell Extract ◽  
Binding Domain ◽  
Full Length ◽  
Truncated Protein ◽  
Content Type ◽  
Heme Peroxidase ◽  
Calcium Binding Domain

ABSTRACTThe manganese (Mn)-oxidizing protein (MopA) fromErythrobactersp. strain SD21 is part of a unique enzymatic family that is capable of oxidizing soluble Mn(II). This enzyme contains two domains, an animal heme peroxidase domain, which contains the catalytic site, followed by a C-terminal calcium binding domain. Different from the bacterial Mn-oxidizing multicopper oxidase enzymes, little is known about MopA. To gain a better understanding of MopA and its role in Mn(II) oxidation, the 238-kDa full-length protein and a 105-kDa truncated protein containing only the animal heme peroxidase domain were cloned and heterologously expressed inEscherichia coli. Despite having sequence similarity to a peroxidase, hydrogen peroxide did not stimulate activity, nor was activity significantly decreased in the presence of catalase. Both pyrroloquinoline quinone (PQQ) and hemin increased Mn-oxidizing activity, and calcium was required. TheKmfor Mn(II) of the full-length protein in cell extract was similar to that of the natively expressed protein, but theKmvalue for the truncated protein in cell extract was approximately 6-fold higher than that of the full-length protein, suggesting that the calcium binding domain may aid in binding Mn(II). Characterization of the heterologously expressed MopA has provided additional insight into the mechanism of bacterial Mn(II) oxidation, which will aid in understanding the role of MopA and Mn oxidation in bioremediation and biogeochemical cycling.

scholarly journals Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

2012 ◽  
Vol 78 (15) ◽  
pp. 5238-5246 ◽  
Author(s):  
Dongfei Han ◽  
Ji-Young Ryu ◽  
Robert A. Kanaly ◽  
Hor-Gil Hur
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Hypothetical Protein ◽  
Aldehyde Group ◽  
Agrobacterium Vitis ◽  
T7 Promoter ◽  
Content Type ◽  
E Coli ◽  
Cell Extracts ◽  
Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

scholarly journals Phyllobacterium loti sp. nov. isolated from nodules of Lotus corniculatus

2014 ◽  
Vol 64 (Pt_3) ◽  
pp. 781-786 ◽  
Author(s):  
Maximo Sánchez ◽  
Martha-Helena Ramírez-Bahena ◽  
Alvaro Peix ◽  
María J. Lorite ◽  
Juan Sanjuán ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Sequence Similarity ◽  
Carbon Sources ◽  
Lotus Corniculatus ◽  
Culture Conditions ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
The 16S Rrna Gene

Strain S658T was isolated from a Lotus corniculatus nodule in a soil sample obtained in Uruguay. Phylogenetic analysis of the 16S rRNA gene and atpD gene showed that this strain clustered within the genus Phyllobacterium . The closest related species was, in both cases, Phyllobacterium trifolii PETP02T with 99.8 % sequence similarity in the 16S rRNA gene and 96.1 % in the atpD gene. The 16S rRNA gene contains an insert at the beginning of the sequence that has no similarities with other inserts present in the same gene in described rhizobial species. Ubiquinone Q-10 was the only quinone detected. Strain S658T differed from its closest relatives through its growth in diverse culture conditions and in the assimilation of several carbon sources. It was not able to reproduce nodules in Lotus corniculatus. The results of DNA–DNA hybridization, phenotypic tests and fatty acid analyses confirmed that this strain should be classified as a representative of a novel species of the genus Phyllobacterium , for which the name Phyllobacterium loti sp. nov. is proposed. The type strain is S658T( = LMG 27289T = CECT 8230T).

scholarly journals 24-Epibrasinolide Modulates the Vase Life of Lisianthus Cut Flowers by Modulating ACC Oxidase Enzyme Activity and Physiological Responses

Plants ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 995
Author(s):  
Mohammad Darvish ◽  
Habib Shirzad ◽  
Mohammadreza Asghari ◽  
Parviz Noruzi ◽  
Abolfazl Alirezalu ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Water Absorption ◽  
Ethylene Production ◽  
Oxidase Activity ◽  
Acc Oxidase ◽  
Postharvest Quality ◽  
Vase Life ◽  
Dependent Manner ◽  
Cut Flowers ◽  
Oxidase Enzyme

Ethylene is the most important factor playing roles in senescence and deterioration of harvested crops including cut flowers. Brassinosteroids (BRs), as natural phytohormones, have been reported to differently modulate ethylene production and related senescence processes in different crops. This study was carried out to determine the effects of different levels of 24-epibrassinolide (EBL) on ACC oxidase enzyme activity, the final enzyme in ethylene biosynthesis pathway, vase life, and senescence rate in lisianthus cut flowers. Harvested flowers were treated with EBL (at 0, 3, 6, and 9 µmol/L) and kept at 25 °C for 15 days. The ACC oxidase activity, water absorption, malondialdehyde (MDA) production and vase solution absorption rates, chlorophyll and anthocyanin contents, and the vase life of the flowers were evaluated during and at the end of storage. EBL at 3 µmol/L significantly (p ≤ 0.01) enhanced the flower vase life by decreasing the ACC oxidase activity, MDA production and senescence rates, and enhancing chlorophyll and anthocyanin biosynthesis and accumulation, relative water content, and vase solution absorption rates. By increasing the concentration, EBL negatively affected the flower vase life and postharvest quality probably via enhancing the ACC oxidase enzyme activity and subsequent ethylene production. EBL at 6 and 9 µmol/L and in a concentration dependent manner, enhanced the ACC oxidase activity and MDA production rate and decreased chlorophyll and anthocyanin accumulation and water absorption rate. The results indicate that the effects of brassinosteroids on ethylene production and physiology of lisianthus cut flowers is highly dose dependent.

scholarly journals Faecalicoccus acidiformans gen. nov., sp. nov., isolated from the chicken caecum, and reclassification of Streptococcus pleomorphus (Barnes et al. 1977), Eubacterium biforme (Eggerth 1935) and Eubacterium cylindroides (Cato et al. 1974) as Faecalicoccus pleomorphus comb. nov., Holdemanella biformis gen. nov., comb. nov. and Faecalitalea cylindroides gen. nov., comb. nov., respectively, within the family Erysipelotrichaceae

2014 ◽  
Vol 64 (Pt_11) ◽  
pp. 3877-3884 ◽  
Author(s):  
Celine De Maesschalck ◽  
Filip Van Immerseel ◽  
Venessa Eeckhaut ◽  
Siegrid De Baere ◽  
Margo Cnockaert ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Species ◽  
Gene Sequence ◽  
Sequence Similarity ◽  
16S Rrna Gene Sequence ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Content Type ◽  
Link Type

Strains LMG 27428T and LMG 27427 were isolated from the caecal content of a chicken and produced butyric, lactic and formic acids as major metabolic end products. The genomic DNA G+C contents of strains LMG 27428T and LMG 27427 were 40.4 and 38.8 mol%. On the basis of 16S rRNA gene sequence similarity, both strains were most closely related to the generically misclassified Streptococcus pleomorphus ATCC 29734T. Strain LMG 27428T could be distinguished from S. pleomorphus ATCC 29734T based on production of more lactic acid and less formic acid in M2GSC medium, a higher DNA G+C content and the absence of activities of acid phosphatase and leucine, arginine, leucyl glycine, pyroglutamic acid, glycine and histidine arylamidases, while strain LMG 27428 was biochemically indistinguishable from S. pleomorphus ATCC 29734T. The novel genus Faecalicoccus gen. nov. within the family Erysipelotrichaceae is proposed to accommodate strains LMG 27428T and LMG 27427. Strain LMG 27428T ( = DSM 26963T) is the type strain of Faecalicoccus acidiformans sp. nov., and strain LMG 27427 ( = DSM 26962) is a strain of Faecalicoccus pleomorphus comb. nov. (type strain LMG 17756T = ATCC 29734T = DSM 20574T). Furthermore, the nearest phylogenetic neighbours of the genus Faecalicoccus are the generically misclassified Eubacterium cylindroides DSM 3983T (94.4 % 16S rRNA gene sequence similarity to strain LMG 27428T) and Eubacterium biforme DSM 3989T (92.7 % 16S rRNA gene sequence similarity to strain LMG 27428T). We present genotypic and phenotypic data that allow the differentiation of each of these taxa and propose to reclassify these generically misnamed species of the genus Eubacterium formally as Faecalitalea cylindroides gen. nov., comb. nov. and Holdemanella biformis gen. nov., comb. nov., respectively. The type strain of Faecalitalea cylindroides is DSM 3983T = ATCC 27803T = JCM 10261T and that of Holdemanella biformis is DSM 3989T = ATCC 27806T = CCUG 28091T.

scholarly journals Relationship of patient shame to working alliance and satisfaction: a preliminary investigation

2019 ◽  
Vol 14 (4) ◽  
pp. 251-263
Author(s):  
Daniel J. Carabellese ◽  
Michael J. Proeve ◽  
Rachel M. Roberts
Keyword(s):  
Patient Satisfaction ◽  
Working Alliance ◽  
Preliminary Investigation ◽  
Community Members ◽  
Content Type ◽  
Mediation Analyses ◽  
External Shame ◽  
Relationship Of ◽  
The Relationship

Purpose The purpose of this paper is to explore the relationship of two distinct variants of dispositional shame (internal and external shame) with collaborative, purpose-driven aspects of the patient–provider relationship (working alliance) and patient satisfaction. The aim of this research was to conduct a preliminary investigation into the relevance of dispositional shame in a general healthcare population. Design/methodology/approach In total, 127 community members (mean age 25.9 years) who reported that they had regularly seen a GP over the past year were recruited at an Australian university. Participants were asked to reflect on their relationship with their GP, and completed instruments assessing various domains of shame, as well as working alliance and patient satisfaction. Findings Non-parametric correlations were examined to determine the direction and strength of relationships, as well as conducting mediation analyses where applicable. Small, negative correlations were evident between external shame and working alliance. Both external and internal shame measures were also negatively correlated with patient satisfaction. Finally, the relationship of external shame to patient satisfaction was partially mediated by working alliance. Practical implications Both the reported quality of patient–provider working alliance, and level of patient satisfaction are related to levels of dispositional shame in patients, and working alliance may act as a mediator for this relationship. Originality/value The findings from this preliminary study suggest that internal and external shame are important factors to consider in the provision of medical care to maximise the quality of patient experience and working alliance.

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