scholarly journals Identification of the miaB Gene, Involved in Methylthiolation of Isopentenylated A37 Derivatives in the tRNA of Salmonella typhimurium andEscherichia coli

1999 ◽  
Vol 181 (23) ◽  
pp. 7256-7265 ◽  
Author(s):  
Birgitta Esberg ◽  
Hon-Chiu Eastwood Leung ◽  
Ho-Ching Tiffany Tsui ◽  
Glenn R. Björk ◽  
Malcolm E. Winkler
Keyword(s):  
Escherichia Coli ◽  
Salmonella Typhimurium ◽  
Binding Site ◽  
Binding Sites ◽  
Open Reading Frame ◽  
Iron Binding ◽  
Reading Frame ◽  
Conserved Motif ◽  
E Coli ◽  
Weak Similarity

ABSTRACT The tRNA of the miaB2508::Tn10dCm mutant of Salmonella typhimurium is deficient in the methylthio group of the modified nucleosideN 6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A37). By sequencing, we found that the Tn10dCm of this strain had been inserted into thef474 (yleA) open reading frame, which is located close to the nag locus in both S. typhimurium and Escherichia coli. By complementation of the miaB2508::Tn10dCm mutation with a minimal subcloned f474 fragment, we showed thatf474 could be identified as the miaB gene, which is transcribed in the counterclockwise direction on the bacterial chromosome. Transcriptional studies revealed two promoters upstream ofmiaB in E. coli and S. typhimurium. A Rho-independent terminator was identified downstream of themiaB gene, at which the majority (96%) of themiaB transcripts terminate in E. coli, showing that the miaB gene is part of a monocistronic operon. A highly conserved motif with three cysteine residues was present in MiaB. This motif resembles iron-binding sites in other proteins. Only a weak similarity to an AdoMet-binding site was found, favoring the idea that the MiaB protein is involved in the thiolation step and not in the methylating reaction of ms2i(o)6A37 formation.

Molecular Cloning and Characterization of a cDNA Encoding a Transferrin Homolog from Bombyx mori

1999 ◽  
Vol 380 (12) ◽  
pp. 1455-1459 ◽  
Author(s):  
Eun Young Yun ◽  
Seok Woo Kang ◽  
Jae Sam Hwang ◽  
Tae Won Goo ◽  
Sang Hyun Kim ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Cdna Sequence ◽  
The Self ◽  
Open Reading Frame ◽  
Iron Binding ◽  
Defense System ◽  
Reading Frame ◽  
E Coli ◽  
Self Defense

Abstract We isolated a cDNA representing a message that was strongly induced by injection with E. coli in Bombyx mori. The 2160 bp cDNA has an open reading frame of 644 amino acids and the deduced product a predicted molecular mass of 71 kDa. The cDNA sequence shared high homology with the transferrins known so far, and its deduced peptide had unique features of transferrins, that is, sites of cystein residues and iron binding. We suggest that the B. mori transferrin plays an important role in the self-defense system.

scholarly journals Characteristics of a New Enantioselective Thermostable Dipeptidase from Brevibacillus borstelensis BCS-1 and Its Application to Synthesis of a d-Amino-Acid-Containing Dipeptide

2004 ◽  
Vol 70 (3) ◽  
pp. 1570-1575 ◽  
Author(s):  
Dae Heoun Baek ◽  
Jae Jun Song ◽  
Seok-Joon Kwon ◽  
Chung Park ◽  
Chang-Min Jung ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Open Reading Frame ◽  
Nucleotide Sequence Analysis ◽  
Reading Frame ◽  
E Coli ◽  
Specificity Constant ◽  
Optimal Ph

ABSTRACT A new thermostable dipeptidase gene was cloned from the thermophile Brevibacillus borstelensis BCS-1 by genetic complementation of the d-Glu auxotroph Escherichia coli WM335 on a plate containing d-Ala-d-Glu. Nucleotide sequence analysis revealed that the gene included an open reading frame coding for a 307-amino-acid sequence with an M r of 35,000. The deduced amino acid sequence of the dipeptidase exhibited 52% similarity with the dipeptidase from Listeria monocytogenes. The enzyme was purified to homogeneity from recombinant E. coli WM335 harboring the dipeptidase gene from B. borstelensis BCS-1. Investigation of the enantioselectivity (E) to the P1 and P1′ site of Ala-Ala revealed that the ratio of the specificity constant (k cat /Km ) for l-enantioselectivity to the P1 site of Ala-Ala was 23.4 � 2.2 [E = (k cat /Km ) l,d /(k cat /Km ) d,d ], while the d-enantioselectivity to the P1′ site of Ala-Ala was 16.4 � 0.5 [E = (k cat /Km ) l,d /(k cat /Km ) l,l ] at 55�C. The enzyme was stable up to 55�C, and the optimal pH and temperature were 8.5 and 65�C, respectively. The enzyme was able to hydrolyze l-Asp-d-Ala, l-Asp-d-AlaOMe, Z-d-Ala-d-AlaOBzl, and Z-l-Asp-d-AlaOBzl, yet it could not hydrolyze d-Ala-l-Asp, d-Ala-l-Ala, d-AlaNH2, and l-AlaNH2. The enzyme also exhibited β-lactamase activity similar to that of a human renal dipeptidase. The dipeptidase successfully synthesized the precursor of the dipeptide sweetener Z-l-Asp-d-AlaOBzl.

scholarly journals Escherichia coli Open Reading Frame 696 Is idi, a Nonessential Gene Encoding Isopentenyl Diphosphate Isomerase

1999 ◽  
Vol 181 (15) ◽  
pp. 4499-4504 ◽  
Author(s):  
Frederick M. Hahn ◽  
Anthony P. Hurlburt ◽  
C. Dale Poulter
Keyword(s):  
Escherichia Coli ◽  
Mevalonate Pathway ◽  
Open Reading Frame ◽  
Restrictive Temperature ◽  
Isopentenyl Diphosphate ◽  
Isopentenyl Diphosphate Isomerase ◽  
Reading Frame ◽  
E Coli ◽  
Isoprene Unit ◽  
Nonessential Gene

ABSTRACT Isopentenyl diphosphate isomerase catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In eukaryotes, archaebacteria, and some bacteria, IPP is synthesized from acetyl coenzyme A by the mevalonate pathway. The subsequent isomerization of IPP to DMAPP activates the five-carbon isoprene unit for subsequent prenyl transfer reactions. In Escherichia coli, the isoprene unit is synthesized from pyruvate and glyceraldehyde-3-phosphate by the recently discovered nonmevalonate pathway. An open reading frame (ORF696) encoding a putative IPP isomerase was identified in the E. coli chromosome at 65.3 min. ORF696 was cloned into an expression vector; the 20.5 kDa recombinant protein was purified in three steps, and its identity as an IPP isomerase was established biochemically. The gene for IPP isomerase, idi, is not clustered with other known genes for enzymes in the isoprenoid pathway. E. coli FH12 was constructed by disruption of the chromosomal idi gene with the aminoglycoside 3′-phosphotransferase gene and complemented by the wild-type idi gene on plasmid pFMH33 with a temperature-sensitive origin of replication. FH12/pFMH33 was able to grow at the restrictive temperature of 44°C and FH12 lacking the plasmid grew on minimal medium, thereby establishing thatidi is a nonessential gene. Although theV max of the bacterial protein was 20-fold lower than that of its yeast counterpart, the catalytic efficiencies of the two enzymes were similar through a counterbalance inKm s. The E. coli protein requires Mg2+ or Mn2+ for activity. The enzyme contains conserved cysteine and glutamate active-site residues found in other IPP isomerases.

Identification of New Delhi metallo-β-lactamase gene (NDM-1) from a clinical isolate of Acinetobacter junii in China

2012 ◽  
Vol 58 (1) ◽  
pp. 112-115 ◽  
Author(s):  
Zhenwen Zhou ◽  
Ruili Guan ◽  
Yiyu Yang ◽  
Ling Chen ◽  
Jie Fu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Clinical Isolate ◽  
Bacterial Resistance ◽  
Open Reading Frame ◽  
New Delhi ◽  
Acinetobacter Junii ◽  
Reading Frame ◽  
E Coli ◽  
Child Patient ◽  
Lactamase Gene

New Delhi metallo-β-lactamase-1 (NDM-1) is a novel type of metallo-β-lactamase (MBL) responsible for bacterial resistance to β-lactam antibiotics. Acinetobacter junii was previously shown to possess a MBL phenotype; however, the genes responsible for this phenotype were not identified. In this study, we reported the identification of NDM-1 gene in a clinical isolate of A. junii from a child patient in China, which was resistant to all β-lactams except aztreonam but sensitive to aminoglycosides and quinolones. The cloned NDM-1 gene contained an open reading frame of 813 bp and had a nucleotide sequence 99.9% identical (812/813) to reported NDM-1 genes carried by Acinetobacter baumannii , Enterococcus faecium , Escherichia coli , and Klebsiella pneumoniae . Recombinant NDM-1 protein was successfully expressed in E. coli BL21, and antibiotic sensitivities of the NDM-1-producing E. coli were largely similar to the A. junii 1454 isolate. The findings of this study raise attention to the emergence and spread of NDM-1-carrying bacteria in China.

scholarly journals Immobilization of Escherichia coli RNA Polymerase and Location of Binding Sites by Use of Chromatin Immunoprecipitation and Microarrays

2005 ◽  
Vol 187 (17) ◽  
pp. 6166-6174 ◽  
Author(s):  
Christopher D. Herring ◽  
Marni Raffaelle ◽  
Timothy E. Allen ◽  
Elenita I. Kanin ◽  
Robert Landick ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Chromatin Immunoprecipitation ◽  
Binding Sites ◽  
Reading Frame ◽  
E Coli ◽  
Genome Wide ◽  
Polymerase Binding ◽  
Chip Chip ◽  
Negative Detection

ABSTRACT The genome-wide location of RNA polymerase binding sites was determined in Escherichia coli using chromatin immunoprecipitation and microarrays (chIP-chip). Cross-linked chromatin was isolated in triplicate from rifampin-treated cells, and DNA bound to RNA polymerase was precipitated with an antibody specific for the β′ subunit. The DNA was amplified and hybridized to “tiled” oligonucleotide microarrays representing the whole genome at 25-bp resolution. A total of 1,139 binding sites were detected and evaluated by comparison to gene expression data from identical conditions and to 961 promoters previously identified by established methods. Of the detected binding sites, 418 were located within 1,000 bp of a known promoter, leaving 721 previously unknown RNA polymerase binding sites. Within 200 bp, we were able to detect 51% (189/368) of the known σ70-specific promoters occurring upstream of an expressed open reading frame and 74% (273/368) within 1,000 bp. Conversely, many known promoters were not detected by chIP-chip, leading to an estimated 26% negative-detection rate. Most of the detected binding sites could be associated with expressed transcription units, but 299 binding sites occurred near inactive transcription units. This map of RNA polymerase binding sites represents a foundation for studies of transcription factors in E. coli and an important evaluation of the chIP-chip technique.

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 A Tetrahydrofolate-Dependent O-Demethylase, LigM, Is Crucial for Catabolism of Vanillate and Syringate in Sphingomonas paucimobilis SYK-6

2005 ◽  
Vol 187 (6) ◽  
pp. 2030-2037 ◽  
Author(s):  
Tomokuni Abe ◽  
Eiji Masai ◽  
Keisuke Miyauchi ◽  
Yoshihiro Katayama ◽  
Masao Fukuda
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Double Mutant ◽  
Open Reading Frame ◽  
Sphingomonas Paucimobilis ◽  
Reading Frame ◽  
E Coli ◽  
Demethylase Activity ◽  
Specific Activities

ABSTRACT Vanillate and syringate are converted into protocatechuate (PCA) and 3-O-methylgallate (3MGA), respectively, by O-demethylases in Sphingomonas paucimobilis SYK-6. PCA is further degraded via the PCA 4,5-cleavage pathway, while 3MGA is degraded through multiple pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and an unidentified 3MGA O-demethylase and gallate dioxygenase are participants. For this study, we isolated a 4.7-kb SmaI fragment that conferred on Escherichia coli the activity required for the conversion of vanillate to PCA. The nucleotide sequence of this fragment revealed an open reading frame of 1,413 bp (ligM), the deduced amino acid sequence of which showed 49% identity with that of the tetrahydrofolate (H4folate)-dependent syringate O-demethylase gene (desA). The metF and ligH genes, which are thought to be involved in H4folate-mediated C1 metabolism, were located just downstream of ligM. The crude LigM enzyme expressed in E. coli converted vanillate and 3MGA to PCA and gallate, respectively, with similar specific activities, and only in the presence of H4folate; however, syringate was not a substrate for LigM. The disruption of ligM led to significant growth retardation on both vanillate and syringate, indicating that ligM is involved in the catabolism of these substrates. The ability of the ligM mutant to transform vanillate was markedly decreased, and this mutant completely lost the 3MGA O-demethylase activity. A ligM desA double mutant completely lost the ability to transform vanillate, thus indicating that desA also contributes to vanillate degradation. All of these results indicate that ligM encodes vanillate/3MGA O-demethylase and plays an important role in the O demethylation of vanillate and 3MGA, respectively.

scholarly journals The apbE Gene Encodes a Lipoprotein Involved in Thiamine Synthesis in Salmonella typhimurium

1998 ◽  
Vol 180 (4) ◽  
pp. 885-891 ◽  
Author(s):  
Brian J. Beck ◽  
Diana M. Downs
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Salmonella Typhimurium ◽  
De Novo ◽  
Open Reading Frame ◽  
Thiamine Pyrophosphate ◽  
Gene Products ◽  
Reading Frame ◽  
Purine Pathway

ABSTRACT Thiamine pyrophosphate is an essential cofactor that is synthesized de novo in Salmonella typhimurium. The biochemical steps and gene products involved in the conversion of aminoimidazole ribotide (AIR), a purine intermediate, to the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine have yet to be elucidated. We have isolated mutations in a new locus (Escherichia coli open reading frame designation yojK) at 49 min on the S. typhimurium chromosome. Two significant phenotypes associated with lesions in this locus (apbE) were identified. First,apbE purF double mutants require thiamine, specifically the HMP moiety. Second, in the presence of adenine, apbE single mutants require thiamine, specifically both the HMP and the thiazole moieties. Together, the phenotypes associated with apbEmutants suggest that flux through the purine pathway has a role in regulating synthesis of the thiazole moiety of thiamine and are consistent with ApbE being involved in the conversion of AIR to HMP. The product of the apbE gene was found to be a 36-kDa membrane-associated lipoprotein, making it the second membrane protein implicated in thiamine synthesis.

Cloning of Acetoacetyl-CoA reductase and Polyhydroxybutyrate synthase genes from the local isolate Bacillus aryabhattai 6N-NRC into Escherichia coli

2021 ◽  
pp. 3299-3306
Author(s):  
Neveen M. El-Metwally ◽  
Abd El-Nasser A. Khattab ◽  
Mona S. Shafei ◽  
Reda F. Allam ◽  
Yasser M. Ragab ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Polyacrylamide Gel Electrophoresis ◽  
Growth Conditions ◽  
Open Reading Frame ◽  
Reading Frame ◽  
E Coli ◽  
Bacillus Aryabhattai ◽  
Local Isolate ◽  
Coa Reductase ◽  
Acetoacetyl Coa Reductase

Polyhydroxybutyrate (PHB) is the most known degradable biopolymer, produced by some genera of bacteria under unfavorable growth conditions. Isolation and cloning of acetoacetyl-CoA reductase (phbB) and polyhydroxybutyrate synthase (phbC) genes from local isolate previously identified as Bacillus aryabhattai 6N-NRC (GenBank accession no. MH997667.1) was achieved. Suitable primers designed for the phbB and phbC PCR approach were used to clone the phbB and phbC genes. The phbB and phbC genes were successfully isolated, cloned and the PCR amplicon 744 bp and 1089 bp corresponding to phbB and phbC genes were identified, cloned with the pET-29a (+) carrying the phbB and phbC genes, transformed and expressed in Escherichia coli BL21. The amplification of the phbB and phbC genes using specific primers of pET-29a (+) plasmid was performed. The open reading frame of phbB sequence was found to be 99.06% identical to the sequence of acetoacetyl-CoA reductase of B. aryabhattai (GenBank accession no. CP024035.1), while the open reading frame of phbC sequence was found to be 87.18% identical to the sequence of polyhydroxybutyrate synthase of B. aryabhattai (Gen Bank accession no. CP024035.1) after DNA sequencing. The analysis of the recombinant proteins from E. coli BL21 recombinant colony by tricine-polyacrylamide gel electrophoresis clarified that the expressed phbB and phbC genes in E. coli BL21 strain showed distinct bands of intensity 26.3 KD and 37.5 KD, respectively.

scholarly journals Functional Expression and Characterization of the Two Cyclic Amidohydrolase Enzymes, Allantoinase and a Novel Phenylhydantoinase, from Escherichia coli

2000 ◽  
Vol 182 (24) ◽  
pp. 7021-7028 ◽  
Author(s):  
Geun Joong Kim ◽  
Dong Eun Lee ◽  
Hak-Sung Kim
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Physical Map ◽  
Open Reading Frame ◽  
Putative Open Reading Frame ◽  
Protein Fusion ◽  
Aromatic Side Chain ◽  
Native Form ◽  
Reading Frame ◽  
E Coli

ABSTRACT A superfamily of cyclic amidohydrolases, including dihydropyrimidinase, allantoinase, hydantoinase, and dihydroorotase, all of which are involved in the metabolism of purine and pyrimidine rings, was recently proposed based on the rigidly conserved structural domains in identical positions of the related enzymes. With these conserved domains, two putative cyclic amidohydrolase genes fromEscherichia coli, flanked by related genes, were identified and characterized. From the genome sequence of E. coli, theallB gene and a putative open reading frame, tentatively designated as a hyuA (for hydantoin-utilizing enzyme) gene, were predicted to express hydrolases. In contrast to allB, high-level expression of hyuA in E. coli of a single protein was unsuccessful even under various induction conditions. We expressed HyuA as a maltose binding protein fusion protein and AllB in its native form and then purified each of them by conventional procedures. allB was found to encode a tetrameric allantoinase (453 amino acids) which specifically hydrolyzes the purine metabolite allantoin to allantoic acid. Another open reading frame, hyuA, located near 64.4 min on the physical map and known as a UUG start, coded for d-stereospecific phenylhydantoinase (465 amino acids) which is a homotetramer. As a novel enzyme belonging to a cyclic amidohydrolase superfamily, E. coli phenylhydantoinase exhibited a distinct activity toward the hydantoin derivative with an aromatic side chain at the 5′ position but did not readily hydrolyze the simple cyclic ureides. The deduced amino acid sequence of the novel phenylhydantoinase shared a significant homology (>45%) with those of allantoinase and dihydropyrimidinase, but its functional role still remains to be elucidated. Despite the unclear physiological function of HyuA, its presence, along with the allantoin-utilizing AllB, strongly suggested that the cyclic ureides might be utilized as nutrient sources in E. coli.

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