Browse
Submit Data
Databases
API
Help

Dataset Information

16 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Cloning and nucleotide sequence of the firA gene and the firA200(Ts) allele from Escherichia coli.

ABSTRACT: The Escherichia coli gene firA, previously reported to code for a small, histonelike DNA-binding protein, has been cloned and found to reside immediately downstream from skp, a gene previously identified as the firA locus. firA encodes a 36-kDa protein. The mutant firA200(Ts) allele was also cloned and shown to contain three mutations, each mutation giving rise to a single amino acid change. Partially purified wild-type FirA (from a firA+ strain) and mutant FirA [from a firA200(Ts) strain] proteins have amino-terminal sequences predicted from their common DNA sequences. Both proteins lack an N-terminal methionine. Modest overexpression of wild-type or mutant FirA restored wild-type growth to firA200(Ts) strains at 43 degrees C, whereas high-level expression of wild-type FirA was required for more complete suppression of the rifampin sensitivity of firA200(Ts) rpoB double mutants. High-level expression of mutant FirA did not suppress this rifampin sensitivity.

SUBMITTER: Dicker IB

PROVIDER: S-EPMC207192 | biostudies-other | 1991 Jan

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Publications

Cloning and nucleotide sequence of the firA gene and the firA200(Ts) allele from Escherichia coli.

Dicker I B IB Seetharam S S

Journal of bacteriology 19910101 1

The Escherichia coli gene firA, previously reported to code for a small, histonelike DNA-binding protein, has been cloned and found to reside immediately downstream from skp, a gene previously identified as the firA locus. firA encodes a 36-kDa protein. The mutant firA200(Ts) allele was also cloned and shown to contain three mutations, each mutation giving rise to a single amino acid change. Partially purified wild-type FirA (from a firA+ strain) and mutant FirA [from a firA200(Ts) strain] prote ...[more]

PMID: 1987124

Similar Datasets

Escherichia coli purB gene: cloning, nucleotide sequence, and regulation by purR.

Project description:Escherichia coli purB encodes adenylosuccinate lyase (ASL), the enzyme that catalyzes step 8 in the pathway for de novo synthesis of IMP and also the final reaction in the two-step sequence from IMP to AMP. Gene purB was cloned and found to encode an ASL protein of 435 amino acids having a calculated molecular weight of 49,225. E. coli ASL is homologous to the corresponding enzymes from Bacillus subtilis and chickens and also to fumarase from B. subtilis. Gene phoP is 232 bp downstream of purB. Gene purB is regulated threefold by the purine pool and purR. Transcriptional regulation of purB involves binding of the purine repressor to the 16-bp conserved pur regulon operator. The purB operator is 224 bp downstream of the transcription start site and overlaps codons 62 to 67 in the protein-coding sequence.

| S-EPMC205686 | biostudies-other

Cloning, nucleotide sequence, and overexpression of the L-rhamnose isomerase gene from Pseudomonas stutzeri in Escherichia coli.

Project description:The gene encoding L-rhamnose isomerase (L-RhI) from Pseudomonas stutzeri was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the L-RhI gene revealed an open reading frame of 1,290 bp coding for a protein of 430 amino acid residues with a predicted molecular mass of 46,946 Da. A comparison of the deduced amino acid sequence with sequences in relevant databases indicated that no significant homology has previously been identified. An amino acid sequence alignment, however, suggested that the residues involved in the active site of L-RhI from E. coli are conserved in that from P. stutzeri. The L-RhI gene was then overexpressed in E. coli cells under the control of the T5 promoter. The recombinant clone, E. coli JM109, produced significant levels of L-RhI activity, with a specific activity of 140 U/mg and a volumetric yield of 20,000 U of soluble enzyme per liter of medium. This reflected a 20-fold increase in the volumetric yield compared to the value for the intrinsic yield. The recombinant L-RhI protein was purified to apparent homogeneity on the basis of three-step chromatography. The purified recombinant enzyme showed a single band with an estimated molecular weight of 42,000 in a sodium dodecyl sulfate-polyacrylamide gel. The overall enzymatic properties of the purified recombinant L-RhI protein were the same as those of the authentic one, as the optimal activity was measured at 60 degrees C within a broad pH range from 5.0 to 11.0, with an optimum at pH 9.0.

| S-EPMC427750 | biostudies-literature

Molecular cloning, nucleotide sequence, and expression in Escherichia coli of a hemolytic toxin (aerolysin) gene from Aeromonas trota.

Project description:Aeromonas trota AK2, which was derived from ATCC 49659 and produces the extracellular pore-forming hemolytic toxin aerolysin, was mutagenized with the transposon mini-Tn5Km1 to generate a hemolysin-deficient mutant, designated strain AK253. Southern blotting data indicated that an 8.7-kb NotI fragment of the genomic DNA of strain AK253 contained the kanamycin resistance gene of mini-Tn5Km1. The 8.7-kb NotI DNA fragment was cloned into the vector pGEM5Zf(-) by selecting for kanamycin resistance, and the resultant clone, pAK71, showed aerolysin activity in Escherichia coli JM109. The nucleotide sequence of the aerA gene, located on the 1.8-kb ApaI-EcoRI fragment, was determined to consist of 1,479 bp and to have an ATG initiation codon and a TAA termination codon. An in vitro coupled transcription-translation analysis of the 1.8-kb region suggested that the aerA gene codes for a 54-kDa protein, in agreement with nucleotide sequence data. The deduced amino acid sequence of the aerA gene product of A. trota exhibited 99% homology with the amino acid sequence of the aerA product of Aeromonas sobria AB3 and 57% homology with the amino acid sequences of the products of the aerA genes of Aeromonas salmonicida 17-2 and A. sobria 33.

| S-EPMC106413 | biostudies-literature

Cloning, nucleotide sequence, and expression of the Escherichia coli gene encoding carnitine dehydratase.

Project description:Carnitine dehydratase from Escherichia coli O44 K74 is an inducible enzyme detectable in cells grown anaerobically in the presence of L-(-)-carnitine or crotonobetaine. The purified enzyme catalyzes the dehydration of L-(-)-carnitine to crotonobetaine (H. Jung, K. Jung, and H.-P. Kleber, Biochim. Biophys. Acta 1003:270-276, 1989). The caiB gene, encoding carnitine dehydratase, was isolated by oligonucleotide screening from a genomic library of E. coli O44 K74. The caiB gene is 1,215 bp long, and it encodes a protein of 405 amino acids with a predicted M(r) of 45,074. The identity of the gene product was first assessed by its comigration in sodium dodecyl sulfate-polyacrylamide gels with the purified enzyme after overexpression in the pT7 system and by its enzymatic activity. Moreover, the N-terminal amino acid sequence of the purified protein was found to be identical to that predicted from the gene sequence. Northern (RNA) analysis showed that caiB is likely to be cotranscribed with at least one other gene. This other gene could be the gene encoding a 47-kDa protein, which was overexpressed upstream of caiB.

| S-EPMC205453 | biostudies-other

Menaquinone (vitamin K2) biosynthesis: cloning, nucleotide sequence, and expression of the menC gene from Escherichia coli.

Project description:The benzenoid aromatic compound o-succinylbenzoic acid is formed by dehydration of the prearomatic compound 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid by the enzyme o-succinylbenzoate synthase, encoded by the menC gene. A 1.3-kb PstI-PvuII fragment was found to complement the menC mutation. The complete nucleotide sequence of this fragment revealed a single open reading frame of 954 bp capable of encoding a 35-kDa protein. A consensus sequence for a ribosomal binding site but no promoter consensus sequences were found. However, the first base of the initiating codon of this open reading frame overlaps the upstream menB gene termination codon, suggesting an operon-like organization for these genes. Consistent with this suggestion, the menB promoter can initiate transcription of the menC gene.

| S-EPMC204947 | biostudies-other

Nucleotide sequence analysis of the enterotoxigenic Escherichia coli Ent plasmid.

Project description:We report here the complete nucleotide sequence of pEntH10407 (65 147 bp), an enterotoxigenic Escherichia coli enterotoxin plasmid (Ent plasmid), which is self-transmissible at low frequency. Within the plasmid, we identified 100 open reading frames (ORFs) which could encode polypeptides. These ORFs included regions encoding heat-labile (LT) and heat-stable (STIa) enterotoxins, regions encoding tools for plasmid replication and an incomplete tra (conjugation) region. The LT and STIa region was located 13.5 kb apart and was surrounded by three IS1s and an IS600 in opposite reading orientations, indicating that the enterotoxin genes may have been horizontally transferred into the plasmid. We identified a single RepFIIA replication region (2.0 kb) including RepA proteins similar to RepA1, RepA2, RepA3 and RepA4. The incomplete tra region was made up of 17 tra genes, which were nearly identical to the corresponding genes of R100, and showed evidence of multiple insertions of ISEc8 and ISEc8-like elements. These data suggest that pEntH10407 has the mosaic nature characteristic of bacterial virulence plasmids, which contains information about its evolution. Although the tra genes might originally have rendered pEntH10407 self-transferable to the same degree as R100, multiple insertion events have occurred in the tra region of pEntH10407 to make it less mobile. Another self-transmissible plasmid might help pEntH10407 to transfer efficiently into H10407 strain. In this paper, we suggest another possibility: that the enterotoxigenic H10407 strain might be formed by auto-transfer of pEntH10407 at a low rate using the incomplete tra region.

| S-EPMC2762410 | biostudies-literature

DNA sequence analysis of the dnaK gene of Escherichia coli B and of two dnaK genes carrying the temperature-sensitive mutations dnaK7(Ts) and dnaK756(Ts).

Project description:The DNA sequence of the dnaK gene of Escherichia coli was analyzed. The nucleotide sequence of the wild-type dnaK gene of E. coli B differed from that of E. coli K-12 in 15 bp, none of which altered the amino acid sequence. Two temperature-sensitive dnaK mutations were examined by cloning and sequence analyses. Results showed that one dnaK mutation, dnaK7(Ts), was a one-base substitution of T for C at nucleotide position 448 in the open reading frame yielding an amber nonsense codon. The other mutation, dnaK756(Ts), consisted of base substitutions (A for G) at three nucleotide positions, 95, 1364, and 1403, in the open reading frame resulting in an aspartic acid codon in place of a glycine codon.

| S-EPMC206061 | biostudies-other

Cloning, nucleotide sequence, and expression of the Escherichia coli fabD gene, encoding malonyl coenzyme A-acyl carrier protein transacylase.

Project description:The Escherichia coli fabD gene encoding malonyl coenzyme A-acyl carrier protein transacylase (MCT) was cloned by complementation of a thermosensitive E. coli fabD mutant (fabD89). Expression of the fabD gene in an appropriate E. coli expression vector resulted in an accumulation of the MCT protein of up to 10% of total soluble protein, which was accompanied by an approximately 1,000-fold increase in the MCT activity. DNA sequence analysis and expression studies revealed that the fabD gene is part of an operon consisting of at least three genes involved in fatty acid biosynthesis. Comparison with available DNA and protein data bases suggest that a 3-ketoacyl-acyl carrier protein synthase and a ketoacyl-acyl carrier protein reductase gene are located immediately upstream and downstream, respectively, of fabD within this fab operon. Western immunoblot analysis with antiserum raised against wild-type E. coli MCT showed that the fabD89 allele encodes a polypeptide with an apparent molecular weight of 27,000 in addition to the normal MCT protein of 32,000. The nature of the temperature-sensitive fabD89 gene product is discussed.

| S-EPMC205936 | biostudies-other

Cloning, nucleotide sequence, and expression in Escherichia coli of the gene for poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis.

Project description:The extracellular poly(3-hydroxybutyrate) depolymerase gene from Alcaligenes faecalis T1 was cloned into Escherichia coli DH1 by using the plasmid pUC8. An A. faecalis T1 genomic library was prepared in E. coli from a partial Sau3AI digest and screened with antibody against the depolymerase. Of the 29 antibody-positive clones, 1 (pDP14), containing about 4 kilobase pairs of A. faecalis T1 DNA, caused expression of a high level of depolymerase activity in E. coli. The enzyme purified from E. coli was not significantly different from the depolymerase of A. faecalis in molecular weight, immunological properties, peptide map, specific activity, or substrate specificity. Most of the expressed enzyme was found to be localized in the periplasmic space of E. coli, although about 10% of the total activity was found in the culture medium. Results of a deletion experiment with pDP14 showed that a large SalI fragment of about 2 kilobase pairs was responsible for expression of the enzyme in E. coli. The nucleotide sequence of the large SalI fragment has been determined. Comparison of the deduced amino terminus with that obtained from sequence analysis of the purified protein indicated that poly(3-hydroxybutyrate) depolymerase exists as a 488-amino-acid precursor with a signal peptide of 27 amino acids.

| S-EPMC209571 | biostudies-other

Cloning and nucleotide sequence of the Streptococcus pneumoniae hyaluronidase gene and purification of the enzyme from recombinant Escherichia coli.

Project description:A gene bank of Sau3A1-generated Streptococcus pneumoniae type 23 DNA fragments was constructed in Escherichia coli K-12 with the low-copy-number cosmid vector pOU61cos. Clone lysates were screened by immunoblotting using a mouse antiserum raised against a crude pneumococcal hyaluronidase preparation. One immunoreactive clone was isolated, and it produced high level of hyaluronidase activity. This clone contained a recombinant cosmid (designated pJCP800) with an approximately 35-kb DNA insert, and the putative hyaluronidase coding sequence was subcloned into pBluescript SK as a 3.8-kb PstI-ClaI fragment (designated pJCP802). The complete nucleotide sequence of this insert was determined. The region included an open reading frame sufficient to encode a polypeptide with an M(r) of 107,751. An active hyaluronidase with an M(r) of approximately 89,000 was purified to homogeneity from E. coli DH5 alpha(pJCP802). N-terminal amino acid sequence analysis of the purified protein suggested that translation initiation was occurring primarily at a TTG codon within the major open reading frame. However, immunoblot analysis using antiserum raised against the purified 89-kDa hyaluronidase indicated that E. coli DH5 alpha(pJCP802) also expressed the 107-kDa form of the enzyme. This antiserum labelled a 107-kDa protein in partially purified hyaluronidase preparations from S. pneumoniae. The hyaluronidase activity in this pneumococcal extract was also neutralized by the antiserum.

| S-EPMC186229 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data