Construction and shuttling of novel bifunctional vectors for Streptomyces spp. and Escherichia coli.

Shuttle vectors for gene transfer between Streptomyces spp. and Escherichia coli have been constructed by fusion of an artificial multicopy E. coli replicon and DNA fragments of pIJ702. Stable transfer to Streptomyces lividans was obtained. Marked differences in transformation efficiency were observed when plasmid DNA isolated from E. coli GM119 was used instead of that from strain HB101.     

Error-prone mutagenesis detected in mammalian cells by a shuttle vector containing the supF gene of Escherichia coli.

When a shuttle vector containing a tyrosine suppressor tRNA (supF) gene as a target for mutagenesis replicated in a monkey kidney cell line, the frequency of SupF+ mutations was 2.3 +/- 0.5 x 10(-3). When the host cells were treated with ethyl methanesulfonate 40 h before transfection, a 10-fold increase in SupF+ mutation frequency was observed. These results supported the hypothesis that a damage-inducible mutagenic pathway exists in mammalian cells and also demonstrated the utility of this shuttle vector for the study of mutagenesis in mammalian cells.     

Cosmid pJAR4, a novel Streptomyces-Escherichia coli shuttle vector for the cloning of Streptomyces operons

A novel shuttle cosmid vector (pJAR4), based on pK505, was constructed for the cloning of Streptomyces DNA. It is a low-copy-number vector which determines hygromycin B-resistance as a selective marker and was used to clone the puromycin biosynthesis pathway from Streptomyces alboniger. Cosmids pJAR4 and pKC505 (which determines apramycin-resistance) stably co-transform both Streptomyces lividans and Streptomyces griseofuscus.     

Construction of a promoter-probe shuttle vector for Escherichia coli and brevibacteria

We constructed a promoter-probe vector, pJUP05, for brevibacteria and Escherichia coli based on the promoterless neomycin-resistance (neoR) gene from Tn5. This gene confers resistance to the aminoglycosides, kanamycin and neomycin. The promoter of the neoR gene was deleted and replaced by a suitable multiple cloning site. There are translation stop codons in all three reading frames upstream from the neoR gene. The plasmid contains functional origins of DNA replication for both brevibacteria and E. coli, and permits selection for chloramphenicol- and/or ampicillin-resistance markers.     

Construction of an Escherichia coli-Rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp.

A plasmid transformation system for Rhodococcus sp. strain H13-A was developed by using an Escherichia coli-Rhodococcus shuttle plasmid constructed in this study. Rhodococcus sp. strain H13-A contains three cryptic indigenous plasmids, designated pMVS100, pMVS200, and pMVS300, of 75, 19.5, and 13.4 kilobases (kb), respectively. A 3.8-kb restriction fragment of pMVS300 was cloned into pIJ30, a 6.3-kb pBR322 derivative, containing the E. coli origin of replication (ori) and ampicillin resistance determinant (bla), as well as a Streptomyces gene for thiostrepton resistance, tsr. The resulting 10.1-kb recombinant plasmid, designated pMVS301, was isolated from E. coli DH1(pMVS301) and transformed into Rhodococcus sp. strain AS-50, a derivative of strain H13-A, by polyethylene glycol-assisted transformation of Rhodococcus protoplasts and selection for thiostrepton-resistant transformants. Thiostrepton-resistant transformants were also ampicillin resistant and were shown to contain pMVS301, which was subsequently isolated and transformed back into E. coli. The cloned 3.8-kb fragment of Rhodococcus DNA in pMVS301 contains a Rhodococcus origin of replication, since the hybrid plasmid was capable of replication in both genera. The plasmid was identical in E. coli and Rhodococcus transformants as determined by restriction analysis and was maintained as a stable, independent replicon in both organisms. Optimization of the transformation procedure resulted in transformation frequencies in the range of 10(5) transformants per micrograms of pMVS301 DNA in Rhodococcus sp. strain H13-A and derivative strains. The plasmid host range extends to strains of Rhodococcus erythropolis, R. globulerus, and R. equi, whereas stable transformants were not obtained with R. rhodochrous or with several coryneform bacteria tested as recipients. A restriction map demonstrated 14 unique restriction sites in pMVS301, some of which are potentially useful for molecular cloning in Rhodococcus spp. and other actinomycetes. This is the first report of plasmid transformation and of heterologous gene expression in a Rhodococcus sp.     

Identification of catalytically essential residues in Escherichia coli esterase by site-directed mutagenesis.

Escherichia coli esterase (EcE) is a member of the hormone-sensitive lipase family. We have analyzed the roles of the conserved residues in this enzyme (His103, Glu128, Gly163, Asp164, Ser165, Gly167, Asp262, Asp266 and His292) by site-directed mutagenesis. Among them, Gly163, Asp164, Ser165, and Gly167 are the components of a G-D/E-S-A-G motif. We showed that Ser165, Asp262, and His292 are the active-site residues of the enzyme. We also showed that none of the other residues, except for Asp164, is critical for the enzymatic activity. The mutation of Asp164 to Ala dramatically reduced the catalytic efficiency of the enzyme by the factor of 10(4) without seriously affecting the substrate binding. This residue is probably structurally important to make the conformation of the active-site functional.     

Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase.

Site-directed mutagenesis was used to examine the catalytic importance of 2 histidine and 4 arginine residues in Escherichia coli periplasmic acid phosphatase (EcAP). The residues that were selected as targets for mutagenesis were those that were also conserved in a number of high molecular weight acid phosphatases from eukaryotic organisms, including human prostatic and lysosomal acid phosphatases. Both wild type EcAP and mutant proteins were overproduced in E. coli using an expression system based on the T7 RNA polymerase promoter, and the proteins were purified to homogeneity. Examination of the purified mutant proteins by circular dichroism and proton NMR spectroscopy revealed no significant conformational changes. The replacement of Arg16 and His17 residues that were localized in a conserved N-terminal RHGXRXP motif resulted in the complete elimination of EcAP enzymatic activity. Critical roles for Arg20, Arg92, and His303 were also established because the corresponding mutant proteins exhibited residual activities that were not higher than 0.4% of that of wild type enzyme. In contrast, the replacement of Arg63 did not cause a significant alteration of the kinetic parameters. The results are in agreement with a previously postulated distant relationship between acid phosphatases, phosphoglycerate mutases, and fructose-2,6-bisphosphatase. These and earlier results are also consistent with the conclusion that 2 histidine residues participate in the catalytic mechanism of acid phosphatases, with His17 playing the role of a nucleophilic acceptor of the phospho group, whereas His303 may act as a proton donor to the alcohol or phenol.     

SiteFind: A software tool for introducing a restriction site as a marker for successful site-directed mutagenesis

Background  

Site-directed mutagenesis is a widely-used technique for introducing mutations into a particular DNA sequence, often with the goal of creating a point mutation in the corresponding amino acid sequence but otherwise leaving the overall sequence undisturbed. However, this method provides no means for verifying its success other than sequencing the putative mutant construct: This can quickly become an expensive method for screening for successful mutations. An alternative to sequencing is to simultaneously introduce a restriction site near the point mutation in manner such that the restriction site has no effect on the translated amino acid sequence. Thus, the novel restriction site can be used as a marker for successful mutation which can be quickly and easily assessed. However, finding a restriction site that does not disturb the corresponding amino acid sequence is a time-consuming task even for experienced researchers. A fast and easy to use computer program is needed for this task.     

A possible model for the concerted allosteric transition in Escherichia coli aspartate transcarbamylase as deduced from site-directed mutagenesis studies

Aspartate transcarbamylase is stabilized in a low-affinity-low-activity state exhibiting no cooperativity by selective perturbation of the Glu-50-Arg-167 and Glu-50-Arg-234 interdomain salt bridges. Similarly, a high-affinity-high-activity state of the enzyme, retaining a significant amount of cooperativity, is obtained by perturbation of the interaction between Tyr-240 and Asp-271. In this work, we show that the rupture of the link between Tyr-240 and Asp-271 in the enzyme already lacking the interdomain salt bridges regenerates the homotropic cooperative interactions between the catalytic sites and substantially increases the activity and affinity of the enzyme for aspartate. These results suggest a possible relationship between these two sets of interactions for the establishment of the cooperative behavior of the enzyme. Another mutation, Glu-239 to Gln, introduced to perturb the Glu-239-Lys-164 and Glu-239-Tyr-165 interactions between the two catalytic subunits, is sufficient to "lock" the enzyme in the R state. These observations emphasize the importance of the interactions at the interface between the catalytic trimers in maintaining the T state of the enzyme and shed light on the role played by this pathway in the communication of homotropic cooperativity between the different sites. A model including all these findings, as well as the interactions stabilizing the T state or the R state in the presence of the natural substrates, is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction of two stable bifunctional plasmids for Streptomyces spp. and Escherichia coli

Two bifunctional plasmid vectors pZG5 (7.45 kb) and pZG6 (6.95 kb), for gene transfer between Streptomyces spp. and Escherichia coli have been constructed by fusion of the multicopy broad-host-range Streptomyces plasmid pIJ350 with E. coli plasmids Bluescribe M13- (pZG5) or pUC18 (pZG6). Both plasmids possess several unique restriction sites suitable for DNA cloning. Stable transformants of Streptomyces rimosus R6 and S. lividans 66 were obtained, harboring intact plasmids regardless of colony age or multiple subculturing. Moreover, pZG5 and pZG6 were successfully used to introduce several homologous transfer RNA genes into S. rimosus.     

Introduction by site-directed mutagenesis of a tryptophan residue as a fluorescent probe for the folding of Escherichia coli phosphofructokinase

The leucine residue at position 178 in the major allosteric phosphofructokinase from Escherichia coli has been replaced by a tryptophan using site-directed mutagenesis. Transformation by the mutated gene of pfk- bacteria results into the expression of a pfk+ phenotype and the production of an active enzyme. The modified protein has been purified and its fluorescence properties show that it contains 2 tryptophan residues, the original Trp 311 and the new Trp 178. During unfolding of the protein by guanidine hydrochloride, the changes in the fluorescence of these 2 residues take place at different steps: Trp 311 becomes exposed to solvent when the dimeric form dissociates into monomers, while Trp 178 is exposed only when a folded chain loses its tertiary structure. The mutant enzyme is stabilized by its substrate fructose-6-phosphate against denaturation induced by heat or guanidine hydrochloride.     

Construction of a shuttle vector for inducible gene expression in Escherichia coli and Bacillus subtilis

The construction of a shuttle vector for inducible gene expression allowing fast and easy cloning in Escherichia coli and subsequent transformation of Bacillus subtilis is presented. The expression is based on the regulation of the tac promoter by the Lac repressor which was assayed with the xylE gene from Pseudomonas putida as a marker gene. The lacIq gene, transcribed by the strong spo promoter, allowed full repression of the weak tac promoter.     

Structure-function studies of Escherichia coli biotin synthase via a chemical modification and site-directed mutagenesis approach.

In Escherichia coli, biotin synthase (bioB gene product) catalyzes the key step in the biotin biosynthetic pathway, converting dethiobiotin (DTB) to biotin. Previous studies have demonstrated that BioB is a homodimer and that each monomer contains an iron-sulfur cluster. The purified BioB protein, however, does not catalyze the formation of biotin in a conventional fashion. The sulfur atom in the iron-sulfur cluster or from the cysteine residues in BioB have been suggested to act as the sulfur donor to form the biotin molecule, and yet unidentified factors were also proposed to be required to regenerate the active enzyme. In order to understand the catalytic mechanism of BioB, we employed an approach involving chemical modification and site-directed mutagenesis. The properties of the modified and mutated BioB species were examined, including DTB binding capability, biotin converting activity, and Fe(2+) content. From our studies, four cysteine residues (Cys 53, 57, 60, and 97) were assigned as the ligands of the iron-sulfur cluster, and Cys to Ala mutations completely abolished biotin formation activity. Two other cysteine residues (Cys 128 and 188) were found to be involved mainly in DTB binding. The tryptophan and histidine residues were suggested to be involved in DTB binding and dimer formation, respectively. The present study also reveals that the iron-sulfur cluster with its ligands are the key components in the formation of the DTB binding site. Based on the current results, a refined model for the reaction mechanism of biotin synthase is proposed.     

Construction of a shuttle vector and expression in Streptomyces lividans of the sulfonamide-resistance gene derived from Escherichia coli plasmid pSAS1206

We have constructed a hybrid plasmid using Streptomyces lividans plasmid p1J101 and Escherichia coli plasmid pSAS1206. This plasmid, designated pFSH102, is able to replicate in both hosts and the sulfonamide-resistance gene encoded by pSAS1206 is phenotypically expressed in S. lividans.     

The active site of Escherichia coli UDP-N-acetylglucosamine acyltransferase. Chemical modification and site-directed mutagenesis.

UDP-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA) catalyzes the reversible transfer of an R-3-hydroxyacyl chain from R-3-hydroxyacyl-acyl carrier protein to the glucosamine 3-OH of UDP-GlcNAc in the first step of lipid A biosynthesis. Lipid A is required for the growth and virulence of most Gram-negative bacteria, making its biosynthetic enzymes intriguing targets for the development of new antibacterial agents. LpxA is a member of a large family of left-handed beta-helical proteins, many of which are acyl- or acetyltransferases. We now demonstrate that histidine-, lysine-, and arginine-specific reagents effectively inhibit LpxA of Escherichia coli, whereas serine- and cysteine-specific reagents do not. Using this information in conjunction with multiple sequence alignments, we constructed site-directed alanine substitution mutations of conserved histidine, lysine, and arginine residues. Many of these mutant LpxA enzymes show severely decreased specific activities under standard assay conditions. The decrease in activity corresponds to decreased k(cat)/K(m,UDP-GlcNAc) values for all the mutants. With the exception of H125A, in which no activity is seen under any assay condition, the decrease in k(cat)/K(m,UDP-GlcNAc) mainly reflects an increased K(m,UDP-GlcNAc). His(125) of E. coli LpxA may therefore function as a catalytic residue, possibly as a general base. LpxA does not catalyze measurable UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc hydrolysis or UDP-GlcNAc/UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc exchange, arguing against a ping-pong mechanism with an acyl-enzyme intermediate.     

A thiostrepton-inducible expression vector for use in Streptomyces spp

A shuttle expression vector containing the thiostrepton-inducible Streptomyces lividans promoter, ptipA, and the origin of transfer from plasmid RP4 was constructed. Cassettes containing a promoterless xylE gene upstream from a hyg gene were used to demonstrate thiostrepton-inducible expression from ptipA in both S. lividans and Streptomyces ambofaciens, ptipA was estimated to be induced 60-fold or more in Streptomyces ambofaciens.     

Production in Escherichia coli and site-directed mutagenesis of a 9-kDa nonspecific lipid transfer protein from wheat.

The sequence encoding a wheat (Triticum durum) nonspecific lipid transfer protein of 9 kDa (nsLTP1) was inserted into an Escherichia coli expression vector, pET3b. The recombinant protein that was expressed accumulated in insoluble cytoplasmic inclusion bodies and was purified and refolded from them. In comparison with the corresponding protein isolated from wheat kernel, the refolded recombinant protein exhibits a methionine extension at its N-terminus but has the same structure and activity as demonstrated by CD, lipid binding and lipid transfer assays. Using the same expression system, four mutants with H5Q, Y16A, Q45R and Y79A replacements were produced and characterized. No significant changes in structure or activity were found for three of the mutants. By contrast, lipid binding experiments with the Y79A mutant did not show any increase of tyrosine fluorescence as observed with the wild-type nsLTP1. Comparison of the two tyrosine mutants suggested that Tyr79 is the residue involved in this phenomenon and thus is located close to the lipid binding site as expected from three-dimensional structure data.