scholarly journals Plasmid pCS1966, a New Selection/Counterselection Tool for Lactic Acid Bacterium Strain Construction Based on the oroP Gene, Encoding an Orotate Transporter from Lactococcus lactis

2008 ◽  
Vol 74 (15) ◽  
pp. 4772-4775 ◽  
Author(s):  
Christian Solem ◽  
Els Defoor ◽  
Peter Ruhdal Jensen ◽  
Jan Martinussen
Keyword(s):  
Lactococcus Lactis ◽  
Triosephosphate Isomerase ◽  
Attachment Site ◽  
Auxotrophic Mutant ◽  
Synthetic Promoter ◽  
Gene Encoding ◽  
E Coli ◽  
Bacterium Strain ◽  
Selection For ◽  
Different Levels

ABSTRACT In this paper we describe the new selection/counterselection vector pCS1966, which is suitable for both sequence-specific integration based on homologous recombination and integration in a bacteriophage attachment site. This plasmid harbors oroP, which encodes a dedicated orotate transporter, and can replicate only in Escherichia coli. Selection for integration is performed primarily by resistance to erythromycin; alternatively, the ability to utilize orotate as a pyrimidine source in a pyrimidine auxotrophic mutant could be utilized. Besides allowing the cell to utilize orotate, the transporter renders the cell sensitive to 5-fluoroorotate. This sensitivity is used to select for loss of the plasmid. When expressed from its own promoter, oroP was toxic to E. coli, whereas in Lactococcus lactis the level of expression of oroP from a chromosomal copy was too low to confer 5-fluoroorotate sensitivity. In order to obtain a plasmid that confers 5-fluoroorotate sensitivity when it is integrated into the chromosome of L. lactis and at the same time can be stably maintained in E. coli, the expression of the oroP gene was controlled from a synthetic promoter conferring these traits. To demonstrate its use, a number of L. lactis strains expressing triosephosphate isomerase (tpiA) at different levels were constructed.

scholarly journals 6-Phosphogluconate dehydrogenase from Lactococcus lactis: a role for arginine residues in binding substrate and coenzyme

1999 ◽  
Vol 338 (1) ◽  
pp. 55-60 ◽  
Author(s):  
Emmanuel TETAUD ◽  
Stefania HANAU ◽  
Jeremy M. WELLS ◽  
Richard W. F. Le PAGE ◽  
Margaret J. ADAMS ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Gene Encoding ◽  
E Coli ◽  
Coenzyme Binding ◽  
Phosphogluconate Dehydrogenase ◽  
Arginine Residues ◽  
Binding Pockets ◽  
Binding Residues ◽  
Cloned Gene ◽  
Binding Substrate

A gene encoding 6-phosphogluconate dehydrogenase (6-PGDH, EC 1.1.1.44) was identified from the homofermentative lactic acid bacterium Lactococcus lactis, by complementation of Escherichia coli mutants. The cloned gene was then expressed to high levels in E. coli and the protein purified for kinetic analysis. The enzyme had a Km for 6-phosphogluconate of 15.4±1.4 µM and for NADP of 1.9±0.2 µM at pH 7.5. Sequence comparison of the L. lactis 6-PGDH with the corresponding enzyme derived from the pathogenic protozoan Trypanosoma brucei and sheep liver revealed the substrate-binding residues to be identical in all three species, although the three coenzyme-binding pockets differed slightly. A totally conserved arginine residue (Arg-447), believed to bind the 6-phosphate of substrate, was mutated to lysine, aspartate, alanine or tryptophan. In each case enzyme activity was lost, confirming an essential role for this residue on activity. A second arginine (Arg-34), believed to be critical in binding the 2´-phosphate of cofactor NADP+, was mutated to a tyrosine residue, as found in one atypical isoform of the enzyme in Bacillus subtilis. This alteration led to decrease in affinity for NADP+ of nearly three orders of magnitude. A second 6-PGDH gene has been identified from the genome of B. subtilis. This second isoform contains an arginine (Arg-34) in this position, suggesting that B. subtilis has two 6-PGDHs with different coenzyme specificities.

Cloning and analysis of the aroB gene encoding dehydroquinate synthase from Corynebacterium glutamicum

1999 ◽  
Vol 45 (10) ◽  
pp. 885-890 ◽  
Author(s):  
Min-Ah Han ◽  
Heung-Shick Lee ◽  
Choong-Ill Cheon ◽  
Kyung-Hee Min ◽  
Myeong-Sok Lee
Keyword(s):  
Escherichia Coli ◽  
Corynebacterium Glutamicum ◽  
Auxotrophic Mutant ◽  
Gene Products ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli ◽  
Dna Library ◽  
Genomic Dna Library ◽  
Dehydroquinate Synthase

The aroB gene encoding dehydroquinate synthase of Corynebacterium glutamicum has been cloned by complementation of an aro auxotrophic mutant of Escherichia coli with the genomic DNA library. The recombinant plasmid contained a 1.4-kb fragment that complemented the Escherichia coli dehydroquinate-synthase-deficient mutant. The nucleotide sequences of the subcloned DNA has been determined. The sequences contain an open reading frame of 360 codons, from which a protein with a molecular mass of about 38 kDa could be predicted. This is consistent with the size of the AroB protein expressed in E. coli. Alignment of different prokaryotic and eukaryotic aroB gene products reveals an overall identity ranging from 29 to 57% and the presence of several highly conserved regions.Key words: Corynebacterium glutamicum, aromatic amino acid biosynthetic gene, dehydroquinate synthase, aroB gene.

scholarly journals Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate

2014 ◽  
Vol 81 (1) ◽  
pp. 130-138 ◽  
Author(s):  
James Kirby ◽  
Minobu Nishimoto ◽  
Ruthie W. N. Chow ◽  
Edward E. K. Baidoo ◽  
George Wang ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Xylose Reductase ◽  
Pentose Phosphate ◽  
Terpene Biosynthesis ◽  
Content Type ◽  
E Coli ◽  
Pathway Expression ◽  
Terpene Synthesis ◽  
Selection For

ABSTRACTTerpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO2. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C5sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of adxsdeletion inEscherichia coligrown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-typeE. coliyajOgene, annotated as a putative xylose reductase, or via various mutations in the nativeribBgene.In vitroanalysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway inE. colifor production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.

Associations of PrP genotypes with live weight and slaughter traits in an experimental flock of Swaledale sheep in Great Britain

2006 ◽  
Vol 82 (5) ◽  
pp. 607-614 ◽  
Author(s):  
W. Y. N. Man ◽  
S. Brotherstone ◽  
B. G. Merrell ◽  
W. A. Murray ◽  
B. Villanueva
Keyword(s):  
Great Britain ◽  
Maternal Effects ◽  
Mixed Models ◽  
Fixed Effects ◽  
Genetic Selection ◽  
Live Weight ◽  
Eradication Programme ◽  
Gene Encoding ◽  
Significant Relationships ◽  
Selection For

AbstractPolymorphisms at codons 136, 154 and 171 of the gene encoding the prion protein (PrP) are associated with susceptibility to classical scrapie in sheep. Genetic selection for scrapie resistance based on PrP genotypes is central to the scrapie eradication programme in Great Britain but there are concerns about how this may affect other economically important traits. The objective of this study was to evaluate associations of PrP genotypes with live weight and slaughter traits in a hill sheep breed in Great Britain. Data used were from an experimental flock of Swaledale sheep in which the alleles ARR, ARQ, AHQ and VRQ were present. About 1450 genotyped lambs with birth, marking and weaning weights, and 620 with slaughter records were used for the study. Mixed models with various fixed effects and random direct genetic and maternal effects were tested to determine the appropriate model to use for each trait. None of the differences in lamb performance between PrP genotypes consistently reached significance. Therefore, this study does not support existence of significant relationships between PrP genotype and lamb performance traits in this breed.

Glycosylated flavones as selective inhibitors of topoisomerase IV.

1997 ◽  
Vol 41 (5) ◽  
pp. 992-998 ◽  
Author(s):  
F X Bernard ◽  
S Sablé ◽  
B Cameron ◽  
J Provost ◽  
J F Desnottes ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Topoisomerase Ii ◽  
Coli Strain ◽  
Selective Inhibitors ◽  
Topoisomerase Iv ◽  
Gene Encoding ◽  
Cottonseed Flour ◽  
Dna Topoisomerase ◽  
E Coli ◽  
Pbr322 Dna

Three flavonoids which promoted Escherichia coli topoisomerase IV-dependent DNA cleavage were isolated from cottonseed flour and identified as quercetin 3-O-beta-D-glucose-[1,6]-O-alpha-L-rhamnose (rutin), quercetin 3-O-beta-D-galactose-[1,6]-O-alpha-L-rhamnose, and quercetin 3-O-beta-D-glucose (isoquercitrin). The most active one (rutin) also inhibited topoisomerase IV-dependent decatenation activity (50% inhibitory concentration, 64 microg/ml) and induced the SOS response of a permeable E. coli strain. Derivatives of quercetin glycosylated at position C-3 were shown to induce two site-specific DNA cleavages of pBR322 DNA, which were mapped by DNA sequence analysis to the gene encoding resistance to tetracycline. Cleavage at these sites was hardly detectable in cleavage reactions with quercetin or fluoroquinolones. None of the three flavonoids isolated from cottonseeds had any stimulatory activity on E. coli DNA gyrase-dependent or calf thymus topoisomerase II-dependent DNA cleavage, and they were therefore specific to topoisomerase IV. These results show that selective inhibitors of topoisomerase IV can be derived from the flavone structure. This is the first report on a DNA topoisomerase inhibitor specific for topoisomerase IV.

A Perturbation Method Based on Singular Value Decomposition and Feature Selection for Privacy Preserving Data Mining

2014 ◽  
Vol 10 (1) ◽  
pp. 55-76 ◽  
Author(s):  
Mohammad Reza Keyvanpour ◽  
Somayyeh Seifi Moradi
Keyword(s):  
Data Mining ◽  
Feature Selection ◽  
Singular Value Decomposition ◽  
Perturbation Method ◽  
Privacy Preserving ◽  
Singular Value ◽  
Privacy Preserving Data Mining ◽  
Selection For ◽  
Value Decomposition ◽  
Different Levels

In this study, a new model is provided for customized privacy in privacy preserving data mining in which the data owners define different levels for privacy for different features. Additionally, in order to improve perturbation methods, a method combined of singular value decomposition (SVD) and feature selection methods is defined so as to benefit from the advantages of both domains. Also, to assess the amount of distortion created by the proposed perturbation method, new distortion criteria are defined in which the amount of created distortion in the process of feature selection is considered based on the value of privacy in each feature. Different tests and results analysis show that offered method based on this model compared to previous approaches, caused the improved privacy, accuracy of mining results and efficiency of privacy preserving data mining systems.

The HiPIP from the acidophilic Acidithiobacillus ferrooxidans is correctly processed and translocated in Escherichia coli, in spite of the periplasm pH difference between these two micro-organisms

Microbiology ◽  
2005 ◽  
Vol 151 (5) ◽  
pp. 1421-1431 ◽  
Author(s):  
Patrice Bruscella ◽  
Laure Cassagnaud ◽  
Jeanine Ratouchniak ◽  
Gaël Brasseur ◽  
Elisabeth Lojou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acidithiobacillus Ferrooxidans ◽  
Biochemical Properties ◽  
Gene Encoding ◽  
Iron Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur ◽  
Sulfur Protein ◽  
Tat System ◽  
Micro Organisms

The gene encoding a putative high-potential iron–sulfur protein (HiPIP) from the strictly acidophilic and chemolithoautotrophic Acidithiobacillus ferrooxidans ATCC 33020 has been cloned and sequenced. This potential HiPIP was overproduced in the periplasm of the neutrophile and heterotroph Escherichia coli. As shown by optical and EPR spectra and by electrochemical studies, the recombinant protein has all the biochemical properties of a HiPIP, indicating that the iron–sulfur cluster was correctly inserted. Translocation of this protein in the periplasm of E. coli was not detected in a ΔtatC mutant, indicating that it is dependent on the Tat system. The genetic organization of the iro locus in strains ATCC 23270 and ATCC 33020 is different from that found in strains Fe-1 and BRGM. Indeed, in A. ferrooxidans ATCC 33020 and ATCC 23270 (the type strain), iro was not located downstream from purA but was instead downstream from petC2, encoding cytochrome c 1 from the second A. ferrooxidans cytochrome bc 1 complex. These findings underline the genotypic heterogeneity within the A. ferrooxidans species. The results suggest that Iro transfers electrons from a cytochrome bc 1 complex to a terminal oxidase, as proposed for the HiPIP in photosynthetic bacteria.

scholarly journals A Novel Gene Encoding Xanthan Lyase of Paenibacillus alginolyticus Strain XL-1

2000 ◽  
Vol 66 (9) ◽  
pp. 3945-3950 ◽  
Author(s):  
Harald J. Ruijssenaars ◽  
Sybe Hartmans ◽  
Jan C. Verdoes
Keyword(s):  
Signal Sequence ◽  
Fold Increase ◽  
Side Chain ◽  
Cloning And Sequencing ◽  
Gene Encoding ◽  
E Coli ◽  
Mature Enzyme ◽  
Locust Bean ◽  
Encoding Gene ◽  
Amino Acid Signal

ABSTRACT Xanthan-modifying enzymes are powerful tools in studying structure-function relationships of this polysaccharide. One of these modifying enzymes is xanthan lyase, which removes the terminal side chain residue of xanthan. In this paper, the cloning and sequencing of the first xanthan lyase-encoding gene is described, i.e., thexalA gene, encoding pyruvated mannose-specific xanthan lyase of Paenibacillus alginolyticus XL-1. ThexalA gene encoded a 100,823-Da protein, including a 36-amino-acid signal sequence. The 96,887-Da mature enzyme could be expressed functionally in Escherichia coli. Like the native enzyme, the recombinant enzyme showed no activity on depyruvated xanthan. Compared to production by P. alginolyticus, a 30-fold increase in volumetric productivity of soluble xanthan lyase was achieved by heterologous production in E. coli. The recombinant xanthan lyase was used to produce modified xanthan, which showed a dramatic loss of the capacity to form gels with locust bean gum.

scholarly journals Phosphorylation of Nucleosides by the Mutated Acid Phosphatase from Morganella morganii

2000 ◽  
Vol 66 (7) ◽  
pp. 2811-2816 ◽  
Author(s):  
Yasuhiro Mihara ◽  
Takashi Utagawa ◽  
Hideaki Yamada ◽  
Yasuhisa Asano
Keyword(s):  
Acid Phosphatase ◽  
Random Mutagenesis ◽  
Reaction Yield ◽  
Mutant Library ◽  
Morganella Morganii ◽  
Gene Encoding ◽  
E Coli ◽  
Acid Phosphatase Gene ◽  
Phosphate Source

ABSTRACT A novel nucleoside phosphorylation process using the food additive pyrophosphate as the phosphate source was investigated. TheMorganella morganii gene encoding a selective nucleoside pyrophosphate phosphotransferase was cloned. It was identical to theM. morganii PhoC acid phosphatase gene. Sequential in vitro random mutagenesis was performed on the gene by error-prone PCR to construct a mutant library. The mutant library was introduced intoEscherichia coli, and the transformants were screened for the production of 5′-IMP. One mutated acid phosphatase with an increased phosphotransferase reaction yield was obtained. With E. coli overproducing the mutated acid phosphatase, 101 g of 5′-IMP per liter (192 mM) was synthesized from inosine in an 88% molar yield. This improvement was achieved with two mutations, Gly to Asp at position 92 and Ile to Thr at position 171. A decreasedKm value for inosine was responsible for the increased productivity.

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