Cloning of erythromycin-resistance determinants and replication origins from indigenous plasmids of Lactobacillus reuteri for potential use in construction of cloning vectors.

Lactobacillus reuteri L1 and N16 strains contain a 7.0-kb plasmid (pTE80) and a 15-kb plasmid (pTE15), respectively, encoding resistance to erythromycin (Em(r)). Physical maps of both plasmids were established. Nucleotide sequences of the genetic determinants encoding Em(r) on pTE80 and pTE15 revealed the existence of a very similar (ca. 99% nucleotide sequence and ca. 98% amino acid sequence identity) open reading frame for an Em(r) transmethylase gene (erm) in both plasmids. These structural erm genes, 753 and 750 bp in length, respectively, were highly related (ca. 98% nucleotide sequence and ca. 97% amino acid sequence identity) to the erm gene of L. fermentum plasmid pLEM3. Sequence analysis showed that these two erm genes from pTE80 and pTE15 could be categorized under the ermB (ermAM) class. These are the first members of the ermB (ermAM) class of Em(r) determinant from L. reuteri to be characterized at the nucleotide sequence level. The Em(r) gene from pTE80 (erm80) was then ligated into pUC18/19 to construct replication origin (RO)-screening vectors pUE80(+) and pUE80(-) (pUE80(+/-)). These plasmids contain the pUC18/19-derived multiple cloning site, ampicillin-resistance trait, and the LacZ' gene, which enable direct screening for recombinants in Escherichia coli. Once the recombinant contains a RO from L. reuteri, the Em(r) trait of erm80 is used as a selection marker for the replication of the chimeric plasmid as it is transformed into L. reuteri using the cloned RO as a replicon. Replication regions from pTE80 and pTE15 were successfully cloned into the constructed vector pUE80(-). The RO cloned from pTE80 was further identified as being highly stable in L. reuteri and also bearing a relatively narrow host range compared with that of pTE15. The Em(r) determinant (erm80) and RO cloned from pTE80 could be used in the future construction of derivatives of cloning vectors for this microbe. Moreover, the pUE80(+/-) and pTE80-RO constructed in this study have the potential to be developed as a suicide vector and an E. coli-L. reuteri shuttle vector, respectively.     

A bridging water anchors the tethered 5-(3-aminopropyl)-2'-deoxyuridine amine in the DNA major groove proximate to the N+2 C.G base pair: implications for formation of interstrand 5'-GNC-3' cross-links by nitrogen mustards

Site-specific insertion of 5-(3-aminopropyl)-2'-deoxyuridine (Z3dU) and 7-deaza-dG into the Dickerson-Drew dodecamers 5'-d(C (1)G (2)C (3)G (4)A (5)A (6)T (7)T (8)C (9) Z (10)C (11)G (12))-3'.5'-d(C (13)G (14)C (15)G (16)A (17)A (18)T (19)T (20)C (21) Z (22)C (23)G (24))-3' (named DDD (Z10)) and 5'-d(C (1)G (2)C (3)G (4)A (5)A (6)T (7) X (8)C (9) Z (10)C (11)G (12))-3'.5'-d(C (13)G (14)C (15)G (16)A (17)A (18)T (19) X (20)C (21) Z (22)C (23)G (24))-3' (named DDD (2+Z10)) (X = Z3dU; Z = 7-deaza-dG) suggests a mechanism underlying the formation of interstrand N+2 DNA cross-links by nitrogen mustards, e.g., melphalan and mechlorethamine. Analysis of the DDD (2+Z10) duplex reveals that the tethered cations at base pairs A (5).X (20) and X (8).A (17) extend within the major groove in the 3'-direction, toward conserved Mg (2+) binding sites located adjacent to N+2 base pairs C (3).Z (22) and Z (10).C (15). Bridging waters located between the tethered amines and either Z (10) or Z (22) O (6) stabilize the tethered cations and allow interactions with the N + 2 base pairs without DNA bending. Incorporation of 7-deaza-dG into the DDD (2+Z10) duplex weakens but does not eliminate electrostatic interactions between tethered amines and Z (10) O (6) and Z (22) O (6). The results suggest a mechanism by which tethered N7-dG aziridinium ions, the active species involved in formation of interstrand 5'-GNC-3' cross-links by nitrogen mustards, modify the electrostatics of the major groove and position the aziridinium ions proximate to the major groove edge of the N+2 C.G base pair, facilitating interstrand cross-linking.     

Specific sequence modifications of a cry3B endotoxin gene result in high levels of expression and insect resistance

Solanum melongena (eggplant) cv. Picentia and the wild species Solanum integrifolium were transformed with both a wild type (wt) and four mutagenized versions of Bacillus thuringiensis (Bt) gene Bt43 belonging to the cry3 class. The Bt gene was partly modified in its nucleotide sequence by replacing four target regions (W: +1 to +170; X: +592 to+1057 ; Y: +1203 to +1376; Z: +1376 to +1984) with synthetic fragments obtained by polymerase chain reaction amplification of crude oligonucleotides. The synthetic Bt genes were designed to avoid, in their modified regions, sequences such as ATTTA sequence, polyadenylation sequences and splicing sites, which might destabilize the messenger RNA. Furthermore, the codon usage was improved for a better expression in the plant system. The amino acid composition was not altered. Four versions of the modified Bt gene were obtained, BtE, BtF, BtH and BtI, with a nucleotide subtitution percentage of 8.2, 8.6, 14, and 16%, respectively, in comparison to the wt gene Bt43. Modified versions contained different subsets of substituted regions: BtE - W+Z, BtF - Y+Z, BtH - X+Y+Z, BtI - W+X+Y+Z. In the final modified version (BtI), overall guanine + cytosine was increased from the 34.1% of the wt gene to 45.5%, and most of the destabilizing sequences were eliminated. Transgenic plants obtained with the more modified versions, BtH and BtI, were fully resistant to Leptinotarsa decemlineata Say first- and third- instar larvae, while Bt43 wt, BtE and BtF genotypes did not cause mortality and did not affect larval development.     

Cloning and sequencing of a gene encoding a glutamate and aspartate carrier of Escherichia coli K-12.

A gene encoding a carrier protein for glutamate and aspartate was cloned into Escherichia coli K-12 strain BK9MDG by using the high-copy-number plasmid pBR322. The gene (designated gltP) is probably identical to a gene recently cloned from E. coli B (Y. Deguchi, I. Yamato, and Y. Anraku, J. Bacteriol. 171:1314-1319). A 1.6-kilobase DNA fragment containing gltP was subcloned into the expression plasmids pT7-5 and pT7-6, and its product was identified by a phage T7 RNA polymerase-T7 promoter coupled system (S. Tabor and C. C. Richardson, Proc. Natl. Acad. Sci. USA 82:1074-1078) as a polypeptide with an apparent mass of 38 kilodaltons. A portion of the gltP polypeptide was associated with the cytoplasmic membrane. The nucleotide sequence of the 1.6-kilobase fragment was determined. It contained an open reading frame capable of encoding a highly hydrophobic polypeptide of 395 amino acids, containing four possible transmembrane segments. Uptake of glutamate and aspartate was increased 5.5- and 4.5-fold, respectively, in strains containing gltP plasmids. Glutamate uptake was insensitive to the concentration of Na+ and was inhibited by L-cysteate and beta-hydroxyaspartate. These results suggest that gltP is a structural gene for a carrier protein of the Na(+)-independent, binding-protein-independent glutamate-aspartate transport system.     

Characteristics of Pseudomonas putida plasmid DNAs

Physico-chemical characteristics of plasmid DNAs isoalted from Pseudomonas putida G7 were studied as well as the behavior of these DNAs in th eourse of chromatography on columns with Sepharose 4B and kieselguhr with methylated albumin (MAC). This strain was found to contain several plasmid DNAs having molecular weights of 33-36X10(6), 15-18X10(6), and 3-5X10(6) dalton. The plasmid DNAs of biodegradation are supposed to be located in the vicinity of chromosomes, and only a small part of them is characterized by extrachromosomal localization.     

1H-NMR studies of synthetic peptide analogues of calcium-binding site III of rabbit skeletal troponin C: effect on the lanthanum affinity of the interchange of aspartic acid and asparagine residues at the metal ion coordinating positions

The present work determines the contribution of liganding aspartic acid (Asp) residues, at the +X, +Y, and +Z metal ion coordinating positions, to the lanthanum(3+) (La3+) ion binding affinity of synthetic analogues of calcium-binding site III of rabbit skeletal troponin C. Eight 13-residue synthetic analogues were prepared by solid-phase synthesis; the primary sequences of these analogues represent all possible combinations having aspartic acid and asparagine at the +X, +Y, and +Z positions. High-field proton nuclear magnetic resonance (NMR) spectroscopy was used to monitor the binding of the La3+ ion to each of the analogues. Comparison of the chemical shift changes showed large variations in the magnitude of the shift; these were reflected in the La3+ ion association constants determined for each analogue. The association constants ranged from 9.1 x 10(2) M-1 to 2.5 x 10(5) M-1. It was observed that those analogues with the larger number of acidic residues to coordinate the La3+ ion yielded the higher association constants. The La3+ ion binding results demonstrate that the Asp residues at the positions of study contribute equally and in an additive manner to the association constant and that the presence of neighboring Asp residues at either the +X and +Y, the +Y and +Z, or the +X and +Y and +Z metal ion coordinating positions introduced dentate-dentate repulsion, which, acts as to detract from the La3+ ion association constant of the analogues.     

Replacement recombination in Lactococcus lactis.

In the pUC18-derived integration plasmid pML336 there is a 5.3-kb chromosomal DNA fragment that carries the X-prolyl dipeptidyl aminopeptidase gene (pepXP). The gene was inactivated by the insertion of an erythromycin resistance determinant into its coding sequence. Covalently closed circular DNA of pML336 was used for the electrotransformation of Lactococcus lactis. In 2% of the erythromycin-resistant transformants the pepXP gene was inactivated by a double-crossover event (replacement recombination) between pML336 and the L. lactis chromosome. The other transformants in which the pepXP gene had not been inactivated carried a Campbell-type integrated copy of the plasmid. Loss of part of the Campbell-type integrated plasmid via recombination between 1.6-kb nontandem repeats occurred with low frequencies that varied between less than 2.8 x 10(-6) and 8.5 x 10(-6), producing cells with a chromosomal structure like that of cells in which replacement recombination had taken place.     

Mechanism of proline transport in Escherichia coli K12. II. Effect of alkaline cations on binding of proline to a H+/proline symport carrier in cytoplasmic membrane vesicles

The substrate binding reaction of the proline carrier was investigated in nonenergized conditions using cytoplasmic membrane vesicles prepared from the proline carrier-overproducing strain MinS/ pLC4 -45 of Escherichia coli K12. The binding activity specifically required both alkaline cations (X+), Na+ and Li+, and protons. The Na+-dependent binding activity was dependent on the proline carrier, which is the product of the putP gene, and was not affected by ionophores and energy transduction inhibitors. The parameters of proline binding were determined by double reciprocal plots in reaction media with various combinations of Na+ and H+ concentrations. The apparent dissociation constant was greatly affected by the Na+ and H+ concentrations of the medium and could be expressed as a combination of the reciprocals of the Na+ and H+ concentrations, while the maximum number of binding sites remained constant. The characteristics of proline binding to the carrier can be explained by a mechanism in which the unloaded carrier forms a carrier/H+/X+ (CH+X+) complex by a random equilibrium and only the CH+X+ complex binds substrate in nonenergized conditions, as proposed for the Na+/H+/glutamate symport carrier of E. coli B ( Fujimura , T., Yamato , I., and Anraku , Y. (1983) Biochemistry 22, 1954-1959).     

Electrotransformation of Thermoanaerobacter ethanolicus JW200

Electrotransformation of Thermoanaerobacter ethanolicus JW200 was achieved using the plasmid, pTE16, and a pUC-based suicide vector, pTEA2. The construct pTE16 is based on the Escherichia coli-Clostridium perfringens shuttle vector pJIR715 and contains a thermostable chloramphenicol (Cm) resistance cassette. Evidence supporting transformation was provided by extracting plasmid pTE16 from presumptive transformants of T. ethanolicus and by PCR specific to the chloramphenicol acetyltransferase (cat) gene on the vector pTEA2. Transformation frequencies of plasmid pTE16 and pTEA2 were 50 ± 7.4 and 30 ± 4.2 transformants per μg plasmid DNA. The results provide the first unequivocal gene transfer method functional in T. ethanolicus.     

Method for the isolation of the replication region of a bacterial replicon: construction of a mini-F'kn plasmid.

A purified fragment of deoxyribonucleic acid (DNA) that determines resistance to kanamycin and is incapable of self-replication was used to select a self-replicating fragment from an EcoRI endonuclease digest of the sex factor F'lac. This F'lac fragment, exhibiting a molecular weight of 6 X 10(6), carries the genes essential for maintenance of the F replicon in Escherichia coli cells. The constructed mini-F'km plasmid also retains the incompatibility properties of the parent F'lac plasmid. Large amounts of the kanamycin resistance fragment of a molecular weight of 4.5 X 10(6) with an EcoRI-cleaved, self-replicating derivative of colicinogenic plasmid E1 that has a molecular weight of 2.2 X 10(6), The recombinant plasmid is able to replicate extensively in E. coli in medium containing chloramphenicol, and, therefore, large quantities of this plasmid DNA can be obtained. The substantial difference in size between the two fragments in the recombinant plasmid greatly facilitates their separation by preparative agarose gel electrophoresis.     

Carcinogens can induce homologous recombination between duplicated chromosomal sequences in mouse L cells.

The ability of a series of DNA-damaging agents to induce homologous intrachromosomal recombination between duplicated genes in the chromosome of mouse cells was investigated. The target cells were the thymidine kinase-deficient mouse L-cell strain 333M, which contains a single integrated copy of a plasmid with two herpes simplex virus thymidine kinase (Htk) genes, each containing an 8-base-pair XhoI linker inserted at a unique site. Expression of a functional Htk enzyme requires a productive recombinational event between the two nonfunctional genes. The spontaneous rate of recombination in this strain is 3 per 10(6) cells per generation. The agents tested represent physical carcinogens (UV and ionizing radiation), a simple alkylating agent (N-methyl-N'-nitro-N-nitrosoguanidine), an alkylating cross-linking agent (mitomycin C), and a reactive metabolite of a polycyclic aromatic hydrocarbon ((+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [BPDE] ). The background frequency of tk+ recombinants in the untreated population averaged 18 X 10(-6) +/- 5 X 10(-6). Ionizing radiation had little or no effect on recombination; exposure to mitomycin C, N-methyl-N'-nitro-N-nitrosoguanidine, BPDE, or UV, at doses that lowered the survival to between 90 and 10% of the control, caused a dose-dependent increase in frequency of recombinants, reaching 50 X 10(-6) to 100 X 10(-6). No tk+ cells could be generated with a control cell line that contained only one mutant copy of the Htk gene. Molecular hybridization analysis showed that 85 to 90% of the tk+ recombinants retained the Htk gene duplication, consistent with nonreciprocal transfer of wild-type genetic information, gene conversion. In the rest, only a single copy of the Htk gene remained, reflecting a single reciprocal exchange within a chromatid or a single unequal exchange between sister chromatids. Each recombinant tested contained an XhoI-resistant (wild-type) Htk gene.     

pMH2, a small plasmid bearing the nif gene cluster of Enterobacter agglomerans 333 as an excisable cassette

A small plasmid containing the entire nif gene cluster of Enterobacter agglomerans 333 as an excisable cassette has been constructed, using pACYC177 as a vector. Two cosmid clones taken from a gene library of E. agglomerans plasmid pEA3 were used as a source of nif genes. A SmaI fragment of peaMS2-2, containing the H,D,K,Y,E,N,X,U,S,V,W,Z,M,L,A and B genes and an ApaI fragment of peaMS2-16 containing nifA,B,Q,F and J were selected to construct pMH2. The resulting plasmid of 33 kb carries the complete nif gene cluster as a nif cassette on a single XbaI fragment. The nif construct pMH2 in Escherichia coli strains has significant nitrogenase activity compared to wild-type E. agglomerans 333. The nif gene cluster construct was found to be very stable.     

Amplification of the lactose carrier protein in Escherichia coli using a plasmid vector.

Summary The isolation and properties of a hybrid plasmid carrying the Y gene of the lac operon of Escherichia coli are described. The lactose carrier protein, coded for by the Y gene, is readily identified upon lac operon induction in strains carrying the plasmid. The protein comprises about 15% of the cytoplasmic membrane protein synthesized in the first generation after induction, compared with a wild type strain induced under the same conditions where lactose carrier protein comprises 1.4% of the cytoplasmic membrane protein.     

pMH2, a small plasmid bearing the nif gene cluster of Enterobacter agglomerans 333 as an excisable cassette

A small plasmid containing the entire nif gene cluster of Enterobacter agglomerans 333 as an excisable cassette has been constructed, using pACYC177 as a vector. Two cosmid clones taken from a gene library of E. agglomerans plasmid pEA3 were used as a source of nif genes. A SmaI fragment of peaMS2-2, containing the H,D,K,Y,E,N,X,U,S,V,W,Z,M,L,A and B genes and an ApaI fragment of peaMS2-16 containing nifA,B,Q,F and J were selected to construct pMH2. The resulting plasmid of 33 kb carries the complete nif gene cluster as a nif cassette on a single XbaI fragment. The nif construct pMH2 in Escherichia coli strains has significant nitrogenase activity compared to wild-type E. agglomerans 333. The nif gene cluster construct was found to be very stable.     

Binding of the yeast phenylalanine tRNA with Escherichia coli ribosomes. Effect of the removal of a modified base from the 3'-end of the anticodon on codon-anticodon interaction

Phe-tRNAPhe+Y and N-acetyl-Phe-tRNAPhe+Y from yeast interact with prokaryotic 30S subunits and 70S ribosomes with slightly lower affinity than respective tRNA's of E. coli (decrease of standard free energy change of interaction less than 10%). The removal of Y-base from Phe-tRNAPhe+Y results in two orders of magnitude decrease of association constant of Phe-tRNAPh-Ye with P site of the 30S X poly(U) complex and one ordef of magnitude or more of that with A site. The same modification decreases the association constants of Phe-tRNAPhe-Y and N-acetyl-Phe-tRNAPhe-Y 60 and 15 times respectively with P site of the 70S X poly(U) complex. In the absence of poly(U) the affinity of N-acetyl-Phe-tRNAPhe-Y to P-site of 70S ribosome was 20-fold lower than that of native N-acetyl-Phe-tRNAPhe+Y. The sign of interaction enthalpy of N-acetyl-Phe-tRNAPhe+/-Y and Phe-tRNAPhe-Y changes below 6-7 degrees C exposing the hydrophobic part of P-site interactions. Similar removal of Y-base does not change both the enthalpy of interaction with P-site and magnesium concentration dependence.     

Mutational analysis of the DRA promoter: cis-acting sequences and trans-acting factors.

Class II major histocompatibility genes are expressed at high levels in B lymphocytes and are gamma interferon (IFN-gamma) inducible in many other cells. Previously, we observed that DRA promoter sequences from positions -150 to +31 determine the tissue specificity of this class II gene. Moreover, Z and X boxes located between positions -145 and -87 conferred B-cell specificity and IFN-gamma inducibility upon a heterologous promoter. In this study, sequences from positions -145 to -35 in the DRA promoter were systematically mutated by using oligonucleotide cassettes. Z (-131 to -125), pyrimidine (-116 to -109), X (-108 to -95), Y (-73 to -61), and octamer (-52 to -45) boxes were required for B-cell specificity and, with the exception of the octamer box, for IFN-gamma inducibility. Z box and sequences flanking Z and X boxes helped to determine low levels of expression in T and uninduced cells. In phenotypically distinct cells, shared and distinct proteins bound to these conserved upstream sequences. However, few correlations between expression and DNA-binding proteins could be made. Similar proteins bound to Z and X boxes, and the Z box most likely represents a duplication of the X box.     

Isolation and characterization of thiodigalactoside-resistant mutants of the lactose permease which possess an enhanced recognition for maltose

In the current study, lactose permease mutants were isolated which exhibited an enhanced recognition for maltose (an alpha-glucoside) but a diminished recognition for thiodigalactoside, TDG (a beta-galactoside). Maltose/TDGR mutants were obtained from four different parental strains encoding either a wild-type permease (pTE18), a mutant lactose permease which recognizes maltose (pB15) or mutant lactose permeases which recognize maltose but are resistant to inhibition by cellobiose (pTG and pBA). A total of 27 independent mutants were isolated: 12 from pTE18, 10 from pB15, 3 from pTG, and 2 from pBA. DNA sequencing of the 27 mutants revealed that the mutants contain single base pair substitutions within the lac Y gene which result in single amino acid substitutions within the lactose permease. All of the mutants obtained from pTE18, pTG, and pBA involved a change of Tyr-236 to histidine, phenylalanine, or asparagine. From pB15, three different types of mutants were obtained: Tyr-236 to histidine, Ile-303 to phenylalanine, or His-322 to asparagine. When assayed for [14C]maltose transport, the maltose/TDGR mutants were seen to transport maltose significantly faster than the wild type. Furthermore, although TDG was shown to inhibit the uptake of maltose in the four parental strains, all of the mutant strains exhibited a dramatic resistance to TDG inhibition. Most of the maltose/TDGR mutants were also shown to be very defective in the transport of lactose. However, certain mutants (i.e., Asn-322) exhibited moderate lactose transport activity. Finally, it was observed that all of the mutant strains were unable to facilitate the uphill accumulation of beta-methylthiogalactopyranoside. The locations of the amino acid substitutions are discussed with regard to their possible role in sugar recognition.     

Isolation of recombinant interleukin-3 produced by E. coli]

A synthetic gene coding for human interleukin-3 (hIL3) was cloned in the plasmid pTE2IL3, the gene expression being controlled by the phage fd PVIII promotor and the phage T7 gene 10 translational enhancer. Under constitutive biosynthesis conditions in E. coli, the accumulation of recombinant hIL3 (in the inclusion bodies) was up to 30-40% of the total cell protein. An effective procedure of the hIL3 isolation is suggested. The hIL3 was solubilized in 5 M guanidinium chloride, renaturated and purified to homogeneity by a single chromatographic step. The protein's yield was 34 mg/g wet cells. The isolated hIL3 showed a specific biological activity.     

Cloning and characterization of a plasmid DNA from anacystis nidulans 6301

A plasmid DNA of Anacystis nidulans 6301 was isolated by CsCl-EtBr centrifugation. The Mr of the plasmid, named pBA1, was estimated to be 5.04 +/- 0.26 X 10(6) by electron microscopic analysis and 5.2 X 10(6) by agarose gel electrophoresis. The pBA1 DNA was opened at a unique site with BamHI and cloned in pBR322 vector propagated in Escherichia coli HB101 cells. The recombinant plasmid, named pBAS18, was digested with various restriction endonucleases and its cleavage map was constructed. Based on this result, the cleavage map of the pBA1 plasmid is presented.