scholarly journals Engineering Desiccation Tolerance inEscherichia coli

2000 ◽  
Vol 66 (4) ◽  
pp. 1680-1684 ◽  
Author(s):  
Daniela Billi ◽  
Deborah J. Wright ◽  
Richard F. Helm ◽  
Todd Prickett ◽  
Malcolm Potts ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Phase Transition ◽  
Freeze Drying ◽  
Fold Increase ◽  
Inescherichia Coli ◽  
Vibration Frequencies ◽  
Wild Type ◽  
Air Drying ◽  
Phase Transition Temperatures ◽  
Cell Dehydration

ABSTRACT Recombinant sucrose-6-phosphate synthase (SpsA) was synthesized inEscherichia coli BL21DE3 by using the spsA gene of the cyanobacterium Synechocystis sp. strain PCC 6803. Transformants exhibited a 10,000-fold increase in survival compared to wild-type cells following either freeze-drying, air drying, or desiccation over phosphorus pentoxide. The phase transition temperatures and vibration frequencies (PO stretch) in phospholipids suggested that sucrose maintained membrane fluidity during cell dehydration.

scholarly journals Conversion of Escherichia coli pyruvate oxidase to an ‘α-ketobutyrate oxidase’

2000 ◽  
Vol 352 (3) ◽  
pp. 717-724 ◽  
Author(s):  
Ying-Ying CHANG ◽  
John E. CRONAN
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Random Mutagenesis ◽  
Fold Increase ◽  
Natural Substrate ◽  
Wild Type ◽  
Pyruvate Oxidase ◽  
Mutant Proteins ◽  
Essentially Normal

Escherichia coli pyruvate oxidase (PoxB), a lipid-activated homotetrameric enzyme, is active on both pyruvate and 2-oxobutanoate (‘α-ketobutyrate’), although pyruvate is the favoured substrate. By localized random mutagenesis of residues chosen on the basis of a modelled active site, we obtained several PoxB enzymes that had a markedly decreased activity with the natural substrate, pyruvate, but retained full activity with 2-oxobutanoate. In each of these mutant proteins Val-380had been replaced with a smaller residue, namely alanine, glycine or serine. One of these, PoxB V380A/L253F, was shown to lack detectable pyruvate oxidase activity in vivo; this protein was purified, studied and found to have a 6-fold increase in Km for pyruvate and a 10-fold lower Vmax with this substrate. In contrast, the mutant had essentially normal kinetic constants with 2-oxobutanoate. The altered substrate specificity was reflected in a decreased rate of pyruvate binding to the latent conformer of the mutant protein owing to the V380A mutation. The L253F mutation alone had no effect on PoxB activity, although it increased the activity of proteins carrying substitutions at residue 380, as it did that of the wild-type protein. The properties of the V380A/L253F protein provide new insights into the mode of substrate binding and the unusual activation properties of this enzyme.

scholarly journals Saccharomyces cerevisiae ribosomes recognize non-AUG initiation codons.

1984 ◽  
Vol 4 (7) ◽  
pp. 1191-1197 ◽  
Author(s):  
R S Zitomer ◽  
D A Walthall ◽  
B C Rymond ◽  
C P Hollenberg
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Fold Increase ◽  
Transcription Unit ◽  
Initiation Codon ◽  
Wild Type ◽  
Protein Coding ◽  
Translational Initiation ◽  
Nonsense Codon ◽  
Initiation Of Translation

A series of Saccharomyces cerevisiae plasmids and mutant derivatives containing fusions of the Escherichia coli galactokinase gene, galK, to the yeast iso-1-cytochrome c CYC1 transcription unit were used to study the sequences affecting the initiation of translation in S. cerevisiae. When the CYC1 AUG initiation codon preceded the galK AUG codon and coding sequence and either the two AUGs were out of frame with each other or a nonsense codon was located between them, the expression of the galK gene was extremely low. Deletion of the CYC1 AUG and its surrounding sequences resulted in a 100-fold increase in galK expression. This dependence of galK expression on the elimination of the CYC1 AUG codon was used to select mutations in that codon. Then the ability of these altered initiation codons to serve in translational initiation was determined by reconstruction of the CYC1 gene 3' to and in frame with them. Initiation was found to occur at the codons UUG and AUA, but not at the codons AAA and AUC. Furthermore the codon UUG, when preceded by an A three nucleotides upstream, served as a better initiation codon than when a U was substituted for the A. The efficiency of translation from these non-AUG codons was quantitated by using a CYC1/galK protein-coding fusion and measuring cellular galactokinase levels. Initiation at the UUG codon was 6.9% as efficient as initiation at the wild-type AUG codon when preceded by an A three nucleotides upstream, but was over 10-fold less efficient when a U was substituted for that A. Initiation at AUA was 0.5% as efficient as at AUG. The effects of the sequences preceding the initiation codon are discussed in light of these results.

Evolving improved Synechococcus Rubisco functional expression in Escherichia coli

2008 ◽  
Vol 414 (2) ◽  
pp. 205-214 ◽  
Author(s):  
Oliver Mueller-Cajar ◽  
Spencer M. Whitney
Keyword(s):  
Escherichia Coli ◽  
Large Subunit ◽  
Fold Increase ◽  
Functional Expression ◽  
Kinetic Properties ◽  
Selection System ◽  
Wild Type ◽  
Bisphosphate Carboxylase ◽  
E Coli ◽  
Enhanced Expression

The photosynthetic CO2-fixing enzyme Rubisco [ribulose-P2 (D-ribulose-1,5-bisphosphate) carboxylase/oxygenase] has long been a target for engineering kinetic improvements. Towards this goal we used an RDE (Rubisco-dependent Escherichia coli) selection system to evolve Synechococcus PCC6301 Form I Rubisco under different selection pressures. In the fastest growing colonies, the Rubisco L (large) subunit substitutions I174V, Q212L, M262T, F345L or F345I were repeatedly selected and shown to increase functional Rubisco expression 4- to 7-fold in the RDE and 5- to 17-fold when expressed in XL1-Blue E. coli. Introducing the F345I L-subunit substitution into Synechococcus PCC7002 Rubisco improved its functional expression 11-fold in XL1-Blue cells but could not elicit functional Arabidopsis Rubisco expression in the bacterium. The L subunit substitutions L161M and M169L were complementary in improving Rubisco yield 11-fold, whereas individually they improved yield ∼5-fold. In XL1-Blue cells, additional GroE chaperonin enhanced expression of the I174V, Q212L and M262T mutant Rubiscos but engendered little change in the yield of the more assembly-competent F345I or F345L mutants. In contrast, the Rubisco chaperone RbcX stimulated functional assembly of wild-type and mutant Rubiscos. The kinetic properties of the mutated Rubiscos varied with noticeable reductions in carboxylation and oxygenation efficiency accompanying the Q212L mutation and a 2-fold increase in Kribulose-P2 (KM for the substrate ribulose-P2) for the F345L mutant, which was contrary to the ∼30% reductions in Kribulose-P2 for the other mutants. These results confirm the RDE systems versatility for identifying mutations that improve functional Rubisco expression in E. coli and provide an impetus for developing the system to screen for kinetic improvements.

scholarly journals In Vivo Increase in Resistance to Ciprofloxacin inEscherichia coli Associated with Deletion of the C-Terminal Part of MarR

2000 ◽  
Vol 44 (7) ◽  
pp. 1865-1868 ◽  
Author(s):  
Hans-Jörg Linde ◽  
Frank Notka ◽  
Michaela Metz ◽  
Bernd Kochanowski ◽  
Peter Heisig ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Northern Blotting ◽  
Pulsed Field Gel Electrophoresis ◽  
Inescherichia Coli ◽  
Wild Type ◽  
Terminal Part ◽  
Causative Relationship

ABSTRACT We recovered two isolates (EP1 and EP2) of Escherichia coli from the same patient that had identical pulsed-field gel electrophoresis patterns but required different MICs of ciprofloxacin (CIP): 16 and 256 mg/liter for EP1 and EP2, respectively. Both isolates had mutations in the quinolone resistance-determining regions of GyrA (Ser83Leu and Asp87Tyr) and ParC (Ser80Ile), but not in those regions of GyrB or ParE. Isolate EP2 was also more resistant to chloramphenicol, tetracyclines, cefuroxime, and organic solvents. A deletion of adenine (A) 1821 was found in marR of isolate EP2, which resulted in an 18-amino-acid C-terminal deletion in the MarR protein. The causative relationship between ΔA1821 and the Mar phenotype was demonstrated both by the replacement of the wild-typemarR by marR ΔA1821 in isolate EP1 and by complementation with the wild-type marR intrans in isolate EP2. In isolate EP2 complemented with wild-type marR, susceptibility to chloramphenicol was restored completely, whereas susceptibility to CIP was restored only incompletely. Northern blotting demonstrated increased expression ofmarA and acrAB but not of soxS in isolate EP2 compared to EP1. In conclusion, the deletion of A1821 inmarR in the clinical isolate EP2 caused an increase in the MICs of CIP and unrelated antibiotics. Presumably, the C-terminal part of MarR is necessary for proper repressor function.

Conjugational Hyperrecombination Achieved by Derepressing the LexA Regulon, Altering the Properties of RecA Protein and Inactivating Mismatch Repair in Escherichia coli K-12

Genetics ◽  
2003 ◽  
Vol 163 (4) ◽  
pp. 1243-1254
Author(s):  
Vladislav A Lanzov ◽  
Irina V Bakhlanova ◽  
Alvin J Clark
Keyword(s):  
Escherichia Coli ◽  
Mismatch Repair ◽  
Reca Protein ◽  
Cumulative Effect ◽  
Fold Increase ◽  
Reca Gene ◽  
Repair System ◽  
Wild Type ◽  
Mismatch Repair System ◽  
K 12

Abstract The frequency of recombinational exchanges (FRE) that disrupt co-inheritance of transferred donor markers in Escherichia coli Hfr by F- crosses differs by up to a factor of two depending on physiological factors and culture conditions. Under standard conditions we found FRE to be 5.01 ± 0.43 exchanges per 100-min units of DNA length for wild-type strains of the AB1157 line. Using these conditions we showed a cumulative effect of various mutations on FRE. Constitutive SOS expression by lexA gene inactivation (lexA71::Tn5) and recA gene mutation (recA730) showed, respectively, ∼4- and 7-fold increases of FRE. The double lexA71 recA730 combination gave an ∼17-fold increase in FRE. Addition of mutS215::Tn10, inactivating the mismatch repair system, to the double lexA recA mutant increased FRE to ∼26-fold above wild-type FRE. Finally, we showed that another recA mutation produced as much SOS expression as recA730 but increased FRE only 3-fold. We conclude that three factors contribute to normally low FRE under standard conditions: repression of the LexA regulon, the properties of wild-type RecA protein, and a functioning MutSHL mismatch repair system. We discuss mechanisms by which the lexA, recA, and mutS mutations may elevate FRE cumulatively to obtain hyperrecombination.

scholarly journals Identification, cloning, and expression of the Escherichia coli pyrazinamidase and nicotinamidase gene, pncA.

1996 ◽  
Vol 40 (6) ◽  
pp. 1426-1431 ◽  
Author(s):  
R Frothingham ◽  
W A Meeker-O'Connell ◽  
E A Talbot ◽  
J W George ◽  
K N Kreuzer
Keyword(s):  
Escherichia Coli ◽  
Genetic Locus ◽  
Fold Increase ◽  
Pyridine Nucleotide ◽  
Cloning And Expression ◽  
Insertion Mutant ◽  
Activity Levels ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
E Coli

Pyrazinamide (PZA) is one of the three most important drugs for treatment of Mycobacterium tuberculosis infections. The antibacterial activity of PZA requires a bacterial enzyme, pyrazinamidase (PZAase), which hydrolyzes PZA to form pyrazinoic acid and ammonia. Most PZA-resistant clinical M. tuberculosis isolates lack PZAase activity. With the goal of eventually identifying and characterizing the M.tuberculosis PZAase gene, we began with the more tractable organism, Escherichia coli, which also has PZAase activity. We screened a transposon-generated E. coli insertion mutant library, using a qualitative PZAase assay. Two PZAase-negative mutants out of 4,000 colonies screened were identified. In each mutant, the transposon interrupted the same 639-bp open reading frame (ORF), ORF1. The expression of ORF1 on a multicopy plasmid complemented a PZAase-negative mutant, leading to PZAase activity levels approximately 10-fold greater than those of the wild type. PZA has a structure similar to that of nicotinamide, a pyridine nucleotide cycle intermediate, so we tested our strains for nicotinamidase activity (EC 3.5.1.19) (genetic locus pncA). The construct with multiple plasmid copies of ORF1 had an approximately 10-fold increase in levels of nicotinamidase activity. This overexpressing strain could utilize nicotinamide as a sole nitrogen source, through wild-type E. coli cannot. We conclude that a single E. coli enzyme accounts for both PZAase and nicotinamidase activities and that ORF1 is the E.coli PZAase and nicotinamidase gene, pncA.

Saccharomyces cerevisiae ribosomes recognize non-AUG initiation codons

1984 ◽  
Vol 4 (7) ◽  
pp. 1191-1197
Author(s):  
R S Zitomer ◽  
D A Walthall ◽  
B C Rymond ◽  
C P Hollenberg
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Fold Increase ◽  
Transcription Unit ◽  
Initiation Codon ◽  
Wild Type ◽  
Protein Coding ◽  
Translational Initiation ◽  
Nonsense Codon ◽  
Initiation Of Translation

A series of Saccharomyces cerevisiae plasmids and mutant derivatives containing fusions of the Escherichia coli galactokinase gene, galK, to the yeast iso-1-cytochrome c CYC1 transcription unit were used to study the sequences affecting the initiation of translation in S. cerevisiae. When the CYC1 AUG initiation codon preceded the galK AUG codon and coding sequence and either the two AUGs were out of frame with each other or a nonsense codon was located between them, the expression of the galK gene was extremely low. Deletion of the CYC1 AUG and its surrounding sequences resulted in a 100-fold increase in galK expression. This dependence of galK expression on the elimination of the CYC1 AUG codon was used to select mutations in that codon. Then the ability of these altered initiation codons to serve in translational initiation was determined by reconstruction of the CYC1 gene 3' to and in frame with them. Initiation was found to occur at the codons UUG and AUA, but not at the codons AAA and AUC. Furthermore the codon UUG, when preceded by an A three nucleotides upstream, served as a better initiation codon than when a U was substituted for the A. The efficiency of translation from these non-AUG codons was quantitated by using a CYC1/galK protein-coding fusion and measuring cellular galactokinase levels. Initiation at the UUG codon was 6.9% as efficient as initiation at the wild-type AUG codon when preceded by an A three nucleotides upstream, but was over 10-fold less efficient when a U was substituted for that A. Initiation at AUA was 0.5% as efficient as at AUG. The effects of the sequences preceding the initiation codon are discussed in light of these results.

scholarly journals Thermus thermophilus as a Cell Factory for the Production of a Thermophilic Mn-Dependent Catalase Which Fails To Be Synthesized in an Active Form in Escherichia coli

2004 ◽  
Vol 70 (7) ◽  
pp. 3839-3844 ◽  
Author(s):  
Aurelio Hidalgo ◽  
Lorena Betancor ◽  
Renata Moreno ◽  
Olga Zafra ◽  
Felipe Cava ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Thermus Thermophilus ◽  
Active Form ◽  
Laboratory Strain ◽  
Fold Increase ◽  
Cell Factory ◽  
Wild Type ◽  
Genes Encoding ◽  
Mutant Derivative

ABSTRACT Thermostable Mn-dependent catalases are promising enzymes in biotechnological applications as H2O2-detoxifying systems. We cloned the genes encoding Mn-dependent catalases from Thermus thermophilus HB27 and HB8 and a less thermostable mutant carrying two amino acid replacements (M129V and E293G). When the wild-type and mutant genes were overexpressed in Escherichia coli, unmodified or six-His-tagged proteins of the expected size were overproduced as inactive proteins. Several attempts to obtain active forms or to activate the overproduced proteins were unsuccessful, even when soluble and thermostable proteins were used. Therefore, a requirement for a Thermus-specific activation factor was suggested. To overcome this problem, the Mn-dependent catalase genes were overexpressed directly in T. thermophilus under the control of the Pnar promoter. This promoter belongs to a respiratory nitrate reductase from of T. thermophilus HB8, whose transcription is activated by the combined action of nitrate and anoxia. Upon induction in T. thermophilus HB8, a 20- to 30-fold increase in catalase specific activity was observed, whereas a 90- to 110-fold increase was detected when the laboratory strain T. thermophilus HB27::nar was used as the host. The thermostability of the overproduced wild-type catalase was identical to that previously reported for the native enzyme, whereas decreased stability was detected for the mutant derivative. Therefore, our results validate the use of T. thermophilus as an alternative cell factory for the overproduction of thermophilic proteins that fail to be expressed in well-known mesophilic hosts.

scholarly journals Human mismatch-repair protein MutL homologue 1 (MLH1) interacts with Escherichia coli MutL and MutS in vivo and in vitro: a simple genetic system to assay MLH1 function

2003 ◽  
Vol 371 (1) ◽  
pp. 183-189 ◽  
Author(s):  
Barbara QUARESIMA ◽  
Pietro ALIFANO ◽  
Pierfrancesco TASSONE ◽  
Enrico V. AVVEDIMENTO ◽  
Francesco S. COSTANZO ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mismatch Repair ◽  
Fold Increase ◽  
Genetic System ◽  
Mutation Rates ◽  
Wild Type ◽  
Mismatched Dna ◽  
Repair Protein

A simple genetic system has been developed to test the effect of over-expression of wild-type or mutated human MutL homologue 1 (hMLH1) proteins on methyl-directed mismatch repair (MMR) in Escherichia coli. The system relies on detection of Lac+ revertants using MMR-proficient or MMR-deficient E. coli strains carrying a lac +1 frameshift mutation expressing hMLH1 proteins. We report that expression of wild-type hMLH1 protein causes an approx. 19-fold increase in mutation rates. The mutator phenotype was due to the ability of hMLH1 protein to interact with bacterial MutL and MutS proteins, thereby interfering with the formation of complexes between MMR proteins and mismatched DNA. Conversely, expression of proteins encoded by alleles deriving from hereditary-non-polyposis-colon-cancer (HNPCC) families decreases mutation rates, depending on the specific amino acid substitutions. These effects parallel the MutL-and MutS-binding and ATP-binding/hydrolysis activities of the mutated proteins.

scholarly journals Site-directed mutagenesis of rat muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: role of Asp-130 in the 2-kinase domain

1994 ◽  
Vol 300 (1) ◽  
pp. 111-115 ◽  
Author(s):  
M H Rider ◽  
K M Crepin ◽  
M De Cloedt ◽  
L Bertrand ◽  
L Hue
Keyword(s):  
Escherichia Coli ◽  
Skeletal Muscle ◽  
Fold Increase ◽  
Kinase Domain ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Wild Type ◽  
Fluorescence Measurements

Asp-130 of the recombinant skeletal-muscle 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase was mutated into Ala in order to study its role in catalysis and/or substrate binding. The D130A mutant displayed a 30- to 140-fold decreased 2-kinase Vmax, depending on the pH, and a 30- and 60-fold increase in Km for MgATP and Fru-6-P respectively at pH 8.5 compared with the wild-type. Mutagenesis of Asp-130 to Ala had no effect on the 2-phosphatase activity, and fluorescence measurements indicated that the changes in kinetic properties of PFK-2 in the D130A mutant were not due to instability. The role of Asp-130 in the 2-kinase reaction is discussed and compared with that of Asp-103 of 6-phosphofructo-1-kinase from Escherichia coli, which binds Mg2+.

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