High-level expression of Bacillus subtilis tryptophanyl-tRNA synthetase in Escherichia coli

1990 ◽  
Vol 68 (2) ◽  
pp. 492-495 ◽  
Author(s):  
Wen Shi ◽  
King-Chuen Chow ◽  
J. Tze-Fei Wong
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Large Scale ◽  
Trna Synthetase ◽  
Start Codon ◽  
Temperature Sensitive ◽  
Gene Encoding ◽  
Scale Preparation ◽  
High Level

The trpS gene encoding Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) was prepared from the pUC8-derived pTSQ2 plasmid, mutagenized to introduce an EcoRI site immediately in front of the ATG start codon, and inserted into the pKK223-3 vector downstream to the tac promoter to yield the pKSW1 plasmid. Upon induction with isopropyl-β-D-thiogalactopyranoside, Escherichia coli JM109[pKSW1] cells synthesized TrpRS to a level corresponding to 45% of total cell proteins. This high level of gene expression facilitates large scale preparation of TrpRS for physical studies, detection of in vivo degradation of mutant forms of TrpRS, and comparative assays of TrpRS by [3H]Trp-tRNA formation and by Trp-hydroxamate formation for the purpose of mutant characterization. Finally, since pKSW1 could complement the temperature-sensitive TrpRS mutation on E. coli trpS 10343 cells, defective mutations of the trpS gene on pKSW1 would be detectible on the basis of complementation testing.Key words: tryptophan-tRNA, aminoacyl-tRNA synthetase, Bacillus subtilis.

The Bacillus subtilis pnbA gene encoding p-nitrobenzyl esterase: cloning, sequence and high-level expression in Escherichia coli

Gene ◽  
1994 ◽  
Vol 151 (1-2) ◽  
pp. 37-43 ◽  
Author(s):  
Joseph Zock ◽  
Cathleen Cantwell ◽  
James Swartling ◽  
Roland Hodges ◽  
Tonya Pohl ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
High Level Expression ◽  
Gene Encoding ◽  
Expression In Escherichia Coli ◽  
High Level

Overproduction of the Bacillus subtilis glutamyl-tRNA synthetase in its host and its toxicity to Escherichia coli

1998 ◽  
Vol 44 (4) ◽  
pp. 378-381 ◽  
Author(s):  
Martin Pelchat ◽  
Lucille Lacoste ◽  
Fu Yang ◽  
Jacques Lapointe
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Recombinant Plasmid ◽  
Specific Activity ◽  
Shuttle Vector ◽  
Fold Increase ◽  
Trna Synthetase ◽  
E Coli ◽  
Downstream Region

The Bacillus subtilis glutamyl-tRNA synthetase (GluRS), encoded by the gltX gene, aminoacylates its homologous tRNAGlu and tRNAGln with glutamate. This gene was cloned with its sigmaA promoter and a downstream region including a rho-independent terminator in the shuttle vector pRB394 for Escherichia coli and B. subtilis. Transformation of B. subtilis with this recombinant plasmid (pMP411) led to a 30-fold increase of glutamyl-tRNA synthetase specific activity in crude extracts. Transformation of E. coli with this plasmid gave no recombinants, but transformation with plasmids bearing an altered gltX was successful. These results indicate that the presence of B. subtilis glutamyl-tRNA synthetase is lethal for E. coli, probably because this enzyme glutamylates tRNA1Gln in vivo as it does in vitro.Key words: glutamyl-tRNA synthetase overproduction, Bacillus subtilis, toxicity, Escherichia coli.

scholarly journals Quinolone resistance locus nfxD of Escherichia coli is a mutant allele of the parE gene encoding a subunit of topoisomerase IV.

1997 ◽  
Vol 41 (1) ◽  
pp. 175-179 ◽  
Author(s):  
D M Breines ◽  
S Ouabdesselam ◽  
E Y Ng ◽  
J Tankovic ◽  
S Shah ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mutant Allele ◽  
Quinolone Resistance ◽  
Temperature Sensitive ◽  
Resistance Locus ◽  
Topoisomerase Iv ◽  
Gene Encoding ◽  
Resistance Phenotype ◽  
E Coli ◽  
High Level

The locus nfxD, which contributes to high-level quinolone resistance in Escherichia coli KF111b (gyrAr nfxB nfxD), is only expressed in the presence of a gyrA mutation, and maps to the region of the parC and parE genes, was outcrossed into strain KF130, creating strain DH161 (gyrAr nfxD). DNA sequence analysis of DH161 revealed no changes in the topoisomerase IV parC quinolone resistance-determining region but did identify a single T-to-A mutation in parE at codon 445, leading to a change from Leu to His. Full-length cloned parE+ partially complemented the resistance phenotype in KF111b and DH161, but did not complement the resistance phenotype in strain KF130 (gyrAr). No complementation was seen with cloned, truncated parE+. To confirm these findings, gyrAr was first outcrossed from KF130 into E. coli W3110parE10 [parE temperature sensitive(Ts)] and KL16. The transduced strains KL16 and W3110parE10 were subsequently transformed with plasmids containing cloned parE from DH161 or KL16. Cloned parE from DH161 increased norfloxacin resistance in the parE(Ts) background twofold at 30 degrees C and fourfold at 42 degrees C compared to those for cloned parE from KL16. The same experiment with a non-Ts background revealed a twofold increase in the norfloxacin MIC at both 30 and 42 degrees C. These data identify the nfxD conditional resistance locus as a mutant allele of parE. This report is the first of a quinolone-resistant parE mutant and confirms the role of topoisomerase IV as a secondary target of norfloxacin in E. coli.

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

scholarly journals LOCALIZED MUTAGENESIS FOR THE ISOLATION OF TEMPERATURE-SENSITIVE MUTANTS OF ESCHERICHIA COLI AFFECTED IN PROTEIN SYNTHESIS

Genetics ◽  
1979 ◽  
Vol 91 (2) ◽  
pp. 215-227
Author(s):  
W Scott Champney
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Temperature Sensitive ◽  
Chromosomal Locus ◽  
Ribosomal Subunits ◽  
Prolonged Incubation ◽  
Temperature Sensitive Mutants ◽  
Inhibition Of Growth ◽  
Localized Mutagenesis

ABSTRACT Two variations of the method of localized mutagenesis were used to introduce mutations into the 72 min region of the Escherichia coli chromosome. Twenty temperature-sensitive mutants, with linkage to markers in this region, have been examined. Each strain showed an inhibition of growth in liquid medium at 44°, and 19 of the mutants lost viability upon prolonged incubation at this temperature. A reduction in the rate of in vivo RNA and protein synthesis was observed for each mutant at 44°, relative to a control strain. Eleven of the mutants were altered in growth sensitivity or resistance to one or more of three ribosomal antibiotics. The incomplete assembly of ribosomal subunits was detected in nine strains grown at 44°. The characteristics of these mutants suggest that many of them are altered in genes for translational or transcriptional components, consistent with the clustering of these genes at this chromosomal locus.

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

A gene encoding a tyrosine tRNA synthetase is located near Sacs in Bacillus subtilis

DNA Sequence ◽  
1991 ◽  
Vol 1 (4) ◽  
pp. 251-261 ◽  
Author(s):  
P. Glaser ◽  
F. Kunst ◽  
M. Débarbouillé ◽  
A. Vertès ◽  
A. Danchin ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Trna Synthetase ◽  
Gene Encoding

scholarly journals Overproduction of SecA Suppresses the Export Defect Caused by a Mutation in the Gene Encoding the Escherichia coli Export Chaperone SecB

1999 ◽  
Vol 181 (10) ◽  
pp. 3010-3017 ◽  
Author(s):  
Heather A. Cook ◽  
Carol A. Kumamoto
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane Proteins ◽  
Protein A ◽  
In Vivo Studies ◽  
Wild Type ◽  
Gene Encoding ◽  
Precursor Proteins ◽  
Additional Support ◽  
Insight Into

ABSTRACT SecB is a cytosolic protein required for rapid and efficient export of particular periplasmic and outer membrane proteins inEscherichia coli. SecB promotes export by stabilizing newly synthesized precursor proteins in a nonnative conformation and by targeting the precursors to the inner membrane. Biochemical studies suggest that SecB facilitates precursor targeting by binding to the SecA protein, a component of the membrane-embedded translocation apparatus. To gain more insight into the functional interaction of SecB and SecA, in vivo, mutations in the secA locus that compensate for the export defect caused by the secBmissense mutation secBL75Q were isolated. Two suppressors were isolated, both of which led to the overproduction of wild-type SecA protein. In vivo studies demonstrated that the SecBL75Q mutant protein releases precursor proteins at a lower rate than does wild-type SecB. Increasing the level of SecA protein in the cell was found to reverse this slow-release defect, indicating that overproduction of SecA stimulates the turnover of SecBL75Q-precursor complexes. These findings lend additional support to the proposed pathway for precursor targeting in which SecB promotes targeting to the translocation apparatus by binding to the SecA protein.

scholarly journals PhhB, a Pseudomonas aeruginosa Homolog of Mammalian Pterin 4a-Carbinolamine Dehydratase/DCoH, Does Not Regulate Expression of Phenylalanine Hydroxylase at the Transcriptional Level

1999 ◽  
Vol 181 (9) ◽  
pp. 2789-2796 ◽  
Author(s):  
Jian Song ◽  
Tianhui Xia ◽  
Roy A. Jensen
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Phenylalanine Hydroxylase ◽  
Transcriptional Level ◽  
Gene Encoding ◽  
Catalytic Function ◽  
Genes Encoding ◽  
Catalytic Role ◽  
Regulatory Effects

ABSTRACT Pterin 4a-carbinolamine dehydratase is bifunctional in mammals. In addition to playing a catalytic role in pterin recycling in the cytoplasm, it plays a regulatory role in the nucleus, where it acts as a dimerization-cofactor component (called DCoH) for the transcriptional activator HNF-1α. A thus far unique operon in Pseudomonas aeruginosa contains a gene encoding a homolog (PhhB) of the regulatory dehydratase, together with genes encoding phenylalanine hydroxylase (PhhA) and aromatic aminotransferase (PhhC). Using complementation of tyrosine auxotrophy in Escherichia colias a functional test, we have found that the in vivo function of PhhA requires PhhB. Strikingly, mammalian DCoH was an effective substitute for PhhB, and either one was effective in trans. Surprisingly, the required presence of PhhB for complementation did not reflect a critical positive regulatory effect of phhB onphhA expression. Rather, in the absence of PhhB, PhhA was found to be extremely toxic in E. coli, probably due to the nonenzymatic formation of 7-biopterin or a similar derivative. However, bacterial PhhB does appear to exert modest regulatory effects in addition to having a catalytic function. PhhB enhances the level of PhhA two- to threefold, as was demonstrated by gene inactivation ofphhB in P. aeruginosa and by comparison of the levels of expression of PhhA in the presence and absence of PhhB inEscherichia coli. Experiments using constructs having transcriptional and translational fusions with a lacZreporter indicated that PhhB activates PhhA at the posttranscriptional level. Regulation of PhhA and PhhB is semicoordinate; both PhhA and PhhB are induced coordinately in the presence of eitherl-tyrosine or l-phenylalanine, but PhhB exhibits a significant basal level of activity that is lacking for PhhA. Immunoprecipitation and affinity chromatography showed that PhhA and PhhB form a protein-protein complex.

scholarly journals Frequent birth-and-death events throughout perforin-1 evolution

2020 ◽  
Author(s):  
Miguel Araujo-Voces ◽  
Victor Quesada
Keyword(s):  
Preliminary Analysis ◽  
Large Scale ◽  
Mammalian Species ◽  
Genomic Data ◽  
Start Codon ◽  
High Similarity ◽  
Gene Encoding ◽  
Coding Sequences ◽  
Copy Numbers ◽  
Multiple Species

Abstract Background Through its ability to open pores in cell membranes, perforin-1 plays a key role in the immune system. Consistent with this role, the gene encoding perforin shows hallmarks of complex evolutionary events, including amplification and pseudogenization, in multiple species. A large proportion of these events occurred in phyla for which scarce genomic data were available. However, recent large-scale genomics projects have added a wealth of information on those phyla. Using this input, we annotated perforin-1 homologs in more than eighty species including mammals, reptiles, birds, amphibians and fishes. Results We have annotated more than 400 perforin genes in all groups studied. Most mammalian species only have one perforin locus, which may contain a related pseudogene. However, we found four independent small expansions in unrelated members of this class. We could reconstruct the full-length coding sequences of only a few avian perforin genes, although we found incomplete and truncated forms of these gene in other birds. In the rest of reptilia, perforin-like genes can be found in at least three different loci containing up to twelve copies. Notably, mammals, non-avian reptiles, amphibians, and possibly teleosts share at least one perforin-1 locus as assessed by flanking genes. Finally, fish genomes contain multiple perforin loci with varying copy numbers and diverse exon/intron patterns. We have also found evidence for shorter genes with high similarity to the C2 domain of perforin in several teleosts. A preliminary analysis suggests that these genes arose at least twice during evolution from perforin-1 homologs. Conclusions The assisted annotation of new genomic assemblies shows complex patterns of birth-and-death events in the evolution of perforin. These events include duplication/pseudogenization in mammals, multiple amplifications and losses in reptiles and fishes and at least one case of partial duplication with a novel start codon in fishes.

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