scholarly journals Genes from Pseudomonas sp. Strain BS Involved in the Conversion of l-2-Amino-Δ2-Thiazolin-4-Carbonic Acid to l-Cysteine

2002 ◽  
Vol 68 (5) ◽  
pp. 2179-2187 ◽  
Author(s):  
Toshikazu Shiba ◽  
Kohji Takeda ◽  
Misako Yajima ◽  
Makoto Tadano
Keyword(s):  
Carbonic Acid ◽  
Open Reading Frames ◽  
Synthetic Activity ◽  
Gene Products ◽  
Gene Encoding ◽  
E Coli ◽  
Cysteine Synthesis ◽  
Molecular Masses ◽  
Dna Fragment ◽  
Derivatives Of

ABSTRACT dl-2-amino-Δ2-thiazolin-4-carbonic acid (dl-ATC) is a substrate for cysteine synthesis in some bacteria, and this bioconversion has been utilized for cysteine production in industry. We cloned a DNA fragment containing the genes involved in the conversion of l-ATC to l-cysteine from Pseudomonas sp. strain BS. The introduction of this DNA fragment into Escherichia coli cells enabled them to convert l-ATC to cysteine via N-carbamyl-l-cysteine (l-NCC) as an intermediate. The smallest recombinant plasmid, designated pTK10, contained a 2.6-kb insert DNA fragment that has l-cysteine synthetic activity. The nucleotide sequence of the insert DNA revealed that two open reading frames (ORFs) encoding proteins with molecular masses of 19.5 and 44.7 kDa were involved in the l-cysteine synthesis from dl-ATC. These ORFs were designated atcB and atcC, respectively, and their gene products were identified by overproduction of proteins encoded in each ORF and by the maxicell method. The functions of these gene products were examined using extracts of E. coli cells carrying deletion derivatives of pTK10. The results indicate that atcB and atcC are involved in the conversion of l-ATC to l-NCC and the conversion of l-NCC to cysteine, respectively. atcB was first identified as a gene encoding an enzyme that catalyzes thiazolin ring opening. AtcC is highly homologous with l-N-carbamoylases. Since both enzymes can only catalyze the l-specific conversion from l-ATC to l-NCC or l-NCC to l-cysteine, it is thought that atcB and atcC encode l-ATC hydrolase and N-carbamyl-l-cysteine amidohydrolase, respectively.

scholarly journals EfrAB, an ABC Multidrug Efflux Pump in Enterococcus faecalis

2003 ◽  
Vol 47 (12) ◽  
pp. 3733-3738 ◽  
Author(s):  
Eun-Woo Lee ◽  
M. Nazmul Huda ◽  
Teruo Kuroda ◽  
Tohru Mizushima ◽  
Tomofusa Tsuchiya
Keyword(s):  
Enterococcus Faecalis ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Multidrug Efflux ◽  
Chromosomal Dna ◽  
Multidrug Efflux Pump ◽  
E Coli ◽  
Dna Fragment

ABSTRACT A DNA fragment responsible for resistance to antimicrobial agents was cloned from the chromosomal DNA of Enterococcus faecalis ATCC 29212 by using drug-hypersensitive mutant Escherichia coli KAM32 as a host cell. Cells of E. coli KAM32 harboring a recombinant plasmid (pAEF82) carrying the DNA fragment became resistant to many structurally unrelated antimicrobial agents, such as norfloxacin, ciprofloxacin, doxycycline, acriflavine, 4′,6-diamidino-2-phenylindole, tetraphenylphosphonium chloride, daunorubicin, and doxorubicin. Since the sequence of the whole genome of E. faecalis is known, we sequenced several portions of the DNA insert in plasmid pAEF82 and identified two open reading frames within the insert. We designated the genes efrA and efrB. A search of the deduced amino acid sequences of EfrA and EfrB revealed that they are similar to each other and that they belong to the ATP-binding cassette (ABC) family of multidrug efflux transporters. Transformed E. coli KAM32 cells harboring efrAB showed energy-dependent efflux of acriflavine. The efflux activity was inhibited by reserpine, verapamil, and sodium-o-vanadate, known inhibitors of ABC efflux pumps.

scholarly journals Microbial Proline 4-Hydroxylase Screening and Gene Cloning

1999 ◽  
Vol 65 (9) ◽  
pp. 4028-4031 ◽  
Author(s):  
Takeshi Shibasaki ◽  
Hideo Mori ◽  
Shigeru Chiba ◽  
Akio Ozaki
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Genomic Library ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Open Reading Frames ◽  
Original Strain ◽  
Degenerate Primers ◽  
E Coli ◽  
Dna Fragment

ABSTRACT Microbial proline 4-hydroxylases, which hydroxylate freel-proline totrans-4-hydroxy-l-proline, were screened in order to establish an industrial system for biotransformation of l-proline totrans-4-hydroxy-l-proline. Enzyme activities were detected in eight strains, including strains ofDactylosporangium spp. and Amycolatopsis spp. The Dactylosporangium sp. strain RH1 enzyme was partially purified 3,300-fold and was estimated to be a monomer polypeptide with an apparent molecular mass of 31 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Degenerate primers based on the N-terminal amino acid sequence of the 31-kDa polypeptide were synthesized in order to amplify the corresponding 71-bp DNA fragment. A 5.5-kbp DNA fragment was isolated by using the 71-bp fragment labeled with digoxigenin as a probe for a genomic library ofDactylosporangium sp. strain RH1 constructed inEscherichia coli. One of the open reading frames found in the cloned DNA, which encoded a 272-amino-acid polypeptide (molecular mass, 29,715 daltons), was thought to be a proline 4-hydroxylase gene. The gene was expressed in E. coli as a fused protein with the N-terminal 34 amino acids of the β-galactosidase α-fragment. The E. coli recombinant exhibited proline 4-hydroxylase activity that was 13.6-fold higher than the activity in the original strain, Dactylosporangium sp. strain RH1. No homology was detected with other 2-oxoglutarate-dependent dioxygenases when databases were searched; however, the histidine motif conserved in 2-oxoglutarate-dependent dioxygenases was found in the gene.

scholarly journals Effect of multiple copies ofrpoBCon the rate of RNA synthesis inEscherichia coli

1986 ◽  
Vol 48 (2) ◽  
pp. 61-64 ◽  
Author(s):  
Elena C. Guzman ◽  
Alfonso Jimenez-Sanchez
Keyword(s):  
Dna Replication ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Synthetic Activity ◽  
Gene Products ◽  
Replication Forks ◽  
E Coli ◽  
Multiple Copies ◽  
Autogenous Regulation ◽  
Rate Of Movement

SummaryThe cloning of therpoBandrpoCgenes in a high copy number vector inE. coliincreased the amount of the encoded gene products, the β and β′ subunits of RNA polymerase. However, this unexpectedly caused a 30–50% decrease in RNA synthetic activity which alternatively induced a reduction of growth rate and enlargement of cell size, and decreased the DNA replication time. The results can be explained by autogenous regulation of the RNA polymerase genes by the ββ′ subunits. A relation between the decrease in number of transcription units and the observed higher rate of movement of DNA replication forks is discussed.

Cloning and analysis of the aroB gene encoding dehydroquinate synthase from Corynebacterium glutamicum

1999 ◽  
Vol 45 (10) ◽  
pp. 885-890 ◽  
Author(s):  
Min-Ah Han ◽  
Heung-Shick Lee ◽  
Choong-Ill Cheon ◽  
Kyung-Hee Min ◽  
Myeong-Sok Lee
Keyword(s):  
Escherichia Coli ◽  
Corynebacterium Glutamicum ◽  
Auxotrophic Mutant ◽  
Gene Products ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli ◽  
Dna Library ◽  
Genomic Dna Library ◽  
Dehydroquinate Synthase

The aroB gene encoding dehydroquinate synthase of Corynebacterium glutamicum has been cloned by complementation of an aro auxotrophic mutant of Escherichia coli with the genomic DNA library. The recombinant plasmid contained a 1.4-kb fragment that complemented the Escherichia coli dehydroquinate-synthase-deficient mutant. The nucleotide sequences of the subcloned DNA has been determined. The sequences contain an open reading frame of 360 codons, from which a protein with a molecular mass of about 38 kDa could be predicted. This is consistent with the size of the AroB protein expressed in E. coli. Alignment of different prokaryotic and eukaryotic aroB gene products reveals an overall identity ranging from 29 to 57% and the presence of several highly conserved regions.Key words: Corynebacterium glutamicum, aromatic amino acid biosynthetic gene, dehydroquinate synthase, aroB gene.

Cloning and characterization of the N-acetylglucosamine operon of Escherichia coli

1990 ◽  
Vol 68 (1) ◽  
pp. 123-137 ◽  
Author(s):  
Krishna G. Peri ◽  
Hughes Goldie ◽  
E. Bruce Waygood
Keyword(s):  
Escherichia Coli ◽  
Genomic Library ◽  
Sodium Dodecyl ◽  
Open Reading Frames ◽  
Open Reading Frame ◽  
Gene Products ◽  
Reading Frame ◽  
E Coli ◽  
Palindromic Sequences ◽  
Repetitive Extragenic Palindromic

Three enzymes are required for N-acetylglucosamine (NAG) utilization in Escherichia coli: enzyme IInag (gene nagE), N-acetylglucosamine-6-phosphate deacetylase (gene nagA), and glucosamine-6-phosphate isomerase (gene nagB). The three genes are located near 16 min on the E. coli chromosome. A strain of E. coli, KPN9, incapable of utilizing N-acetylglucosamine, was used to screen a genomic library of E. coli for a complementing recombinant colicin E1 plasmid that allowed for growth on N-acetylglucosamine. Plasmid pLC5-21 was found to contain all three known nag genes on a 5.7-kilobase (5.7-kb) fragment of DNA. The products of these nag genes were identified by complementation of E. coli strains with mutations in nagA, nagB, and nagE. The gene products from the 5.7-kb fragment were identified by [35S]methionine-labelled maxicells and autoradiography of sodium dodecyl sulphate – polyacrylamide electrophoresis gels. The gene products had the following relative masses (Mrs: nagE, 62 000; nagA, 45 000; nagB, 29 000. In addition, another product of Mr 44 000 was detected. The genes have been sequenced to reveal an additional open reading frame (nagC), a putative catabolite activator protein binding site that may control nagB and nagE, putative rho-independent terminator sites for nagB and nagE, and sequence homologies for RNA polymerase binding sites preceding each of the open reading frames, except for nagA. The calculated molecular weights (MWs) of the gene products derived from the sequence are as follows: nagA, 40 954; nagB, 29 657; nagC, 44 664; nagE, 68 356. No role is known for nagC, although a number of regulatory roles appear to be plausible. No obvious transcriptional termination site distal to nagC was found and another open reading frame begins after nagC. This gene, nagD, was isolated separately from pLC5-21, and the sequence revealed a protein with a calculated MW of 27 181. The nagD gene is followed by repetitive extragenic palindromic sequences. The nag genes appear to be organized in an operon: [Formula: see text]Key words: N-acetylglucosamine, N-acetylglucosamine-6-P deacetylase, glucosamine-6-P isomerase, repetitive extragenic palindromic sequences, catabolite repression.

scholarly journals Gene Cluster Responsible for Validamycin Biosynthesis in Streptomyces hygroscopicus subsp. jinggangensis 5008

2005 ◽  
Vol 71 (9) ◽  
pp. 5066-5076 ◽  
Author(s):  
Yi Yu ◽  
Linquan Bai ◽  
Kazuyuki Minagawa ◽  
Xiaohong Jian ◽  
Lei Li ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Control Agent ◽  
Open Reading Frames ◽  
Streptomyces Hygroscopicus ◽  
E Coli ◽  
Sheath Blight Disease ◽  
Dna Fragment ◽  
Blight Disease

ABSTRACT A gene cluster responsible for the biosynthesis of validamycin, an aminocyclitol antibiotic widely used as a control agent for sheath blight disease of rice plants, was identified from Streptomyces hygroscopicus subsp. jinggangensis 5008 using heterologous probe acbC, a gene involved in the cyclization of d-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone of the acarbose biosynthetic gene cluster originated from Actinoplanes sp. strain SE50/110. Deletion of a 30-kb DNA fragment from this cluster in the chromosome resulted in loss of validamycin production, confirming a direct involvement of the gene cluster in the biosynthesis of this important plant protectant. A sequenced 6-kb fragment contained valA (an acbC homologue encoding a putative cyclase) as well as two additional complete open reading frames (valB and valC, encoding a putative adenyltransferase and a kinase, respectively), which are organized as an operon. The function of ValA was genetically demonstrated to be essential for validamycin production and biochemically shown to be responsible specifically for the cyclization of d-sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone in vitro using the ValA protein heterologously overexpressed in E. coli. The information obtained should pave the way for further detailed analysis of the complete biosynthetic pathway, which would lead to a complete understanding of validamycin biosynthesis.

scholarly journals DNA-FACE™ - An Escherichia coli-based DNA Amplification-Expression Technology for Automatic Assembly of Concatemeric ORFs and Proteins

2021 ◽  
Author(s):  
Piotr M. Skowron ◽  
Agnieszka Zylicz-Stachula
Keyword(s):  
Escherichia Coli ◽  
Animal Studies ◽  
Immunological Response ◽  
Dna Amplification ◽  
Open Reading Frames ◽  
Biological Drugs ◽  
E Coli ◽  
Potential Applications ◽  
Dna Fragment ◽  
High Level

DNA-FACE™ (DNA Fragment Amplification & Concatemeric Expressed Nucleic Acids and Proteins) is a universal biotechnological platform, developed as Escherichia coli (E. coli) system. It is based on the ordered, head-to-tail directional ligation of the amplified DNA fragments. The technology enables the construction of targeted biomolecules - genetically programmed, concatemeric DNA, RNA, and proteins, designed to fit a particular task. The constructed, “artificial” (never seen in Nature) tandem repeat macromolecules, with specialized functions, may contain up to 500 copies of monomeric units. The technology greatly exceeds the current capabilities of chemical gene synthesis. The vector-enzymatic DNA fragment amplification assembles the DNA segments, forming continuous Open Reading Frames (ORFs). The obtained ORFs are ready for high-level expression in E. coli without a need for subcloning. The presented method has potential applications in pharmaceutical industry and tissue engineering, including vaccines, biological drugs, drug delivery systems, mass-production of peptide-derived biomaterials, industrial and environmental processes. The technology has been patented worldwide and used successfully in the construction of anti-HBV vaccines, pro-regenerative biological drugs and, recently, the anti-SARS-CoV-2 vaccine. The anti-SARS-CoV-2 vaccine, developed using the DNA-FACE™ technology, is nontoxic and induces strong immunological response to recombinant human spike and nucleocapsid proteins, as shown in animal studies.

scholarly journals Chlamydia trachomatis Serovar L2 Can Utilize Exogenous Lipoic Acid through the Action of the Lipoic Acid Ligase LplA1

2010 ◽  
Vol 192 (23) ◽  
pp. 6172-6181 ◽  
Author(s):  
Aishwarya V. Ramaswamy ◽  
Anthony T. Maurelli
Keyword(s):  
Chlamydia Trachomatis ◽  
Lipoic Acid ◽  
Open Reading Frames ◽  
Sequence Motifs ◽  
Gene Encoding ◽  
E Coli ◽  
Genes Encoding ◽  
Surrogate Host ◽  
Lipoate Synthase

ABSTRACT Lipoic acid is an essential protein bound cofactor that is vital for the functioning of several important enzymes involved in central metabolism. Genomes of all sequenced chlamydiae show the presence of two genes encoding lipoic acid ligases and one gene encoding a lipoate synthase. However, the roles of these proteins in lipoic acid utilization or biosynthesis have not yet been characterized. The two distinct lipoic acid ligases in Chlamydia trachomatis serovar L2, LplA1Ct and LplA2Ct (encoded by the open reading frames ctl0537 and ctl0761) display moderate identity with Escherichia coli LplA (30 and 27%, respectively) but possess amino acid sequence motifs that are well conserved among all lipoyl protein ligases. The putative lipoic acid synthase LipACt, encoded by ctl0815, is ca. 43% identical to the E. coli LipA homolog. We demonstrate here the presence of lipoylated proteins in C. trachomatis serovar L2 and show that the lipoic acid ligase LplA1Ct is capable of utilizing exogenous lipoic acid for the lipoylation Therefore, host-derived lipoic acid may be important for intracellular growth and development. Based on genetic complementation in a surrogate host, our study also suggests that the C. trachomatis serovar L2 LipA homolog may not be functional in vivo.

scholarly journals A Gene Encoding l-Methionine γ-Lyase Is Present in Enterobacteriaceae Family Genomes: Identification and Characterization of Citrobacter freundiil-Methionine γ-Lyase

2005 ◽  
Vol 187 (11) ◽  
pp. 3889-3893 ◽  
Author(s):  
Ilya V. Manukhov ◽  
Daria V. Mamaeva ◽  
Sergei M. Rastorguev ◽  
Nicolai G. Faleev ◽  
Elena A. Morozova ◽  
...  
Keyword(s):  
Shigella Flexneri ◽  
Open Reading Frames ◽  
Gene Encoding ◽  
High Sequence Identity ◽  
E Coli ◽  
The Family ◽  
Serovar Typhimurium ◽  
Identification And Characterization ◽  
Reading Frames

ABSTRACT Citrobacter freundii cells produce l-methionine γ-lyase when grown on a medium containing l-methionine. The nucleotide sequence of the hybrid plasmid with a C. freundii EcoRI insert of about 3.0 kbp contained two open reading frames, consisting of 1,194 nucleotides and 1,296 nucleotides, respectively. The first one (denoted megL) encoded l-methionine γ-lyase. The enzyme was overexpressed in Escherichia coli and purified. The second frame encoded a protein belonging to the family of permeases. Regions of high sequence identity with the 3′-terminal part of the C. freundii megL gene located in the same regions of Salmonella enterica serovar Typhimurium, Shigella flexneri, E. coli, and Citrobacter rodentium genomes were found.

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