scholarly journals sal Genes Determining the Catabolism of Salicylate Esters Are Part of a Supraoperonic Cluster of Catabolic Genes in Acinetobacter sp. Strain ADP1

2000 ◽  
Vol 182 (7) ◽  
pp. 2018-2025 ◽  
Author(s):  
Rheinallt M. Jones ◽  
Vassilis Pagmantidis ◽  
Peter A. Williams
Keyword(s):  
Esterase Activity ◽  
Carbon Sources ◽  
Open Reading Frames ◽  
Fatty Acid Transport ◽  
Gene Products ◽  
Hydroxylase Activity ◽  
Cell Extracts ◽  
Catabolic Genes ◽  
Hydrocarbon Transport ◽  
Reading Frames

ABSTRACT A 5-kbp region upstream of the are-ben-cat genes was cloned from Acinetobacter sp. strain ADP1, extending the supraoperonic cluster of catabolic genes to 30 kbp. Four open reading frames, salA, salR, salE, andsalD, were identified from the nucleotide sequence. Reverse transcription-PCR studies suggested that these open reading frames are organized into two convergent transcription units, salARand salDE. The salE gene, encoding a protein of 239 residues, was ligated into expression vector pET5a. Its product, SalE, was shown to have esterase activity against short-chain alkyl esters of 4-nitrophenol but was also able to hydrolyze ethyl salicylate to ethanol and salicylic acid. A mutant of ADP1 with a Kmrcassette introduced into salE had lost the ability to utilize only ethyl and methyl salicylates of the esters tested as sole carbon sources, and no esterase activity against ethyl salicylate could be detected in cell extracts. SalE was induced during growth on ethyl salicylate but not during growth on salicylate itself. salDencoded a protein of undetermined function with homologies to theEscherichia coli FadL membrane protein, which is involved in facilitating fatty acid transport, and a number of other proteins detected during aromatic catabolism, which may also function in hydrocarbon transport or uptake processes. A Kmr cassette insertion in salD deleteriously affected cell growth and viability. The salA and salR gene products closely resemble two Pseudomonas proteins, NahG and NahR, respectively encoding salicylate hydroxylase and the LysR family regulator of both salicylate and naphthalene catabolism.salA was cloned into pUC18 together with salRand salE, and its gene product showed salicylate-inducible hydroxylase activity against a range of substituted salicylates, with the same relative specific activities as found in wild-type ADP1 grown on salicylate. Mutations involving insertion of Kmrcassettes into salA and salR eliminated expression of salicylate hydroxylase activity and the ability to grow on either salicylate or ethyl salicylate. Studies of mutants with disruptions of genes of the β-ketoadipate pathway with or without an additional salE mutation confirmed that ethyl salicylate and salicylate were channeled into the β-ketoadipate pathway at the level of catechol and thence dissimilated by the cat gene products. SalR appeared to regulate expression of salA but not salE.

scholarly journals A Novel DNA Polymerase Family Found inArchaea

1998 ◽  
Vol 180 (8) ◽  
pp. 2232-2236 ◽  
Author(s):  
Yoshizumi Ishino ◽  
Kayoko Komori ◽  
Isaac K. O. Cann ◽  
Yosuke Koga
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Pyrococcus Furiosus ◽  
Dna Polymerases ◽  
Open Reading Frames ◽  
Gene Products ◽  
Polymerase Gene ◽  
Hyperthermophilic Archaeon ◽  
Dna Polymerase Ii ◽  
Reading Frames

ABSTRACT One of the most puzzling results from the complete genome sequence of the methanogenic archaeon Methanococcus jannaschii was that the organism may have only one DNA polymerase gene. This is because no other DNA polymerase-like open reading frames (ORFs) were found besides one ORF having the typical α-like DNA polymerase (family B). Recently, we identified the genes of DNA polymerase II (the second DNA polymerase) from the hyperthermophilic archaeonPyrococcus furiosus, which has also at least one α-like DNA polymerase (T. Uemori, Y. Sato, I. Kato, H. Doi, and Y. Ishino, Genes Cells 2:499–512, 1997). The genes in M. jannaschiiencoding the proteins that are homologous to the DNA polymerase II ofP. furiosus have been located and cloned. The gene products of M. jannaschii expressed in Escherichia colihad both DNA polymerizing and 3′→5′ exonuclease activities. We propose here a novel DNA polymerase family which is entirely different from other hitherto-described DNA polymerases.

scholarly journals The Characteristics and Genome Analysis of vB_AviM_AVP, the First Phage Infecting Aerococcus viridans

Viruses ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 104 ◽  
Author(s):  
Hengyu Xi ◽  
Jiaxin Dai ◽  
Yigang Tong ◽  
Mengjun Cheng ◽  
Feiyang Zhao ◽  
...  
Keyword(s):  
Opportunistic Pathogen ◽  
Nucleotide Metabolism ◽  
Open Reading Frames ◽  
Gene Products ◽  
New Class ◽  
Aerococcus Viridans ◽  
Double Stranded Dna ◽  
The Family ◽  
Reading Frames ◽  
Human And Animal Diseases

Aerococcus viridans is an opportunistic pathogen that is clinically associated with various human and animal diseases. In this study, the first identified A. viridans phage, vB_AviM_AVP (abbreviated as AVP), was isolated and studied. AVP belongs to the family Myoviridae. AVP harbors a double-stranded DNA genome with a length of 133,806 bp and a G + C content of 34.51%. The genome sequence of AVP showed low similarity (<1% identity) to those of other phages, bacteria, or other organisms in the database. Among 165 predicted open reading frames (ORFs), there were only 69 gene products exhibiting similarity (≤65% identity) to proteins of known functions in the database. In addition, the other 36 gene products did not match any viral or prokaryotic sequences in any publicly available database. On the basis of the putative functions of the ORFs, the genome of AVP was divided into three modules: nucleotide metabolism and replication, structural components, and lysis. A phylogenetic analysis of the terminase large subunits and capsid proteins indicated that AVP represents a novel branch of phages. The observed characteristics of AVP indicate that it represents a new class of phages.

Detection of potential suberinase-encoding genes in Streptomyces scabiei strains and other actinobacteria

2013 ◽  
Vol 59 (5) ◽  
pp. 294-303 ◽  
Author(s):  
Doaa Komeil ◽  
Anne-Marie Simao-Beaunoir ◽  
Carole Beaulieu
Keyword(s):  
Esterase Activity ◽  
Common Scab ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Open Reading Frames ◽  
Streptomyces Scabiei ◽  
Polymerase Chain ◽  
Encoding Genes ◽  
Reading Frames ◽  
Important Disease

Streptomyces scabiei causes common scab, an economically important disease of potato tubers. Some authors have previously suggested that S. scabiei penetration into host plant tissue is facilitated by secretion of esterase enzymes degrading suberin, a lipidic biopolymer of the potato periderm. In the present study, S. scabiei EF-35 showed high esterase activity in suberin-containing media. This strain also exhibited esterase activity in the presence of other biopolymers, such as lignin, cutin, or xylan, but at a much lower level. In an attempt to identify the esterases involved in suberin degradation, translated open reading frames of S. scabiei 87-22 were examined for the presence of protein sequences corresponding to extracellular esterases of S. scabiei FL1 and of the fungus Coprinopsis cinerea VTT D-041011, which have previously been shown to be produced in the presence of suberin. Two putative extracellular suberinase genes, estA and sub1, were identified. The presence of these genes in several actinobacteria was investigated by Southern blot hybridization, and both genes were found in most common-scab-inducing strains. Moreover, reverse transcription – polymerase chain reaction performed with S. scabiei EF-35 showed that estA was expressed in the presence of various biopolymers, including suberin, whereas the sub1 gene appeared to be specifically expressed in the presence of suberin and cutin.

scholarly journals Experimental Confirmation of Global Murine Cytomegalovirus Open Reading Frames by Transcriptional Detection and Partial Characterization of Newly Described Gene Products

2006 ◽  
Vol 80 (14) ◽  
pp. 6873-6882 ◽  
Author(s):  
Qiyi Tang ◽  
Eain A. Murphy ◽  
Gerd G. Maul
Keyword(s):  
Dna Microarray ◽  
Small Animal ◽  
Immunohistochemical Localization ◽  
Open Reading Frames ◽  
Site Directed Mutagenesis ◽  
Murine Cytomegalovirus ◽  
Gene Products ◽  
System A ◽  
Human Cmv ◽  
Reading Frames

ABSTRACT Murine cytomegalovirus (MCMV) and human CMV (HCMV) share many features making the mouse system a potential small-animal model for HCMV. Although the genomic DNA sequence and the predicted open reading frames (ORFs) of MCMV have been determined, experimental evidence that the ORFs are actually transcribed has been lacking. We developed an MCMV global-DNA microarray that includes all previously predicted ORFs and 14 potential ones. A total of 172 ORFs were confirmed to be transcribed, including 7 newly discovered ORFs not previously predicted. No gene products from 10 previously predicted ORFs were detected by either DNA microarray analysis or reverse transcriptase PCR in MCMV-infected mouse fibroblasts, although 2 of those were expressed in a macrophage cell line, suggesting that potential gene products from these open reading fames are silenced in fibroblasts and required in macrophages. Immunohistochemical localization of the six newly described ORF products and three recently identified ones in cells transfected with the respective construct revealed four of the products in the nucleus and five in mitochondria. Analysis of two ORFs using site-directed mutagenesis showed that deletion of one of the mitochondrion-localized gene products led to significantly decreased replication in fibroblasts.

scholarly journals Translation Initiation from Conserved Non-AUG Codons Provides Additional Layers of Regulation and Coding Capacity

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Ivaylo P. Ivanov ◽  
Jiajie Wei ◽  
Stephen Z. Caster ◽  
Kristina M. Smith ◽  
Audrey M. Michel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Neurospora Crassa ◽  
Open Reading Frames ◽  
Reading Frame ◽  
Coding Sequence ◽  
Cell Extracts ◽  
Upstream Open Reading Frames ◽  
Reading Frames

ABSTRACT Neurospora crassa cpc-1 and Saccharomyces cerevisiae GCN4 are homologs specifying transcription activators that drive the transcriptional response to amino acid limitation. The cpc-1 mRNA contains two upstream open reading frames (uORFs) in its >700-nucleotide (nt) 5′ leader, and its expression is controlled at the level of translation in response to amino acid starvation. We used N. crassa cell extracts and obtained data indicating that cpc-1 uORF1 and uORF2 are functionally analogous to GCN4 uORF1 and uORF4, respectively, in controlling translation. We also found that the 5′ region upstream of the main coding sequence of the cpc-1 mRNA extends for more than 700 nucleotides without any in-frame stop codon. For 100 cpc-1 homologs from Pezizomycotina and from selected Basidiomycota, 5′ conserved extensions of the CPC1 reading frame are also observed. Multiple non-AUG near-cognate codons (NCCs) in the CPC1 reading frame upstream of uORF2, some deeply conserved, could potentially initiate translation. At least four NCCs initiated translation in vitro . In vivo data were consistent with initiation at NCCs to produce N-terminally extended N. crassa CPC1 isoforms. The pivotal role played by CPC1, combined with its translational regulation by uORFs and NCC utilization, underscores the emerging significance of noncanonical initiation events in controlling gene expression. IMPORTANCE There is a deepening and widening appreciation of the diverse roles of translation in controlling gene expression. A central fungal transcription factor, the best-studied example of which is Saccharomyces cerevisiae GCN4, is crucial for the response to amino acid limitation. Two upstream open reading frames (uORFs) in the GCN4 mRNA are critical for controlling GCN4 synthesis. We observed that two uORFs in the corresponding Neurospora crassa cpc-1 mRNA appear functionally analogous to the GCN4 uORFs. We also discovered that, surprisingly, unlike GCN4, the CPC1 coding sequence extends far upstream from the presumed AUG start codon with no other in-frame AUG codons. Similar extensions were seen in homologs from many filamentous fungi. We observed that multiple non-AUG near-cognate codons (NCCs) in this extended reading frame, some conserved, initiated translation to produce longer forms of CPC1, underscoring the significance of noncanonical initiation in controlling gene expression.

scholarly journals Genetic Characterization of vanG, a Novel Vancomycin Resistance Locus of Enterococcus faecalis

2000 ◽  
Vol 44 (11) ◽  
pp. 3224-3228 ◽  
Author(s):  
Stuart J. McKessar ◽  
Anne M. Berry ◽  
Jan M. Bell ◽  
John D. Turnidge ◽  
James C. Paton
Keyword(s):  
Enterococcus Faecalis ◽  
Amino Acid Sequence Identity ◽  
Vancomycin Resistance ◽  
Open Reading Frames ◽  
Pcr Analysis ◽  
Resistance Locus ◽  
Gene Products ◽  
The Individual ◽  
Reading Frames

ABSTRACT Enterococcus faecalis strain WCH9 displays a moderate level of resistance to vancomycin (MIC = 16 μg/ml) and full susceptibility to teicoplanin but is negative by PCR analysis using primers specific for all known enterococcal vancomycin resistance genotypes (vanA, vanB,vanC, vanD, and vanE). We have isolated and sequenced a novel putative vancomycin resistance locus (designated vanG), which contains seven open reading frames, from this strain. These are organized differently from those of all the other enterococcal vanloci, and, furthermore, the individual vanG gene products exhibit less than 50% amino acid sequence identity to othervan gene products.

scholarly journals Alkane Hydroxylase from Acinetobactersp. Strain ADP1 Is Encoded by alkM and Belongs to a New Family of Bacterial Integral-Membrane Hydrocarbon Hydroxylases

1998 ◽  
Vol 64 (4) ◽  
pp. 1175-1179 ◽  
Author(s):  
Andreas Ratajczak ◽  
Walter Geißdörfer ◽  
Wolfgang Hillen
Keyword(s):  
Insertional Mutagenesis ◽  
Carbon Sources ◽  
Open Reading Frames ◽  
Alkane Hydroxylase ◽  
Sequence Comparisons ◽  
New Family ◽  
Sequence Homologies ◽  
Acyl Coenzyme A ◽  
Encoding Genes ◽  
Reading Frames

ABSTRACT Degradation of long-chain alkanes by Acinetobacter sp. strain ADP1 involves rubredoxin and rubredoxin reductase. We complemented a mutant deficient in alkane utilization and sequenced four open reading frames (ORFs) on the complementing DNA. Each of these ORFs was disrupted by insertional mutagenesis on the chromosome. As determined from sequence comparisons, ORF1 and ORF4 seem to encode a rotamase of the PpiC type and an acyl coenzyme A dehydrogenase, respectively. Disruption of these ORFs does not affect alkane utilization. In contrast, the two other ORFs, alkR andalkM, are essential for growth on alkanes as sole carbon sources. alkR encodes a polypeptide with extensive homology to AraC-XylS-like transcriptional regulators. It is located next toalkM, which encodes the terminal alkane hydroxylase, but is in the opposite orientation. Sequence homologies with other bacterial integral-membrane hydrocarbon hydroxylases suggest that AlkM may be the first member of a new protein family. The genes identified here are not linked to the rubredoxin- and rubredoxin reductase-encoding genes on the Acinetobacter sp. strain ADP1 chromosome.

scholarly journals 2-Oxoglutarate:NADP+ Oxidoreductase in Azoarcus evansii: Properties and Function in Electron Transfer Reactions in Aromatic Ring Reduction

2003 ◽  
Vol 185 (20) ◽  
pp. 6119-6129 ◽  
Author(s):  
Christa Ebenau-Jehle ◽  
Matthias Boll ◽  
Georg Fuchs
Keyword(s):  
Binding Site ◽  
Electron Donor ◽  
Tricarboxylic Acid Cycle ◽  
Large Subunit ◽  
Open Reading Frames ◽  
Anaerobic Growth ◽  
Thiamine Diphosphate ◽  
Α Subunit ◽  
Cell Extracts ◽  
Reading Frames

ABSTRACT The conversion of [14C]benzoyl-coenzyme A (CoA) to nonaromatic products in the denitrifying β-proteobacterium Azoarcus evansii grown anaerobically on benzoate was investigated. With cell extracts and 2-oxoglutarate as the electron donor, benzoyl-CoA reduction occurred at a rate of 10 to 15 nmol min−1 mg−1. 2-Oxoglutarate could be replaced by dithionite (200% rate) and by NADPH (∼10% rate); in contrast NADH did not serve as an electron donor. Anaerobic growth on aromatic compounds induced 2-oxoglutarate:acceptor oxidoreductase (KGOR), which specifically reduced NADP+, and NADPH:acceptor oxidoreductase. KGOR was purified by a 76-fold enrichment. The enzyme had a molecular mass of 290 ± 20 kDa and was composed of three subunits of 63 (γ), 62 (α), and 37 (β) kDa in a 1:1:1 ratio, suggesting an (αβγ)2 composition. The native enzyme contained Fe (24 mol/mol of enzyme), S (23 mol/mol), flavin adenine dinucleotide (FAD; 1.4 mol/mol), and thiamine diphosphate (0.95 mol/mol). KGOR from A. evansii was highly specific for 2-oxoglutarate as the electron donor and accepted both NADP+ and oxidized viologens as electron acceptors; in contrast NAD+ was not reduced. These results suggest that benzoyl-CoA reduction is coupled to the complete oxidation of the intermediate acetyl-CoA in the tricarboxylic acid cycle. Electrons generated by KGOR can be transferred to both oxidized ferredoxin and NADP+, depending on the cellular needs. N-terminal amino acid sequence analysis revealed that the open reading frames for the three subunits of KGOR are similar to three adjacently located open reading frames in Bradyrhizobium japonicum. We suggest that these genes code for a very similar three-subunit KGOR, which may play a role in nitrogen fixation. The α-subunit is supposed to harbor one FAD molecule, two [4Fe-4S] clusters, and the NADPH binding site; the β-subunit is supposed to harbor one thiamine diphosphate molecule and one further [4Fe-4S] cluster; and the γ-subunit is supposed to harbor the CoA binding site. This is the first study of an NADP+-specific KGOR. A similar NADP+-specific pyruvate oxidoreductase, which contains all domains in one large subunit, has been reported for the mitochondrion of the protist Euglena gracilis and the apicomplexan Cryptosporidium parvum.

In-silico evidence of a pAO1 encoded pathway for the catabolism of tagatose derivatives in Arthrobacter nicotinovorans

Biologia ◽  
2010 ◽  
Vol 65 (5) ◽  
Author(s):  
Marius Mihăşan
Keyword(s):  
In Silico ◽  
Homology Modelling ◽  
Open Reading Frames ◽  
Gene Products ◽  
Catabolic Pathway ◽  
Arthrobacter Nicotinovorans ◽  
Similarity Searches ◽  
General Organization ◽  
Reading Frames

AbstractBased on similarity searches, two putative pathways were previously described as being encoded by the pAO1 megaplasmid of Arthrobacter nicotinovorans: an almost fully established nicotine-degrading pathway and a yet unknown putative sugar-catabolic pathway. The general organization of the open reading frames (ORFs) of the latter indicated possible gene products as targets for docking experiments, aimed at identifying possible sugar substrates of this pathway. Homology modelling and docking results with the deduced proteins of three ORFs of the putative sugar catabolic pathway indicated D-tagatose-1,6-bisphosphate as a common ligand and thus as substrate of the pathway.

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