scholarly journals Distinct Actions by Paenibacillus sp. Strain E18 α-l-Arabinofuranosidases and Xylanase in Xylan Degradation

2013 ◽  
Vol 79 (6) ◽  
pp. 1990-1995 ◽  
Author(s):  
Pengjun Shi ◽  
Xiaoyan Chen ◽  
Kun Meng ◽  
Huoqing Huang ◽  
Yingguo Bai ◽  
...  
Keyword(s):  
High Performance ◽  
Synergistic Effects ◽  
Amino Acid Sequences ◽  
Water Soluble ◽  
Open Reading Frames ◽  
Content Type ◽  
Xylan Degradation ◽  
Wheat Arabinoxylan ◽  
Paenibacillus Sp ◽  
Family 10 Xylanase

ABSTRACTWe cloned aPaenibacillussp. strain E18 5.3-kb xylanolytic gene cluster that contains three open reading frames encoding two family 43 α-l-arabinofuranosidases (Abf43A and Abf43B) and one family 10 xylanase (XynBE18). The deduced amino acid sequences of Abf43A and Abf43B were at most 68% and 63% identical to those of two putative family 43 proteins fromClostridiumsp. strain DL-VIII (EHI98634.1 and EHI98635.1), respectively, but were only 11% identical to each other. Recombinant Abf43A and Abf43B had similar activities at 45°C and pH 6.0 but varied in thermostabilities and substrate specificities. Abf43B was active against only 4-nitrophenyl α-l-arabinofuranoside, whereas Abf43A acted on 4-nitrophenyl α-l-arabinofuranoside, wheat arabinoxylan, 4-nitrophenyl α-d-xylopyranoside, and sugar beet arabinan. The sequential and combined effects on xylan degradation by XynBE18, Abf43A, and Abf43B were characterized. For beechwood, birchwood, and oat spelt xylans as the substrates, synergistic effects were found when XynBE18 and Abf43A or Abf43B were incubated together and when the substrates were first incubated with Abf43A or Abf43B and then with XynBE18. Further high-performance liquid chromatography (HPLC) analysis showed that the amounts of xylobiose and xylose increased sharply in the aforementioned reactions. For water-soluble wheat arabinoxylan as the substrate, Abf43A not only released arabinose but also had a synergistic effect with XynBE18. Synergy may arise as the result of removal of arabinose residues from xylans by α-l-arabinofuranosidases, which eliminates steric hindrance caused by the arabinose side chains and which allows xylanases to then degrade the xylan backbone, producing short xylooligosaccharides.

scholarly journals Establishment of anIn Vitrod-Cycloserine-Synthesizing System by UsingO-Ureido-l-Serine Synthase and d-Cycloserine Synthetase Found in the Biosynthetic Pathway

2013 ◽  
Vol 57 (6) ◽  
pp. 2603-2612 ◽  
Author(s):  
Narutoshi Uda ◽  
Yasuyuki Matoba ◽  
Takanori Kumagai ◽  
Kosuke Oda ◽  
Masafumi Noda ◽  
...  
Keyword(s):  
Gene Cluster ◽  
High Performance ◽  
Sequence Similarity ◽  
Open Reading Frames ◽  
Homocysteine Thiolactone ◽  
Putative Gene ◽  
Content Type ◽  
Dna Fragment ◽  
Layer Chromatography

ABSTRACTWe have recently cloned a DNA fragment containing a gene cluster that is responsible for the biosynthesis of an antituberculosis antibiotic,d-cycloserine. The gene cluster is composed of 10 open reading frames, designateddcsAtodcsJ. Judging from the sequence similarity between each putative gene product and known proteins, DcsC, which displays high homology to diaminopimelate epimerase, may catalyze the racemization ofO-ureidoserine. DcsD is similar toO-acetylserine sulfhydrylase, which generatesl-cysteine usingO-acetyl-l-serine with sulfide, and therefore, DcsD may be a synthase to generateO-ureido-l-serine usingO-acetyl-l-serine and hydroxyurea. DcsG, which exhibits similarity to a family of enzymes with an ATP-grasp fold, may be an ATP-dependent synthetase convertingO-ureido-d-serine intod-cycloserine. In the present study, to characterize the enzymatic functions of DcsC, DcsD, and DcsG, each protein was overexpressed inEscherichia coliand purified to near homogeneity. The biochemical function of each of the reactions catalyzed by these three proteins was verified by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and, in some cases, mass spectrometry. The results from this study demonstrate that by using a mixture of the three purified enzymes and the two commercially available substratesO-acetyl-l-serine and hydroxyurea, synthesis ofd-cycloserine was successfully attained. Thesein vitrostudies yield the conclusion that DcsD and DcsG are necessary for the syntheses ofO-ureido-l-serine andd-cycloserine, respectively. DcsD was also able to catalyze the synthesis ofl-cysteine when sulfide was added instead of hydroxyurea. Furthermore, the present study shows that DcsG can also form other cyclicd-amino acid analogs, such asd-homocysteine thiolactone.

scholarly journals Cloning and Expression Analysis of Genes Encoding Lytic Endopeptidases L1 and L5 from Lysobacter sp. Strain XL1

2012 ◽  
Vol 78 (19) ◽  
pp. 7082-7089 ◽  
Author(s):  
Y. S. Lapteva ◽  
O. E. Zolova ◽  
M. G. Shlyapnikov ◽  
I. M. Tsfasman ◽  
T. A. Muranova ◽  
...  
Keyword(s):  
Stop Codon ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Cloning And Expression ◽  
Start Codon ◽  
Lytic Enzymes ◽  
Homologous Proteins ◽  
Logarithmic Growth Phase ◽  
Transcription Terminator ◽  
Content Type

ABSTRACTLytic enzymes are the group of hydrolases that break down structural polymers of the cell walls of various microorganisms. In this work, we determined the nucleotide sequences of theLysobactersp. strain XL1alpAandalpBgenes, which code for, respectively, secreted lytic endopeptidases L1 (AlpA) and L5 (AlpB).In silicoanalysis of their amino acid sequences showed these endopeptidases to be homologous proteins synthesized as precursors similar in structural organization: the mature enzyme sequence is preceded by an N-terminal signal peptide and a pro region. On the basis of phylogenetic analysis, endopeptidases AlpA and AlpB were assigned to the S1E family [clan PA(S)] of serine peptidases. Expression of thealpAandalpBopen reading frames (ORFs) inEscherichia coliconfirmed that they code for functionally active lytic enzymes. Each ORF was predicted to have the Shine-Dalgarno sequence located at a canonical distance from the start codon and a potential Rho-independent transcription terminator immediately after the stop codon. ThealpAandalpBmRNAs were experimentally found to be monocistronic; transcription start points were determined for both mRNAs. The synthesis of thealpAandalpBmRNAs was shown to occur predominantly in the late logarithmic growth phase. The amount ofalpAmRNA in cells ofLysobactersp. strain XL1 was much higher, which correlates with greater production of endopeptidase L1 than of L5.

Characteristic features and ligand specificity of the two olfactory receptor classes from Xenopus laevis

2001 ◽  
Vol 204 (17) ◽  
pp. 2987-2997
Author(s):  
Mario Mezler ◽  
Jörg Fleischer ◽  
Heinz Breer
Keyword(s):  
Xenopus Laevis ◽  
Olfactory Receptors ◽  
Functional Expression ◽  
Class Ii ◽  
Amino Acid Sequences ◽  
Water Soluble ◽  
Open Reading Frames ◽  
Class I ◽  
Characteristic Features ◽  
Intracellular Loops

SUMMARY Amphibia have two classes of olfactory receptors (ORs), class I (fish-like receptors) and class II (mammalian-like receptors). These two receptor classes correspond to the two classes identified in other vertebrates, and amphibians thus provide a unique opportunity to compare olfactory receptors of both classes in one animal species, without the constraints of evolutionary distance between different vertebrate orders, such as fish and mammals. We therefore identified the complete open reading frames of class I and class II ORs in Xenopus laevis. In addition to allowing a representative comparison of the deduced amino acid sequences between both receptor classes, we were also able to perform differential functional analysis. These studies revealed distinct class-specific motifs, particularly in the extracellular loops 2 and 3, which might be of importance for the interaction with odorants, as well as in the intracellular loops 2 and 3, which might be responsible for interactions with specific G-proteins. The results of functional expression studies in Xenopus oocytes, comparing distinct receptor types, support the idea that class I receptors are activated by water-soluble odorants, whereas class II receptors are activated by volatile compounds.

scholarly journals Metagenome-Derived Clones Encoding Two Novel Lactonase Family Proteins Involved in Biofilm Inhibition in Pseudomonas aeruginosa

2008 ◽  
Vol 75 (1) ◽  
pp. 224-233 ◽  
Author(s):  
C. Schipper ◽  
C. Hornung ◽  
P. Bijtenhoorn ◽  
M. Quitschau ◽  
S. Grond ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
High Performance ◽  
Homoserine Lactone ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Biofilm Inhibition ◽  
Soil Dna ◽  
Isolation And Characterization ◽  
Bacterial Quorum

ABSTRACT Here we report the isolation and characterization of three metagenome-derived clones that interfere with bacterial quorum sensing and degrade N-(3-oxooctanoyl)-l-homoserine lactone (3-oxo-C8-HSL). By using a traI-lacZ gene fusion, the metagenome-derived clones were identified from a soil DNA library and analyzed. The open reading frames linked to the 3-oxo-C8-HSL-degrading activities were designated bpiB01, bpiB04, and bpiB07. While the BpiB07 protein was similar to a known lactonase, no significant similarities were observed for the BpiB01 and BpiB04 proteins or the deduced amino acid sequences. High-performance liquid chromatography-mass spectrometry analyses confirmed that the identified genes encode novel lactone-hydrolyzing enzymes. The original metagenome-derived clones were expressed in Pseudomonas aeruginosa and employed in motility and biofilm assays. All clones were able to reproducibly inhibit motility in P. aeruginosa. Furthermore, these genes clearly inhibited biofilm formation in P. aeruginosa when expressed in P. aeruginosa PAO1. Thus, this is the first study in which metagenome-derived proteins have been expressed in P. aeruginosa to successfully inhibit biofilm formation.

Controlled release and synergistic effects of polyvinyl alcohol (PVA) on phosphate corrosion inhibitor at the interface of thermal insulation and carbon steel

2015 ◽  
Vol 62 (2) ◽  
pp. 109-115
Author(s):  
Zhouyang Lian ◽  
Lirui Yuan ◽  
Wuji Wei ◽  
Qing Zhou ◽  
Juncheng Jiang
Keyword(s):  
Controlled Release ◽  
Carbon Steel ◽  
Polyvinyl Alcohol ◽  
Corrosion Inhibitor ◽  
Thermal Insulation ◽  
Synergistic Effects ◽  
Water Soluble ◽  
Protective Film ◽  
Content Type ◽  
Rock Wool

Purpose – This paper aims to study the controlled release and synergistic effect of water-soluble polyvinyl alcohol (PVA) on phosphate corrosion inhibitor at the interface of thermal insulation cotton/carbon steel. Design/methodology/approach – This study was carried out using a coating method, scanning electron microscopy, energy dispersive spectroscopy and AC impedance. Findings – The single-phase phosphate particles were coated/adsorbed on the PVA film, which was formed on the fiber surface of corrosion inhibitor/PVA-impregnated rock wool sample. On the surface of Q235 steel, an effective protective film was formed by the corrosion inhibitor with partially dissolved PVA that can significantly increase the polarization resistance of corrosion reaction, and reduce the capacitive reactance of electric double layer. The rock wool impregnated with the phosphate corrosion inhibitor and 1.5 per cent PVA showed obvious controlled release and inhibition synergism. Originality/value – The rock wool impregnated with the phosphate corrosion inhibitor and 1.5 per cent PVA showed the following advantages: the adsorption and release quantities of the corrosion inhibitor increased by 3.3 and 2.9 times, respectively; the release-adsorption equilibrium time increased from 2 to 6 h; and the corrosion inhibition efficiency increased from 61.55 per cent to 94.6 per cent.

scholarly journals Transcriptional and Translational Landscape of Equine Torovirus

2018 ◽  
Vol 92 (17) ◽  
Author(s):  
Hazel Stewart ◽  
Katherine Brown ◽  
Adam M. Dinan ◽  
Nerea Irigoyen ◽  
Eric J. Snijder ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Rna Synthesis ◽  
Gastrointestinal Disease ◽  
Ribosome Profiling ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Comparative Genomic ◽  
Human Pathogens ◽  
Content Type ◽  
Reading Frames

ABSTRACT The genus Torovirus (subfamily Torovirinae, family Coronaviridae, order Nidovirales) encompasses a range of species that infect domestic ungulates, including cattle, sheep, goats, pigs, and horses, causing an acute self-limiting gastroenteritis. Using the prototype species equine torovirus (EToV), we performed parallel RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq) to analyze the relative expression levels of the known torovirus proteins and transcripts, chimeric sequences produced via discontinuous RNA synthesis (a characteristic of the nidovirus replication cycle), and changes in host transcription and translation as a result of EToV infection. RNA sequencing confirmed that EToV utilizes a unique combination of discontinuous and nondiscontinuous RNA synthesis to produce its subgenomic RNAs (sgRNAs); indeed, we identified transcripts arising from both mechanisms that would result in sgRNAs encoding the nucleocapsid. Our ribosome profiling analysis revealed that ribosomes efficiently translate two novel CUG-initiated open reading frames (ORFs), located within the so-called 5′ untranslated region. We have termed the resulting proteins U1 and U2. Comparative genomic analysis confirmed that these ORFs are conserved across all available torovirus sequences, and the inferred amino acid sequences are subject to purifying selection, indicating that U1 and U2 are functionally relevant. This study provides the first high-resolution analysis of transcription and translation in this neglected group of livestock pathogens. IMPORTANCE Toroviruses infect cattle, goats, pigs, and horses worldwide and can cause gastrointestinal disease. There is no treatment or vaccine, and their ability to spill over into humans has not been assessed. These viruses are related to important human pathogens, including severe acute respiratory syndrome (SARS) coronavirus, and they share some common features; however, the mechanism that they use to produce sgRNA molecules differs. Here, we performed deep sequencing to determine how equine torovirus produces sgRNAs. In doing so, we also identified two previously unknown open reading frames “hidden” within the genome. Together these results highlight the similarities and differences between this domestic animal virus and related pathogens of humans and livestock.

scholarly journals Identification of a Genetic Locus Essential for Serotype b-Specific Antigen Synthesis in Actinobacillus actinomycetemcomitans

1998 ◽  
Vol 66 (1) ◽  
pp. 107-114 ◽  
Author(s):  
Yasuo Yoshida ◽  
Yoshio Nakano ◽  
Yoshihisa Yamashita ◽  
Toshihiko Koga
Keyword(s):  
High Performance ◽  
Genetic Locus ◽  
Specific Antigen ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Actinobacillus Actinomycetemcomitans ◽  
E Coli ◽  
Chromatography Analysis ◽  
Large Gene ◽  
Serotype B

ABSTRACT A large gene cluster associated with the biosynthesis of the serotype-specific polysaccharide antigen (SPA) of Actinobacillus actinomycetemcomitans Y4 (serotype b) was cloned and characterized. Western blot analysis showed that Escherichia coli DH5α, containing a plasmid carrying this cluster, produced a polysaccharide which reacted with a monoclonal antibody directed against the SPA of A. actinomycetemcomitans Y4. High-performance liquid chromatography analysis indicated that the polysaccharide produced by an E. coli transformant, as well as A. actinomycetemcomitans Y4 SPA, was composed of rhamnose and fucose. Furthermore, using various derivatives of the plasmid, we demonstrated that the cloned 13-kbBssHII-BspHI fragment was indispensable for SPA synthesis in E. coli DH5α. The 24,909-bp nucleotide sequence, which included this fragment and its flanking regions, was determined. In the sequenced area, 24 open reading frames (ORFs) with the same orientation were found. Most of these were located sequentially within a short distance of each other. Many of the deduced amino acid sequences were similar to the gene products of the polysaccharide synthetic genes of other bacteria. The average G+C content (37.7%) of all 24 ORFs in the sequenced area was lower than that (45.6%) of the whole chromosome of A. actinomycetemcomitans Y4. It is noteworthy the average G+C content of the nine ORFs in the 8.5-kb central region of the 13-kbBssHII-BspHI fragment indispensable for SPA synthesis in E. coli was found to be especially low (27.0%).

scholarly journals Functional Identification of Two Novel Genes from Pseudomonas sp. Strain HZN6 Involved in the Catabolism of Nicotine

2012 ◽  
Vol 78 (7) ◽  
pp. 2154-2160 ◽  
Author(s):  
Jiguo Qiu ◽  
Yun Ma ◽  
Yuezhong Wen ◽  
Liansheng Chen ◽  
Lifei Wu ◽  
...  
Keyword(s):  
Amine Oxidase ◽  
Bartonella Henselae ◽  
Insertion Site ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Functional Identification ◽  
Novel Genes ◽  
Content Type ◽  
Transposon Insertion ◽  
Arthrobacter Nicotinovorans

ABSTRACTNicotine is a natural alkaloid produced by tobacco plants, and the mechanisms of its catabolism by microorganisms are diverse. In the present study, we reported the mutation, cloning, and identification of two novel genes involved in nicotine degradation from the newly isolatedPseudomonassp. strain HZN6. Transposon mutagenesis identified a HZN6 mutant in which the nicotine-degrading pathway was blocked at pseudooxynicotine. A 3,874-bp DNA fragment flanking the transposon insertion site was obtained through self-formed adaptor PCR. Two open reading frames (designatedpaoandsap) were analyzed, and the deduced amino acid sequences shared 29% identity with 6-hydroxy-l-nicotine oxidase fromArthrobacter nicotinovoransand 49% identity with an aldehyde dehydrogenase fromBartonella henselae. Bothpaoandsapwere cloned and functionally expressed in recombinantEscherichia coliBL21. Thepaogene encoded a novel pseudooxynicotine amine oxidase with noncovalently bound flavin adenine dinucleotide (FAD) and exhibited substrate specificity removing the methylamine from pseudooxynicotine with the formation of 3-succinoylsemialdehyde-pyridine and hydrogen dioxide. Thesapgene encoded a NADP+-dependent 3-succinoylsemialdehyde-pyridine dehydrogenase that catalyzed the dehydrogenation of 3-succinoylsemialdehyde-pyridine to 3-succinoyl-pyridine. Genetic analyses indicated that thepaogene played an essential role in nicotine or pseudooxynicotine mineralization in strain HZN6, whereas thesapgene did not. This study provides novel insight into the nicotine-degrading mechanism at the genetic level inPseudomonasspp.

scholarly journals Involvement of Multiple Loci in Quorum Quenching of Autoinducer I Molecules in the Nitrogen-Fixing Symbiont Rhizobium (Sinorhizobium) sp. Strain NGR234

2011 ◽  
Vol 77 (15) ◽  
pp. 5089-5099 ◽  
Author(s):  
D. Krysciak ◽  
C. Schmeisser ◽  
S. Preuß ◽  
J. Riethausen ◽  
M. Quitschau ◽  
...  
Keyword(s):  
High Performance ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Open Reading Frames ◽  
Nitrogen Fixing ◽  
Content Type ◽  
Multiple Loci ◽  
Genome Wide ◽  
A Genome

ABSTRACTRhizobiumsp. strain NGR234 is a unique alphaproteobacterium (orderRhizobiales) that forms nitrogen-fixing nodules with more legumes than any other microsymbiont. Since we have previously described the complete genome sequence of NGR234, we now report on a genome-wide functional analysis of the genes and enzymes involved in autoinducer I hydrolysis in this microbe. Altogether we identified five cosmid clones that repeatedly gave a positive result in our function-based approach for the detection of autoinducer I hydrolase genes. Of these five cosmid clones, two were located on pNGR234band three were on cNGR234. Subcloning andin vitromutagenesis in combination with BLAST analyses identified the corresponding open reading frames (ORFs) of all cosmid clones:dlhR,qsdR1,qsdR2,aldR, andhitR-hydR. Analyses of recombinant DlhR and QsdR1 proteins by using high-performance liquid chromatography-mass spectrometry (HPLC-MS) demonstrate that these enzymes function as acyl homoserine lactone (AHL) lactonases. Furthermore, we showed that these enzymes inhibited biofilm formation and other quorum-sensing-dependent processes inPseudomonas aeruginosa,Chromobacterium violaceum, andAgrobacterium tumefaciens. Finally, our experimental data suggest that competitive colonization of roots in the rhizospheres of cowpea plants is affected by DlhR and QsdR1.

scholarly journals F420H2Is Required for Phthiocerol Dimycocerosate Synthesis in Mycobacteria

2016 ◽  
Vol 198 (15) ◽  
pp. 2020-2028 ◽  
Author(s):  
Endang Purwantini ◽  
Lacy Daniels ◽  
Biswarup Mukhopadhyay
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Protein A ◽  
Mycobacterium Leprae ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Hydroxyl Group ◽  
Host Immune System ◽  
Content Type ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Phthiocerol Dimycocerosates

ABSTRACTPhthiocerol dimycocerosates (PDIM) are a group of cell surface-associated apolar lipids ofMycobacterium tuberculosisand closely related mycobacteria, such asMycobacterium bovisandMycobacterium leprae. A characteristic methoxy group of these lipids is generated from the methylation of a hydroxyl group of the direct precursors, the phthiotriols. The precursors arise from the reduction of phthiodiolones, the keto intermediates, by a ketoreductase. The putative phthiodiolone ketoreductase (PKR) is encoded by Rv2951c inM. tuberculosisand BCG_2972c inM. bovisBCG, and these open reading frames (ORFs) encode identical amino acid sequences. We investigated the cofactor requirement of the BCG_2972c protein. A comparative analysis based on the crystallographic structures of similar enzymes identified structural elements for binding of coenzyme F420and hydrophobic phthiodiolones in PKR. Coenzyme F420is a deazaflavin coenzyme that serves several key functions in pathogenic and nonpathogenic mycobacteria. We found that anM. bovisBCG mutant lacking F420-dependent glucose-6-phosphate dehydrogenase (Fgd), which generates F420H2(glucose-6-phosphate + F420→ 6-phosphogluconate + F420H2), was devoid of phthiocerols and accumulated phthiodiolones. When the mutant was provided with F420H2, a broken-cell slurry of the mutant converted accumulated phthiodiolones to phthiocerols; F420H2was generatedin situfrom F420and glucose-6-phosphate by the action of Fgd. Thus, the reaction mixture was competent in reducing phthiodiolones to phthiotriols (phthiodiolones + F420H2→ phthiotriols + F420), which were then methylated to phthiocerols. These results established the mycobacterialphthiodioloneketoreductase as anF420H2-dependent enzyme (fPKR). A phylogenetic analysis of close homologs of fPKR revealed potential F420-dependent lipid-modifying enzymes in a broad range of mycobacteria.IMPORTANCEMycobacterium tuberculosisis the causative agent of tuberculosis, and phthiocerol dimycocerosates (PDIM) protect this pathogen from the early innate immune response of an infected host. Thus, the PDIM synthesis system is a potential target for the development of effective treatments for tuberculosis. The current study shows that a PDIM synthesis enzyme is dependent on the coenzyme F420. F420is universally present in mycobacteria and absent in humans. This finding expands the number of experimentally validated F420-dependent enzymes inM. tuberculosisto six, each of which helps the pathogen to evade killing by the host immune system, and one of which activates an antituberculosis drug, PA-824. This work also has relevance to leprosy, since similar waxy lipids are found inMycobacterium leprae.

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