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.

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.

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 Biosynthesis of the Pyrimidine Moiety of Thiamine Independent of the PurF Enzyme (Phosphoribosylpyrophosphate Amidotransferase) in Salmonella typhimurium: Incorporation of Stable Isotope-Labeled Glycine and Formate

1999 ◽  
Vol 181 (3) ◽  
pp. 841-848 ◽  
Author(s):  
Jodi L. Enos-Berlage ◽  
Diana M. Downs
Keyword(s):  
Stable Isotope ◽  
Salmonella Typhimurium ◽  
Isotope Labeling ◽  
De Novo ◽  
Thiamine Pyrophosphate ◽  
Gas Chromatography Mass Spectrometry ◽  
Alternative Route ◽  
Purine Pathway ◽  
Phosphoribosylpyrophosphate Amidotransferase ◽  
Pyrimidine Moiety

ABSTRACT Genetic analyses have suggested that the pyrimidine moiety of thiamine can be synthesized independently of the first enzyme of de novo purine synthesis, phosphoribosylpyrophosphate amidotransferase (PurF), in Salmonella typhimurium. To obtain biochemical evidence for and to further define this proposed synthesis, stable isotope labeling experiments were performed with two compounds, [2-13C]glycine and [13C]formate. These compounds are normally incorporated into thiamine pyrophosphate (TPP) via steps in the purine pathway subsequent to PurF. Gas chromatography-mass spectrometry analyses indicated that both of these compounds were incorporated into the pyrimidine moiety of TPP in apurF mutant. This result clearly demonstrated that the pyrimidine moiety of thiamine was being synthesized in the absence of the PurF enzyme and strongly suggested that this synthesis utilized subsequent enzymes of the purine pathway. These results were consistent with an alternative route to TPP that bypassed only the first enzyme in the purine pathway. Experiments quantitating cellular thiamine monophosphate (TMP) and TPP levels suggested that the alternative route to TPP did not function at the same capacity as the characterized pathway and determined that levels of TMP and TPP in the wild-type strain were significantly altered by the presence of purines in the medium.

scholarly journals Regulation of the nfsA Gene in Escherichia coli by SoxS

2002 ◽  
Vol 184 (1) ◽  
pp. 51-58 ◽  
Author(s):  
E. Suzanne Paterson ◽  
Sherri E. Boucher ◽  
I. B. Lambert
Keyword(s):  
Escherichia Coli ◽  
Promoter Sequence ◽  
Open Reading Frame ◽  
Base Sequence ◽  
Start Site ◽  
Transcription Start ◽  
Band Shift ◽  
Reading Frame ◽  
Shift Analysis ◽  
Small Open Reading Frame

ABSTRACT In Escherichia coli, the response to oxidative stress due to elevated levels of superoxide is mediated, in part, by the soxRS regulon. One member of the soxRS regulon, nfsA, encodes the major oxygen-insensitive nitroreductase in Escherichia coli which catalyzes the reduction of nitroaromatic and nitroheterocyclic compounds by NADPH. In this study we investigate the regulation of nfsA in response to the superoxide generating compound paraquat. The transcription start site (TSS) of nfsA was located upstream of the ybjC gene, a small open reading frame of unknown function located directly upstream of nfsA, suggesting that these two genes form an operon. The activity of the promoter associated with this TSS was confirmed with lacZ fusions and was shown to be inducible by paraquat. Footprinting and band shift analysis showed that purified His-tagged SoxS protein binds to a 20-base sequence 10 bases upstream of the −35 promoter sequence in the forward orientation, suggesting that the ybjC-nfsA promoter is a class I SoxS-dependent promoter.

scholarly journals Identification and Function of the pdxY Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5′-Phosphate Biosynthesis in Escherichia coli K-12

1998 ◽  
Vol 180 (7) ◽  
pp. 1814-1821 ◽  
Author(s):  
Yong Yang ◽  
Ho-Ching Tiffany Tsui ◽  
Tsz-Kwong Man ◽  
Malcolm E. Winkler
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Specific Activity ◽  
Salvage Pathway ◽  
Pyridoxal Kinase ◽  
Triple Mutant ◽  
Reading Frame ◽  
E Coli ◽  
Specific Activities ◽  
K 12

ABSTRACT pdxK encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5′-phosphate (PLP) coenzyme biosynthesis. The observation that pdxK null mutants still contain PL kinase activity led to the hypothesis that Escherichia coli K-12 contains at least one other B6-vitamer kinase. Here we support this hypothesis by identifying the pdxY gene (formally, open reading frame f287b) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete E. coli genome. Minimal clones of pdxY + overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette intopdxY and crossed the resultingpdxY::ΩKanr mutation into the bacterial chromosome of a pdxB mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts ofpdxK and pdxY single and double mutants. Significantly, the requirement of the pdxB pdxK pdxY triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B6vitamer kinases in E. coli and that their function is confined to the PLP salvage pathway. Last, we show thatpdxY is located downstream from pdxH (encoding PNP/PMP oxidase) and essential tyrS (encoding aminoacyl-tRNATyr synthetase) in a multifunctional operon.pdxY is completely cotranscribed with tyrS, but about 92% of tyrS transcripts terminate at a putative Rho-factor-dependent attenuator located in thetyrS-pdxY intercistronic region.

scholarly journals Identification by Subtractive Hybridization of a Novel Insertion Sequence Specific for Virulent Strains ofPorphyromonas gingivalis

1999 ◽  
Vol 67 (11) ◽  
pp. 5621-5625 ◽  
Author(s):  
Koichi Sawada ◽  
Susumu Kokeguchi ◽  
Hiroshi Hongyo ◽  
Satoko Sawada ◽  
Manabu Miyamoto ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Subtractive Hybridization ◽  
Open Reading Frame ◽  
Specific Marker ◽  
Avirulent Strain ◽  
Target Sequence ◽  
Dna Fragments ◽  
Reading Frame

ABSTRACT Subtractive hybridization was employed to isolate specific genes from virulent Porphyromonas gingivalis strains that are possibly related to abscess formation. The genomic DNA from the virulent strain P. gingivalis W83 was subtracted with DNA from the avirulent strain ATCC 33277. Three clones unique to strain W83 were isolated and sequenced. The cloned DNA fragments were 885, 369, and 132 bp and had slight homology with only Bacillus stearothermophilus IS5377, which is a putative transposase. The regions flanking the cloned DNA fragments were isolated and sequenced, and the gene structure around the clones was revealed. These three clones were located side-by-side in a gene reported as an outer membrane protein. The three clones interrupt the open reading frame of the outer membrane protein gene. This inserted DNA, consisting of three isolated clones, was designated IS1598, which was 1,396 bp (i.e., a 1,158-bp open reading frame) in length and was flanked by 16-bp terminal inverted repeats and a 9-bp duplicated target sequence. IS1598 was detected inP. gingivalis W83, W50, and FDC 381 by Southern hybridization. All three P. gingivalis strains have been shown to possess abscess-forming ability in animal models. However, IS1598 was not detected in avirulent strains of P. gingivalis, including ATCC 33277. The IS1598 may interrupt the synthesis of the outer membrane protein, resulting in changes in the structure of the bacterial outer membrane. The IS1598 isolated in this study is a novel insertion element which might be a specific marker for virulent P. gingivalisstrains.

The extrachromosomal replication of Dictyostelium plasmid Ddp2 requires a cis-acting element and a plasmid-encoded trans-acting factor

1990 ◽  
Vol 10 (7) ◽  
pp. 3727-3736
Author(s):  
B Leiting ◽  
I J Lindner ◽  
A A Noegel
Keyword(s):  
Escherichia Coli ◽  
Copy Number ◽  
Wild Strain ◽  
Open Reading Frame ◽  
Mobility Shift ◽  
Reading Frame ◽  
Cis Acting ◽  
Transformation Vector ◽  
Cis And Trans ◽  
Electrophoretic Mobility Shift Assays

Dictyostelium discoideum plasmid Ddp2 from the wild strain WS380B is a 5.8-kilobase (kb) supercoiled circle with a copy number of 300 per haploid genome. We previously described the construction of an extrachromosomally replicating transformation vector pnDeI carrying 4.7 kb of Ddp2 sequences (B. Leiting, and A. Noegel, Plasmid 20:241-248, 1988). In order to reduce the sequences required for extrachromosomal maintenance in D. discoideum, we characterized Ddp2 by sequence analysis, by deletion experiments, by transcription mapping, by electrophoretic mobility shift assays, and by expression of its single open reading frame in Escherichia coli. Two elements were involved in replication of Ddp2: a cis-acting sequence located on a 592-base-pair (bp) fragment that consisted of 220 bp of essential and 372 bp of auxiliary sequences, and a 2.7-kb open reading frame which most likely encodes a trans-acting factor. The cis- and trans-acting elements did not overlap and were shown to act independently from the location of the sequences encoding the trans-acting factor.

scholarly journals Identification of the ubiD Gene on theEscherichia coli Chromosome

2000 ◽  
Vol 182 (21) ◽  
pp. 6243-6246 ◽  
Author(s):  
Haitao Zhang ◽  
George T. Javor
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Gene Product ◽  
Mutant Strain ◽  
Amino Acid Residue ◽  
Open Reading Frame ◽  
Reading Frame

ABSTRACT The open reading frame at 86.7 min on the Escherichia coli chromosome, “yigC,” complemented aubiD mutant strain, AN66, indicating that yigCis the ubiD gene. The gene product, a 497-amino-acid-residue protein, showed extensive homology to the UPF 00096 family of proteins in the Swiss-Prot database.

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