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 Two C—P Lyase Operons in Pseudomonas stutzeri and Their Roles in the Oxidation of Phosphonates, Phosphite, and Hypophosphite

2004 ◽  
Vol 186 (14) ◽  
pp. 4730-4739 ◽  
Author(s):  
Andrea K. White ◽  
William W. Metcalf
Keyword(s):  
Insertion Site ◽  
Pseudomonas Stutzeri ◽  
Gene Organization ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
E Coli ◽  
Reverse Transcription Pcr ◽  
Transposon Insertion ◽  
Intergenic Sequences ◽  
Reading Frames

ABSTRACT DNA sequencing and analysis of two distinct C—P lyase operons in Pseudomonas stutzeri WM88 were completed. The htxABCDEFGHIJKLMN operon encodes a hypophosphite-2-oxoglutarate dioxygenase (HtxA), whereas the predicted amino acid sequences of HtxB to HtxN are each homologous to the components of the Escherichia coli phn operon, which encodes C—P lyase, although homologs of E. coli phnF and phnO are absent. The genes in the htx operon are cotranscribed based on gene organization, and the presence of the intergenic sequences is verified by reverse transcription-PCR with total RNA. Deletion of the htx locus does not affect the ability of P. stutzeri to grow on phosphonates, indicating the presence of an additional C—P lyase pathway in this organism. To identify the genes comprising this pathway, a Δhtx strain was mutagenized and one mutant lacking the ability to grow on methylphosphonate as the sole P source was isolated. A ca.-10.6-kbp region surrounding the transposon insertion site of this mutant was sequenced, revealing 13 open reading frames, designated phnCDEFGHIJKLMNP, which were homologous to the E. coli phn genes. Deletion of both the htx and phn operons of P. stutzeri abolishes all growth on methylphosphonate and aminoethylphosphonate. Both operons individually support growth on methylphosphonate; however, the phn operon supports growth on aminoethylphosphonate and phosphite, as well. The substrate ranges of both C—P lyases are limited, as growth on other phosphonate compounds, including glyphosate and phenylphosphonate, was not observed.

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.

scholarly journals Transposon Insertion Site Sequencing of Providencia stuartii: Essential Genes, Fitness Factors for Catheter-Associated Urinary Tract Infection, and the Impact of Polymicrobial Infection on Fitness Requirements

mSphere ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Alexandra O. Johnson ◽  
Valerie Forsyth ◽  
Sara N. Smith ◽  
Brian S. Learman ◽  
Aimee L. Brauer ◽  
...  
Keyword(s):  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Insertion Site ◽  
Single Species ◽  
Polymicrobial Infection ◽  
Common Cause ◽  
Content Type ◽  
Transposon Insertion ◽  
Providencia Stuartii ◽  
The Impact

ABSTRACT Providencia stuartii is a common cause of polymicrobial catheter-associated urinary tract infection (CAUTI), and yet literature describing the molecular mechanisms of its pathogenesis is limited. To identify factors important for colonization during single-species infection and during polymicrobial infection with a common cocolonizer, Proteus mirabilis, we created a saturating library of ∼50,000 transposon mutants and conducted transposon insertion site sequencing (Tn-Seq) in a murine model of CAUTI. P. stuartii strain BE2467 carries 4,398 genes, 521 of which were identified as essential for growth in laboratory medium and therefore could not be assessed for contribution to infection. Using an input/output fold change cutoff value of 20 and P values of <0.05, 340 genes were identified as important for establishing single-species infection only and 63 genes as uniquely important for polymicrobial infection with P. mirabilis, and 168 genes contributed to both single-species and coinfection. Seven mutants were constructed for experimental validation of the primary screen that corresponded to flagella (fliC mutant), twin arginine translocation (tatC), an ATP-dependent protease (clpP), d-alanine-d-alanine ligase (ddlA), type 3 secretion (yscI and sopB), and type VI secretion (impJ). Infection-specific phenotypes validated 6/7 (86%) mutants during direct cochallenge with wild-type P. stuartii and 3/5 (60%) mutants during coinfection with P. mirabilis, for a combined validation rate of 9/12 (75%). Tn-Seq therefore successfully identified genes that contribute to fitness of P. stuartii within the urinary tract, determined the impact of coinfection on fitness requirements, and added to the identification of a collection of genes that may contribute to fitness of multiple urinary tract pathogens. IMPORTANCE Providencia stuartii is a common cause of polymicrobial catheter-associated urinary tract infections (CAUTIs), particularly during long-term catheterization. However, little is known regarding the pathogenesis of this organism. Using transposon insertion site sequencing (Tn-Seq), we performed a global assessment of P. stuartii fitness factors for CAUTI while simultaneously determining how coinfection with another pathogen alters fitness requirements. This approach provides four important contributions to the field: (i) the first global estimation of P. stuartii genes essential for growth in laboratory medium, (ii) identification of novel fitness factors for P. stuartii colonization of the catheterized urinary tract, (iii) identification of core fitness factors for both single-species and polymicrobial CAUTI, and (iv) assessment of conservation of fitness factors between common uropathogens. Genomewide assessment of the fitness requirements for common uropathogens during single-species and polymicrobial CAUTI thus elucidates complex interactions that contribute to disease severity and will uncover conserved targets for therapeutic intervention.

scholarly journals The tet39 Determinant and the msrE-mphE Genes in Acinetobacter Plasmids Are Each Part of Discrete Modules Flanked by Inversely Oriented pdif (XerC-XerD) Sites

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Grace A. Blackwell ◽  
Ruth M. Hall
Keyword(s):  
Insertion Sequence ◽  
Insertion Site ◽  
Tetracycline Resistance ◽  
Open Reading Frames ◽  
Gene Encoding ◽  
Content Type ◽  
System A ◽  
Genes Encoding ◽  
Putative Toxin ◽  
Reading Frames

ABSTRACT The tet39 tetracycline resistance determinant and the macrolide resistance genes msrE and mphE were found in an 18.2-kb plasmid, pS30-1, recovered from a global clone 2 (GC2) Acinetobacter baumannii isolate from Singapore, that conferred resistance to tetracycline and erythromycin. pS30-1 also contains mobA and mobC genes encoding MOBQ family proteins, but attempts to mobilize pS30-1 utilizing a coresident conjugative repAci6 plasmid were unsuccessful. Eight pdif sites, consisting of inversely oriented binding sites for the XerC and XerD recombinases separated by 6 bp, were detected in pS30-1. The tet39 determinant and the msrE-mphE gene pair are each surrounded by two pdif sites in inverse orientation. Identical regions in different contexts and many previously unnoticed pdif sites were found in a number of different plasmids in GenBank, showing that the tet39 and msrE-mphE dif modules are mobile. A putative toxin/antitoxin system, a gene encoding a serine recombinase, and open reading frames of unknown function were also part of dif modules in pS30-1. In general, modules with internal XerC or XerD sites alternate. Two copies of ISAjo2-1 (94% identical to ISAjo2) in pS30-1 were inserted 5 bp from a XerC site, and this appears to be the preferred insertion site for this insertion sequence (IS) group. Apparently, Acinetobacter plasmids exploit the Acinetobacter XerC-XerD recombinases to mobilize DNA units containing resistance and other genes, via an uncharacterized mechanism. The tet39 and msrE-mphE dif modules add to the oxa24 module and the oxa58 module redefined here, bringing the total of resistance gene-containing dif modules in Acinetobacter plasmids to four.

scholarly journals Identification of Genes Relevant to Symbiosis and Competitiveness in Sinorhizobium meliloti Using Signature-Tagged Mutants

2008 ◽  
Vol 21 (2) ◽  
pp. 219-231 ◽  
Author(s):  
Nataliya Pobigaylo ◽  
Silke Szymczak ◽  
Tim W. Nattkemper ◽  
Anke Becker
Keyword(s):  
Sinorhizobium Meliloti ◽  
Insertion Site ◽  
Nitrogen Fixing ◽  
In Planta ◽  
Novel Genes ◽  
Transposon Insertion

Sinorhizobium meliloti enters an endosymbiosis with alfalfa plants through the formation of nitrogen-fixing nodules. In order to identify S. meliloti genes required for symbiosis and competitiveness, a method of signature-tagged mutagenesis was used. Two sets, each consisting of 378 signature-tagged mutants with a known transposon insertion site, were used in an experiment in planta. As a result, 67 mutants showing attenuated symbiotic phenotypes were identified, including most of the exo, fix, and nif mutants in the sets. For 38 mutants in genes previously not described to be involved in competitiveness or symbiosis in S. meliloti, attenuated competitiveness phenotypes were tested individually. A large part of these phenotypes was confirmed. Moreover, additional symbiotic defects were observed for mutants in several novel genes such as infection deficiency phenotypes (ilvI and ilvD2 mutants) or delayed nodulation (pyrE, metA, thiC, thiO, and thiD mutants).

scholarly journals Plasmid-Encoded MCP Is Involved in Virulence, Motility, and Biofilm Formation of Cronobacter sakazakii ATCC 29544

2014 ◽  
Vol 83 (1) ◽  
pp. 197-204 ◽  
Author(s):  
Younho Choi ◽  
Seongok Kim ◽  
Hyelyeon Hwang ◽  
Kwang-Pyo Kim ◽  
Dong-Hyun Kang ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Protein Gene ◽  
Open Reading Frames ◽  
Insertion Mutant ◽  
Biological Processes ◽  
Cronobacter Sakazakii ◽  
Content Type ◽  
Rat Pups ◽  
Transposon Insertion ◽  
Reading Frames

The aim of this study was to elucidate the function of the plasmid-bornemcp(methyl-accepting chemotaxis protein) gene, which plays pleiotropic roles inCronobacter sakazakiiATCC 29544. By searching for virulence factors using a random transposon insertion mutant library, we identified and sequenced a new plasmid, pCSA2, inC. sakazakiiATCC 29544. Anin silicoanalysis of pCSA2 revealed that it included six putative open reading frames, and one of them wasmcp. Themcpmutant was defective for invasion into and adhesion to epithelial cells, and the virulence of themcpmutant was attenuated in rat pups. In addition, we demonstrated that putative MCP regulates the motility ofC. sakazakii, and the expression of the flagellar genes was enhanced in the absence of a functionalmcpgene. Furthermore, a lack of themcpgene also impaired the ability ofC. sakazakiito form a biofilm. Our results demonstrate a regulatory role for MCP in diverse biological processes, including the virulence ofC. sakazakiiATCC 29544. To the best of our knowledge, this study is the first to elucidate a potential function of a plasmid-encoded MCP homolog in theC. sakazakiisequence type 8 (ST8) lineage.

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 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 Biological Role of Pigment Production for the Bacterial Phytopathogen Pantoea stewartii subsp. stewartii

2012 ◽  
Vol 78 (19) ◽  
pp. 6859-6865 ◽  
Author(s):  
Mojtaba Mohammadi ◽  
Lindsey Burbank ◽  
M. Caroline Roper
Keyword(s):  
Sweet Corn ◽  
Insertion Site ◽  
Carotenoid Biosynthesis ◽  
Carotenoid Pigment ◽  
In Planta ◽  
Gene Encoding ◽  
Content Type ◽  
Pantoea Stewartii ◽  
Transposon Insertion ◽  
Stewart's Wilt

ABSTRACTPantoea stewartiisubsp.stewartii, the causal agent of Stewart's wilt of sweet corn, produces a yellow carotenoid pigment. A nonpigmented mutant was selected from a bank of mutants generated by random transposon mutagenesis. The transposon insertion site was mapped to thecrtBgene, encoding a putative phytoene synthase, an enzyme involved in the early steps of carotenoid biosynthesis. We demonstrate here that the carotenoid pigment imparts protection against UV radiation and also contributes to the complete antioxidant pathway ofP. stewartii. Moreover, production of this pigment is regulated by the EsaI/EsaR quorum-sensing system and significantly contributes to the virulence of the pathogenin planta.

Ingi, a 5.2-kb dispersed sequence element from Trypanosoma brucei that carries half of a smaller mobile element at either end and has homology with mammalian LINEs

1987 ◽  
Vol 7 (4) ◽  
pp. 1465-1475 ◽  
Author(s):  
B E Kimmel ◽  
O K ole-MoiYoi ◽  
J R Young
Keyword(s):  
Trypanosoma Brucei ◽  
Repetitive Element ◽  
Insertion Site ◽  
Mobile Element ◽  
Protozoan Parasite ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Sequence Element ◽  
Reverse Transcriptases ◽  
Reading Frames

A dispersed repetitive element named ingi, which is present in the genome of the protozoan parasite Trypanosoma brucei, is described. One complete 5.2-kilobase element and the ends of two others were sequenced. There were no direct or inverted terminal repeats. Rather, the ends consisted of two halves of a previously described 512-base-pair transposable element (G. Hasan, M.J. Turner, and J.S. Cordingley, Cell 37:333-341, 1984). Oligo(dA) tails and possible insertion site duplications suggested that ingi is a retroposon. The sequenced element appears to be a pseudogene copy of an original retroposon with one or more open reading frames occupying most of its length. Significant homologies of the encoded amino acid sequences with reverse transcriptases and mammalian long interpersed nuclear element sequences suggest a remote evolutionary origin for this kind of retroposon.

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