scholarly journals Molybdenum-Containing Nicotine Hydroxylase Genes in a Nicotine Degradation Pathway That Is a Variant of the Pyridine and Pyrrolidine Pathways

2015 ◽  
Vol 81 (24) ◽  
pp. 8330-8338 ◽  
Author(s):  
Hao Yu ◽  
Hongzhi Tang ◽  
Yangyang Li ◽  
Ping Xu
Keyword(s):  
Inductively Coupled Plasma ◽  
Large Subunit ◽  
Sole Source ◽  
Small Subunit ◽  
Binding Domain ◽  
Mass Spectrometry Analysis ◽  
Resting Cells ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Nicotine Degradation

ABSTRACTOchrobactrumsp. strain SJY1 utilizes nicotine as a sole source of carbon, nitrogen, and energy via a variant of the pyridine and pyrrolidine pathways (the VPP pathway). Several strains and genes involved in the VPP pathway have recently been reported; however, the first catalyzing step for enzymatic turnover of nicotine is still unclear. In this study, a nicotine hydroxylase for the initial hydroxylation step of nicotine degradation was identified and characterized. The nicotine hydroxylase (VppA), which converts nicotine to 6-hydroxynicotine in the strain SJY1, is encoded by two open reading frames (vppASandvppAL[subunits S and L, respectively]). ThevppAgenes were heterologously expressed in the non-nicotine-degrading strainsEscherichia coliDH5α andPseudomonas putidaKT2440; only thePseudomonasstrain acquired the ability to degrade nicotine. The small subunit of VppA contained a [2Fe-2S] cluster-binding domain, and the large subunit of VppA contained a molybdenum cofactor-binding domain; however, an FAD-binding domain was not found in VppA. Resting cells cultivated in a molybdenum-deficient medium had low nicotine transformation activity, and excess molybdenum was detected in the purified VppA by inductively coupled plasma-mass spectrometry analysis. Thus, it is demonstrated that VppA is a two-component molybdenum-containing hydroxylase.

scholarly journals Haloalkylphosphorus Hydrolases Purified from Sphingomonas sp. Strain TDK1 and Sphingobium sp. Strain TCM1

2014 ◽  
Vol 80 (18) ◽  
pp. 5866-5873 ◽  
Author(s):  
Katsumasa Abe ◽  
Satoshi Yoshida ◽  
Yuto Suzuki ◽  
Junichi Mori ◽  
Yuka Doi ◽  
...  
Keyword(s):  
Inductively Coupled Plasma ◽  
Divalent Cations ◽  
Sequence Similarity ◽  
Mass Spectrometry Analysis ◽  
Amino Acid Residues ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Inductively Coupled ◽  
Trialkyl Phosphates ◽  
Monomeric Proteins

ABSTRACTPhosphotriesterases catalyze the first step of organophosphorus triester degradation. The bacterial phosphotriesterases purified and characterized to date hydrolyze mainly aryl dialkyl phosphates, such as parathion, paraoxon, and chlorpyrifos. In this study, we purified and cloned two novel phosphotriesterases fromSphingomonassp. strain TDK1 andSphingobiumsp. strain TCM1 that hydrolyze tri(haloalkyl)phosphates, and we named these enzymes haloalkylphosphorus hydrolases (TDK-HAD and TCM-HAD, respectively). Both HADs are monomeric proteins with molecular masses of 59.6 (TDK-HAD) and 58.4 kDa (TCM-HAD). The enzyme activities were affected by the addition of divalent cations, and inductively coupled plasma mass spectrometry analysis suggested that zinc is a native cofactor for HADs. These enzymes hydrolyzed not only chlorinated organophosphates but also a brominated organophosphate [tris(2,3-dibromopropyl) phosphate], as well as triaryl phosphates (tricresyl and triphenyl phosphates). Paraoxon-methyl and paraoxon were efficiently degraded by TCM-HAD, whereas TDK-HAD showed weak activity toward these substrates. Dichlorvos was degraded only by TCM-HAD. The enzymes displayed weak or no activity against trialkyl phosphates and organophosphorothioates. The TCM-HAD and TDK-HAD genes were cloned and found to encode proteins of 583 and 574 amino acid residues, respectively. The primary structures of TCM-HAD and TDK-HAD were very similar, and the enzymes also shared sequence similarity with fenitrothion hydrolase (FedA) ofBurkholderiasp. strain NF100 and organophosphorus hydrolase (OphB) ofBurkholderiasp. strain JBA3. However, the substrate specificities and quaternary structures of the HADs were largely different from those of FedA and OphB. These results show that HADs from sphingomonads are novel members of the bacterial phosphotriesterase family.

scholarly journals Molecular Diversity of the Syndinean Genus Euduboscquella Based on Single-Cell PCR Analysis

2011 ◽  
Vol 78 (2) ◽  
pp. 334-345 ◽  
Author(s):  
Tsvetan R. Bachvaroff ◽  
Sunju Kim ◽  
Laure Guillou ◽  
Charles F. Delwiche ◽  
D. Wayne Coats
Keyword(s):  
Large Subunit ◽  
Small Subunit ◽  
Base Change ◽  
Pcr Analysis ◽  
Ssu Rrna ◽  
Compensatory Base Change ◽  
Content Type ◽  
Single Cell Pcr ◽  
Group I ◽  
Lsu Rrna

ABSTRACTThe genusEuduboscquellais one of a few described genera within the syndinean dinoflagellates, an enigmatic lineage with abundant diversity in marine environmental clone libraries based on small subunit (SSU) rRNA. The region composed of the SSU through to the partial large subunit (LSU) rRNA was determined from 40 individual tintinnid ciliate loricae infected withEuduboscquellasampled from eight surface water sites in the Northern Hemisphere, producing seven distinct SSU sequences. The corresponding host SSU rRNA region was also amplified from eight host species. The SSU tree ofEuduboscquellaand syndinean group I sequences from environmental clones had seven well-supported clades and one poorly supported clade across data sets from 57 to 692 total sequences. The genusEuduboscquellaconsistently formed a supported monophyletic clade within a single subclade of group I sequences. For most parasites with identical SSU sequences, the more variable internal transcribed spacer (ITS) to LSU rRNA regions were polymorphic at 3 to 10 sites. However, inE. cachonithere was variation between ITS to LSU copies at up to 20 sites within an individual, while in a parasite ofTintinnopsisspp., variation between different individuals ranged up to 19 polymorphic sites. However, applying the compensatory base change model to the ITS2 sequences suggested no compensatory changes within or between individuals with the same SSU sequence, while one to four compensatory changes between individuals with similar but not identical SSU sequences were found. Comparisons between host and parasite phylogenies do not suggest a simple pattern of host or parasite specificity.

scholarly journals Thiol Peroxidase Is an Important Component of Streptococcus pneumoniae in Oxygenated Environments

2012 ◽  
Vol 80 (12) ◽  
pp. 4333-4343 ◽  
Author(s):  
Barak Hajaj ◽  
Hasan Yesilkaya ◽  
Rachel Benisty ◽  
Maayan David ◽  
Peter W. Andrew ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Mass Spectrometry Analysis ◽  
Activity Levels ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Cellular Processes ◽  
Thiol Peroxidase ◽  
Fine Tune

ABSTRACTStreptococcus pneumoniaeis an aerotolerant Gram-positive bacterium that causes an array of diseases, including pneumonia, otitis media, and meningitis. During aerobic growth,S. pneumoniaeproduces high levels of H2O2. SinceS. pneumoniaelacks catalase, the question of how it controls H2O2levels is of critical importance. Thepsalocus encodes an ABC Mn2+-permease complex (psaBCA) and a putative thiol peroxidase,tpxD. This study shows thattpxDencodes a functional thiol peroxidase involved in the adjustment of H2O2homeostasis in the cell. Kinetic experiments showed that recombinant TpxD removed H2O2efficiently. However,in vivoexperiments revealed that TpxD detoxifies only a fraction of the H2O2generated by the pneumococcus. Mass spectrometry analysis demonstrated that TpxD Cys58undergoes selective oxidationin vivo, under conditions where H2O2is formed, confirming the thiol peroxidase activity. Levels of TpxD expression and synthesisin vitrowere significantly increased in cells grown under aerobic versus anaerobic conditions. The challenge with D39 and TIGR4 with H2O2resulted intpxDupregulation, whilepsaBCAexpression was oppositely affected. However, the challenge of ΔtpxDmutants with H2O2did not affectpsaBCA, implying that TpxD is involved in the regulation of thepsaoperon, in addition to its scavenging activity. Virulence studies demonstrated a notable difference in the survival time of mice infected intranasally with D39 compared to that of mice infected intranasally with D39ΔtpxD. However, when bacteria were administered directly into the blood, this difference disappeared. The findings of this study suggest that TpxD constitutes a component of the organism's fundamental strategy to fine-tune cellular processes in response to H2O2.

scholarly journals Characterization of the 3-O-Methylgallate Dioxygenase Gene and Evidence of Multiple 3-O-Methylgallate Catabolic Pathways in Sphingomonas paucimobilis SYK-6

2004 ◽  
Vol 186 (15) ◽  
pp. 4951-4959 ◽  
Author(s):  
Daisuke Kasai ◽  
Eiji Masai ◽  
Keisuke Miyauchi ◽  
Yoshihiro Katayama ◽  
Masao Fukuda
Keyword(s):  
Sole Source ◽  
Cell Extract ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Insertion Mutant ◽  
Sphingomonas Paucimobilis ◽  
Spectrometry Analysis ◽  
Dioxygenase Gene ◽  
Transformation Activity ◽  
Extradiol Dioxygenases

ABSTRACT Sphingomonas paucimobilis SYK-6 is able to grow on various lignin-derived biaryls as the sole source of carbon and energy. These compounds are degraded to vanillate and syringate by the unique and specific enzymes in this strain. Vanillate and syringate are converted to protocatechuate (PCA) and 3-O-methylgallate (3MGA), respectively, by the tetrahydrofolate-dependent O-demethylases. Previous studies have suggested that these compounds are further degraded via the PCA 4,5-cleavage pathway. However, our subsequent analysis of the ligB insertion mutant, which encodes the β subunit of PCA 4,5-dioxygenase, suggested that at least one alternative route is involved in 3MGA degradation. In the present study, we isolated the desZ gene, which confers 3MGA degradation activity on Escherichia coli. The deduced amino acid sequence of desZ showed ca. 20 to 43% identity with the type II extradiol dioxygenases. Gas chromatography-mass spectrometry analysis suggested that DesZ catalyzes the 3,4-cleavage of 3MGA. Disruption of both desZ and ligB in SYK-6 resulted in loss of the dioxygen-dependent 3MGA transformation activity, but the resulting mutant retained the ability to grow on syringate. We found that the cell extract of the desZ ligB double mutant was able to convert 3MGA to gallate when tetrahydrofolate was added to the reaction mixture, and the cell extract of this mutant degraded gallate to the same degree as the wild type did. All these results suggest that syringate is degraded through multiple 3MGA degradation pathways in which ligAB, desZ, 3MGA O-demethylase, and gallate dioxygenase are participants.

scholarly journals Cloning of a Novel Nicotine Oxidase Gene from Pseudomonas sp. Strain HZN6 Whose Product Nonenantioselectively Degrades Nicotine to Pseudooxynicotine

2013 ◽  
Vol 79 (7) ◽  
pp. 2164-2171 ◽  
Author(s):  
Jiguo Qiu ◽  
Yun Ma ◽  
Jing Zhang ◽  
Yuezhong Wen ◽  
Weiping Liu
Keyword(s):  
Amine Oxidase ◽  
Sole Source ◽  
Site Directed Mutagenesis ◽  
Broad Host Range ◽  
Upstream Sequence ◽  
Oxidase Gene ◽  
Content Type ◽  
Reading Frame ◽  
Degrading Bacteria ◽  
Nicotine Degradation

ABSTRACTPseudomonassp. strain HZN6 utilizes nicotine as its sole source of carbon, nitrogen, and energy. However, its catabolic mechanism has not been elucidated. In this study, self-formed adaptor PCR was performed to amplify the upstream sequence of the pseudooxynicotine amine oxidase gene. A 1,437-bp open reading frame (designatednox) was found to encode a nicotine oxidase (NOX) that shows 30% amino acid sequence identity with 6-hydroxy-l-nicotine oxidase fromArthrobacter nicotinovorans. Thenoxgene was cloned into a broad-host-range cloning vector and transferred into the non-nicotine-degrading bacteriaEscherichia coliDH5α (DH-nox) andPseudomonas putidaKT2440 (KT-nox). The transconjugant KT-nox obtained nicotine degradation ability and yielded an equimolar amount of pseudooxynicotine, while DH-nox did not. Reverse transcription-PCR showed that thenoxgene is expressed in both DH5α and KT2440, suggesting that additional factors required for nicotine degradation are present in aPseudomonasstrain(s), but not inE. coli. The mutant of strain HZN6 withnoxdisrupted lost the ability to degrade nicotine, but not pseudooxynicotine. These results suggested that thenoxgene is responsible for the first step of nicotine degradation. The (RS)-nicotine degradation results showed that the two enantiomers were degraded at approximately the same rate, indicating that NOX does not show chiral selectivity. Site-directed mutagenesis revealed that both the conserved flavin adenine dinucleotide (FAD)-binding GXGXXG motif and His456 are essential for nicotine degradation activity.

scholarly journals Biodesulfurization of Naphthothiophene and Benzothiophene through Selective Cleavage of Carbon-Sulfur Bonds by Rhodococcus sp. Strain WU-K2R

2002 ◽  
Vol 68 (8) ◽  
pp. 3867-3872 ◽  
Author(s):  
Kohtaro Kirimura ◽  
Toshiki Furuya ◽  
Rika Sato ◽  
Yoshitaka Ishii ◽  
Kuniki Kino ◽  
...  
Keyword(s):  
Sole Source ◽  
Diesel Oil ◽  
The Other ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Structural Isomer ◽  
Degradation Pathways ◽  
Spectrometry Analysis ◽  
Specific Degradation ◽  
Rhodococcus Sp

ABSTRACT Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and in addition to DBT derivatives, NTH derivatives can also be detected in diesel oil following hydrodesulfurization treatment. Rhodococcus sp. strain WU-K2R was newly isolated from soil for its ability to grow in a medium with NTH as the sole source of sulfur, and growing cells of WU-K2R degraded 0.27 mM NTH within 7 days. WU-K2R could also grow in the medium with NTH sulfone, benzothiophene (BTH), 3-methyl-BTH, or 5-methyl-BTH as the sole source of sulfur but could not utilize DBT, DBT sulfone, or 4,6-dimethyl-DBT. On the other hand, WU-K2R did not utilize NTH or BTH as the sole source of carbon. By gas chromatography-mass spectrometry analysis, desulfurized NTH metabolites were identified as NTH sulfone, 2′-hydroxynaphthylethene, and naphtho[2,1-b]furan. Moreover, since desulfurized BTH metabolites were identified as BTH sulfone, benzo[c][1,2]oxathiin S-oxide, benzo[c][1,2]oxathiin S,S-dioxide, o-hydroxystyrene, 2-(2′-hydroxyphenyl)ethan-1-al, and benzofuran, it was concluded that WU-K2R desulfurized NTH and BTH through the sulfur-specific degradation pathways with the selective cleavage of carbon-sulfur bonds. Therefore, Rhodococcus sp. strain WU-K2R, which could preferentially desulfurize asymmetric heterocyclic sulfur compounds such as NTH and BTH through the sulfur-specific degradation pathways, is a unique desulfurizing biocatalyst showing properties different from those of DBT-desulfurizing bacteria.

scholarly journals Dissection of the Role of Pili and Type 2 and 3 Secretion Systems in Adherence and Biofilm Formation of an Atypical Enteropathogenic Escherichia coli Strain

2013 ◽  
Vol 81 (10) ◽  
pp. 3793-3802 ◽  
Author(s):  
Rodrigo T. Hernandes ◽  
Miguel A. De la Cruz ◽  
Denise Yamamoto ◽  
Jorge A. Girón ◽  
Tânia A. T. Gomes
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Coli Strain ◽  
Mass Spectrometry Analysis ◽  
Secretion Systems ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Rigid Rod

ABSTRACTAtypical enteropathogenicEscherichia coli(aEPEC) strains are diarrheal pathogens that lack bundle-forming pilus production but possess the virulence-associated locus of enterocyte effacement. aEPEC strain 1551-2 produces localized adherence (LA) on HeLa cells; however, its isogenic intimin (eae) mutant produces a diffuse-adherence (DA) pattern. In this study, we aimed to identify the DA-associated adhesin of the 1551-2eaemutant. Electron microscopy of 1551-2 identified rigid rod-like pili composed of an 18-kDa protein, which was identified as the major pilin subunit of type 1 pilus (T1P) by mass spectrometry analysis. Deletion offimAin 1551-2 affected biofilm formation but had no effect on adherence properties. Analysis of secreted proteins in supernatants of this strain identified a 150-kDa protein corresponding to SslE, a type 2 secreted protein that was recently reported to be involved in biofilm formation of rabbit and human EPEC strains. However, neither adherence nor biofilm formation was affected in a 1551-2sslEmutant. We then investigated the role of the EspA filament associated with the type 3 secretion system (T3SS) in DA by generating a doubleeae espAmutant. This strain was no longer adherent, strongly suggesting that the T3SS translocon is the DA adhesin. In agreement with these results, specific anti-EspA antibodies blocked adherence of the 1551-2eaemutant. Our data support a role for intimin in LA, for the T3SS translocon in DA, and for T1P in biofilm formation, all of which may act in concert to facilitate host intestinal colonization by aEPEC strains.

scholarly journals Bacteriophage φEf11 ORF28 Endolysin, a Multifunctional Lytic Enzyme with Properties Distinct from All Other IdentifiedEnterococcus faecalisPhage Endolysins

2019 ◽  
Vol 85 (13) ◽  
Author(s):  
Hongming Zhang ◽  
Bettina A. Buttaro ◽  
Derrick E. Fouts ◽  
Salar Sanjari ◽  
Bradley S. Evans ◽  
...  
Keyword(s):  
Cell Wall ◽  
Enterococcus Faecalis ◽  
Amino Acid Identity ◽  
Lytic Infection ◽  
Mass Spectrometry Analysis ◽  
Lytic Enzyme ◽  
Lytic Enzymes ◽  
Content Type ◽  
Spectrometry Analysis ◽  
Wide Range

ABSTRACTϕEf11 is a temperateSiphoviridaebacteriophage that infects strains ofEnterococcus faecalis. The ϕEf11 genome, encompassing 65 open reading frames (ORFs), is contained within 42,822 bp of DNA. Within this genome, a module of six lysis-related genes was identified. Based upon sequence homology, one of these six genes, ORF28, was predicted to code for anN-acetylmuramoyl-l-alanine amidase endolysin of 46.133 kDa, composed of 421 amino acids. The PCR-amplified ORF28 was cloned and expressed, and the resulting gene product was affinity purified to homogeneity. The purified protein was obtained from a fusion protein that exhibited a molecular mass of 72.5 kDa, consistent with a 46.1-kDa protein combined with a fused 26.5-kDa glutathioneS-transferase tag. It produced rapid, profound lysis inE. faecalispopulations and was active against 73 of 103 (71%)E. faecalisstrains tested. In addition, it caused substantial destruction ofE. faecalisbiofilms. The lysin was quite stable, retaining its activity for three years in refrigerated storage, was stable over a wide range of pHs, and was unaffected by the presence of a reducing agent; however, it was inhibited by increasing concentrations of Ca2+. Liquid chromatography-mass spectrometry analysis ofE. faecaliscell wall digestion products produced by the ORF28 endolysin indicated that the lysin acted as anN-acetylmuramidase, an endo-β-N-acetylglucosaminidase, and an endopeptidase, rather than anN-acetylmuramoyl-l-alanine amidase. The ϕEf11 ORF28 lysin shared 10% to 37% amino acid identity with the lytic enzymes of all other characterizedE. faecalisbacteriophages.IMPORTANCEThe emergence of multidrug-resistant pathogenic microorganisms has brought increasing attention to the urgent need for the development of alternative antimicrobial strategies. One such alternative to conventional antibiotics employs lytic enzymes (endolysins) that are produced by bacteriophages in the course of lytic infection. During lytic infection by a bacteriophage, these enzymes hydrolyze the cell wall peptidoglycan, resulting in the lysis of the host cell. However, external endolysin application can result in lysis from without. In this study, we have cloned, expressed, purified, and characterized an endolysin produced by a bacteriophage infecting strains ofEnterococcus faecalis. The lysin is broadly active against most of the testedE. faecalisstrains and exhibits multifunctional enzymatic specificities that differ from all other characterized endolysins produced byE. faecalisbacteriophages.

The Occurrence and Origin of Pentlandite-Chalcopyrite-Pyrrhotite Loop Textures in Magmatic Ni-Cu Sulfide Ores

2020 ◽  
Vol 115 (8) ◽  
pp. 1777-1798 ◽  
Author(s):  
Stephen J. Barnes ◽  
Valentina Taranovic ◽  
Louise E. Schoneveld ◽  
Eduardo T. Mansur ◽  
Margaux Le Vaillant ◽  
...  
Keyword(s):  
Grain Boundary ◽  
Peritectic Reaction ◽  
Inductively Coupled Plasma ◽  
Mass Spectrometry Analysis ◽  
Sulfide Liquid ◽  
Monosulfide Solid Solution ◽  
Spectrometry Analysis ◽  
Inductively Coupled ◽  
Regional Deformation ◽  
Wide Range

Abstract Pentlandite is the dominant Ni-hosting ore mineral in most magmatic sulfide deposits and has conventionally been interpreted as being entirely generated by solid-state exsolution from the high-temperature monosulfide solid solution (MSS) (Fe,Ni)1–xS. This process gives rise to the development of loops of pentlandite surrounding pyrrhotite grains. Recently it has been recognized that not all pentlandite forms by exsolution. Some may form as the result of peritectic reaction between early formed MSS and residual Ni-Cu–rich sulfide liquid during differentiation of the sulfide melt, such that at least some loop textures may be genuinely magmatic in origin. Testing this hypothesis involved microbeam X-ray fluorescence mapping to image pentlandite-pyrrhotite-chalcopyrite intergrowths from a range of different deposits. These deposits exemplify slowly cooled magmatic environments (Nova, Western Australia; Sudbury, Canada), globular ores from shallow-level intrusions (Norilsk, Siberia), extrusive komatiite-hosted ores from low and high metamorphic-grade terranes, and a number of other deposits. Our approach was complemented by laser ablation-inductively coupled plasma-mass spectrometry analysis of palladium in varying textural types of pentlandite within these deposits. Pentlandite forming coarse granular aggregates, together with loop-textured pentlandite where chalcopyrite also forms part of the loop framework, consistently has the highest Pd content compared with pentlandite clearly exsolved as lamellae from MSS or pyrrhotite. This is consistent with much of granular and loop pentlandite being formed by peritectic reaction between Pd-rich residual sulfide liquid and early crystallized MSS, rather than forming entirely by subsolidus grain boundary exsolution from MSS, as has hitherto been assumed. The wide range of Pd contents in pentlandite in individual samples reflects a continuum of processes between peritectic reaction and grain boundary exsolution. Textures in metamorphically recrystallized ores are distinctly different from loop-textured ores, implying that loop textures cannot be regenerated (except in special circumstances) by metamorphic recrystallization of original magmatic-textured ores. The presence of loop textures can therefore be taken as evidence of a lack of penetrative deformation and remobilization at submagmatic temperatures, a conclusion of particular significance to the interpretation of the Nova deposit as having formed synchronously with the peak of regional deformation at temperatures within the sulfide melting range.

Sign in / Sign up

Export Citation Format

Share Document