Cloning and expression of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity.

A gene of Staphylococcus aureus PS47 encoding lytic activity was cloned and expressed in Escherichia coli. Deletion analysis of a recombinant plasmid carrying a 7.4-kilobase-pair fragment (kbp) of S. aureus DNA suggested that the gene was located within a 2.5-kbp EcoRI-XbaI fragment. Analysis of extracts of E. coli harboring recombinant plasmids on denaturing polyacrylamide gels containing purified cell walls of S. aureus showed a clearing zone by a polypeptide of apparent Mr 23,000. The release of dinitrophenylalanine but not reducing groups from purified cell walls by a cell extract of recombinant E. coli suggested that we had cloned an N-acetylmuramyl-L-alanine amidase.     

Transduction of plasmid determinants in Staphylococcus aureus and Escherichia coli.

Buoyant density analysis of transducing lysates derived from Staphylococcus aureus and Escherichia coli indicated that phage particles bearing plasmid determinants contain a quantity of DNA equivalent to that found in the lytic particles. Transducing particles that bear plasmid determinants smaller than viral DNA must therefore contain a quantity of DNA in excess of a single plasmid genome. In the E. coli P1vir system, a dependence upon host-mediated recombination for the transduction of small plasmids, but not for large R factors or chromosomal genes, was observed. However, no evidence for the involvement of such functions in the transduction of S. aureus plasmids was obtained. Although the origin of the additional DNA in plasmid transducing particles has not been identified, circumstantial evidence has been presented in the staphylococcal system indicating that transducing particles carrying a small tetracycline plasmid are not formed by the wrapping of multiple copies of this plasmid DNA.     

Molecular cloning of the structural genes of the Escherichia coli deo-operon in plasmid RSF2124]

A specialized phage lambda ddeo carrying the deo operon of Escherichia coli is analyzed by exposing the DNA to the specific restriction endonucleases EcoRI and BamHI. Using the lambda ddeo DNA fragment, obtained by digestion with BamHI and plasmid RSF2124 as a vehicle, the hybrid plasmid pAM1 carrying all the genes of the deo operon is constructed and cloned in E. coli cells. It is shown that the activity of thymidine phosphorylase in the strain AM061, which contains hybrid plasmid pAM1 is 30-fold greater than that in strains of E. coli with chromosomal localization of the deo operon.     

Mapping of three plasmids from Lactobacillus helveticus ATCC 15009

A 1.4 kb DNA fragment from the chromosomal DNA of Penicillium nalgiovense was isolated which confers proteolytic activity to E. coli DH5α cells when cloned under the control of the E. coli lacZ promoter. The protein was excreted by the cells as was shown by the formation of a clearing zone in skim milk medium. A retransformation of the plasmid carrying the protease gene into P. nalgiovense leads to transformants with both increased and with nearly no proteolytic activity under neutral conditions. Southern blotting experiments revealed that the transforming plasmid had apparently integrated into the homologous locus and thereby inactivated the residual gene.     

Cloning of a gene from Pseudomonas sp. strain PG2982 conferring increased glyphosate resistance

A plasmid carrying a 2.4-kilobase-pair fragment of DNA from Pseudomonas sp. strain PG2982 has been isolated which was able to increase the glyphosate resistance of Escherichia coli cells. The increase in resistance was dependent on the presence of a plasmid-encoded protein with a molecular weight of approximately 33,000, the product of a translational fusion between a gene on the vector, pACYC184, and the insert DNA. An overlapping region of the PG2982 chromosome carrying the entire gene (designated igrA) was cloned, and a plasmid (pPG18) carrying the gene was also able to increase glyphosate resistance in E. coli. A protein with a molecular weight of approximately 40,000 was encoded by the PG2982 DNA contained in pPG18. This plasmid was not able to complement a mutation in the gene for 5-enolpyruvylshikimate-3-phosphate synthase (aroA) in E. coli, and modification of glyphosate by E. coli cells containing the plasmid could not be demonstrated. The nucleotide sequence of the PG2982 DNA contained an open reading frame able to encode a protein with a calculated molecular weight of 39,396.     

Cloning and expression of the thermostable pullulanase gene from Thermus sp. strain AMD-33 in Escherichia coli

Abstract The gene coding for a thermostable pullulanase from a thermophile, Thermus sp. strain AMD-33, was cloned in Escherichia coli using pDR540 as a vector. A restriction map was determined for the plasmid pTPS131 which contained the fragment carrying the pullulanase gene. DNA-DNA hybridisation analysis showed that the DNA fragment contained the gene from Thermus sp. strain AMD-33. The strain of E. coli harbouring the plasmid pTPS131 produced most of the pullulanase protein cellularly, whereas Thermus sp. strain AMD-33 produced pullulanase extracellularly. Comparative studies of the enzyme from the thermophile and the plasmid-encoded enzyme in E. coli demonstrated that the optimum temperature and pH of the enzymes were closely similar.     

Cloning of a gene from Pseudomonas sp. strain PG2982 conferring increased glyphosate resistance.

A plasmid carrying a 2.4-kilobase-pair fragment of DNA from Pseudomonas sp. strain PG2982 has been isolated which was able to increase the glyphosate resistance of Escherichia coli cells. The increase in resistance was dependent on the presence of a plasmid-encoded protein with a molecular weight of approximately 33,000, the product of a translational fusion between a gene on the vector, pACYC184, and the insert DNA. An overlapping region of the PG2982 chromosome carrying the entire gene (designated igrA) was cloned, and a plasmid (pPG18) carrying the gene was also able to increase glyphosate resistance in E. coli. A protein with a molecular weight of approximately 40,000 was encoded by the PG2982 DNA contained in pPG18. This plasmid was not able to complement a mutation in the gene for 5-enolpyruvylshikimate-3-phosphate synthase (aroA) in E. coli, and modification of glyphosate by E. coli cells containing the plasmid could not be demonstrated. The nucleotide sequence of the PG2982 DNA contained an open reading frame able to encode a protein with a calculated molecular weight of 39,396.     

Molecular cloning and overexpression of the glucosamine synthetase gene from Escherichia coli

A recombinant plasmid carrying a 4.6 kg restriction endonuclease NcoI-ClaI fragment of genomic DNA from Escherichia coli K12 was constructed. This plasmid complements the glmS mutation. Subcloning into pUC18 gave plasmid pGM10 encoding the structural gene of glucosamine synthetase, as judged by overexpression of enzyme activity and the isolation in high yield of the pure protein.     

The identification and partial characterization of a plasmid containing the gene for the membrane-associated hydrogenase from E. coli

Escherichia coli DNA was digested with restriction endonuclease PstI and ligated into the PstI site of plasmid pBR322. Recombinant plasmids that were constructed in this manner were used to transform E. coli H61, a mutant with a decreased level of hydrogenase activity. Complementation of this hydrogenase mutation identified a bacterial clone carrying the gene for the membrane-associated E. coli hydrogenase in plasmid pBL101. In E. coli minicells, the pBL101 DNA directed the synthesis of a protein of a size corresponding to that of the precursor of the E. coli membrane-associated hydrogenase, which appears to contain an uncleaved leader peptide. A restriction map of the cloned DNA was determined for 14 endonucleases.     

Cloning and functional characterization of an NAD(+)-dependent DNA ligase from Staphylococcus aureus

A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus ligA gene. Orthologues of the putative S. aureus NAD(+)-dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli, and the enzyme was purified to near homogeneity. NAD(+)-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of (32)P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD(+)-dependent DNA ligase from B. stearothermophilus, two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.     

Escherichia coli DnaA protein: specific biochemical defects of mutant DnaAs reduce initiation frequency to suppress a temperature-sensitive dnaX mutation

The Escherichia coli dnaA73, dnaA721, and dnaA71 alleles, which encode A213D, R432L, T435K substitutions, respectively, were originally isolated as extragenic suppressors of a temperature-sensitive dnaX mutant. As the A213D substitution resides in a domain that functions in ATP binding and the R432L and T435K substitutions affect residues that recognize the DnaA box motif, they might be expected to reduce ATP and specific DNA binding, respectively. Therefore, a major objective was to quantify the biochemical defects of the mutant DnaAs to understand how the altered proteins suppress the temperature-sensitive phenotype of a dnaX mutant. A second purpose was to address the paradox that mutant proteins with substitutions of amino acids essential for recognition of the DnaA box motifs within the E. coli replication origin (oriC) may well be inactive in initiation, yet chromosomal dnaA mutants expressing DnaA proteins with the R432L and T435K substitutions are viable at temperatures from 30 to 39 degrees C. We show biochemically that mutant DnaAs carrying R432L and T435K substitutions fail to bind to the DnaA box sequence. The A213D mutant is sevenfold reduced in its affinity for ATP compared to wild-type DnaA, and its affinity for the DnaA box sequence is also reduced. However, the reduced activity of the A213D mutant in oriC plasmid replication appears to arise from a defect in DnaA oligomerization. Although the T435K mutant fails to bind to the DnaA box sequence, other results suggest that DnaA oligomerization stabilizes the binding of the mutant DnaA to oriC to support its partial activity in initiation in vitro. These results support a model that suppression of dnaX occurs by reducing the frequency of initiation to a manageable level for the mutant DnaX so that viability is maintained.     

Strategy to approach stable production of recombinant nattokinase in Bacillus subtilis

Bacillus subtilis (B. subtilis) is widely accepted as an excellent host cell for the secretory production of recombinant proteins. In this study, a shuttle vector was constructed by fusion of Staphylococcus aureus (S. aureus) plasmid pUB110 with Escherichia coli (E. coli) plasmid pUC18 and used for the expression of nattokinase in B. subtilis. The pUB110/pUC-based plasmid was found to exhibit high structural instability with the identification of a DNA deletion between two repeated regions. An initial attempt was made to eliminate the homologous site in the plasmid, whereas the stability of the resulting plasmid was not improved. In an alternative way, the pUC18-derived region in this hybrid vector was replaced by the suicidal R6K plasmid origin of E. coli. As a consequence, the pUB110/R6K-based plasmid displayed full structural stability, leading to a high-level production of recombinant nattokinase in the culture broth. This was mirrored by the detection of a very low level of high molecular weight DNAs generated by the plasmid. Moreover, 2-fold higher nattokinase production was obtained by B. subtilis strain carrying the pUB110/R6K-based plasmid as compared to the cell with the pAMbeta1-derived vector, a plasmid known to have high structural stability. Overall, it indicates the feasibility of the approach by fusing two compatible plasmid origins for stable and efficient production of recombinant nattokinase in B. subtilis.     

Molecular cloning and sequencing of pheU, a gene for Escherichia coli tRNAPhe.

A recombinant plasmid (designated pID2) carrying the E. coli gene for tRNAPhe has been isolated from a plasmid bank constructed by the ligation of a total EcoRI digest of E. coli K12 DNA into the EcoRI site of pACYC184 DNA. The plasmid was selected by virtue of its ability to complement a temperature-sensitive lesion in the gene (PheS) for the alpha-subunit of phenylalanyl-tRNA synthetase. Crude tRNA isolated from such transformants exhibited elevated levels of phenylalanine acceptor activity. The tRNAPhe gene has been localized within the first 300 base pairs of a 3.6 kb SalI fragment of pID2. The sequence of the gene and its flanking regions is presented.     

Cloning and expression of the staphylokinase gene of Staphylococcus aureus in Escherichia coli

Restriction fragments of DNA from bacteriophage S phi-C of Staphylococcus aureus which carries the gene for staphylokinase, one of the plasminogen activators, were cloned onto plasmid pBR322. Recombinant plasmids carrying the 2.5 kilobase pair segment of S phi-C DNA confer on Escherichia coli cells the capacity to synthesize staphylokinase. The enzyme is synthesized in amounts comparable to that found in S. aureus, and irrespective of the orientation of cloned fragments and their insertion site on pBR322. The active enzyme produced by E. coli cells is preferentially recovered from the periplasmic space and in part excreted into the culture medium. It is indistinguishable from the enzyme produced by S. aureus in molecular weight, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and in antigenicity, as determined by the micro-Ouchterlony precipitation test.     

A new method of plasmid DNA preparation by sucrose-mediated detergent lysis from Escherichia coli (gram-negative) and Staphylococcus aureus (gram-positive)

A simple and cheap method of plasmid DNA preparation from both gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) organism is presented here. In this method, in place of the high-priced chemicals lysostaphin and lysozyme which are commonly used for removal of cell-wall during plasmid DNA preparation from gram-positive and gram-negative bacteria, respectively, only sucrose has been used. Firstly, bacteria is treated with Trizma (pH 8.0) containing 100% sucrose (hypertonic solution). Due to this osmotic shock, protoplasm covered by the plasma membrane of bacteria possibly shrinks and becomes detached from the cell-wall. Osmotically sensitive cells thus formed, from gram-positive (S. aureus) and gram-negative (E. coli) bacteria, are finally lysed by the lysis mixture, containing brij 58 and sodium deoxycholate. The lysate is centrifuged at 15,000 rpm for 30 min to pellet the cell debris. The supernatant containing plasmid DNA is treated with either polyethylene glycol or isopropanol. The precipitate which contains plasmid DNA is dissolved in a buffer containing Tris, EDTA, NaCl, and sodium dodecyl sulfate (pH 8.0); thus protein is denatured and removed. Finally, RNA is removed by RNase treatment. The average yield of staphylococcal plasmid DNA as well as plasmid pBR322 from E. coli HB101 in 100% sucrose-treated preparations is greater than that of lysostaphin- and lysozyme-treated preparations. This method is applicable for both large-scale and small-scale preparations. The substrate activity for restriction enzyme, cloning, transforming ability, and electron microscopic profile of the plasmid DNA prepared by this method remains unaltered.     

Cloning, nucleotide sequence and expression of a hemolysin gene of Clostridium septicum

Abstract A genomic library of Clostridium septicum NCTC547 strain was made in Escherichia coli by means of λgt10. The DNA insert of a hemolysin-positive (Hly⁺) λ-clone was transferred into pUC19. The resulting plasmid, pCS21, confers a Hly⁺ phenotype on E. coli . Crude lysates of E. coli (pCS21) possessed a strong lytic activity on human erythrocytes and also a lethal effect on mice, characteristic of an α toxin. Nucleotide sequence analysis revealed that the insert DNA (5.2 kb) in pCS21 included at least one open reading frame of 1380 bp. The coding frame for hemolysin was predicted to be 1329 bp in size and to encode a protein of 49.8 kDa. It coincided with the molecular mass (48 kDa) of the α toxin secreted by C. septicum . Taken together, the data indicated that plasmid pCS21 indeed encoded an α toxin gene of C. septicum .     

Cloning and expression of the thermostable α-amylase gene from Clostridium thermosulfurogenes (DSM 3896) in Escherichia coli

Abstract The gene coding for a thermostable α-amylase from Clostridium thermosulfurogenes (DSM 3896) was cloned in Escherichia coli using pUC18 as a vector. The recombinant plasmid pCT2 of an amylolytic positive transformant of E. coli contained a 2.9 kbp fragment of chromosomal DNA of C. thermosulforogenes carrying the α-amylase gene. In E. coli the gene was apparently transcribed by its own promoter. Comparative studies showed no difference between the original and the heterologously in E. coli expressed enzyme. The latter was not secreted into the medium.     

Directed modification of the Tcr gene region of the plasmid pBR322 using complementary single-stranded DNA fragments carrying alkylating groups

A method is suggested for chemical modification of preselected regions of plasmid DNA by complementary single-stranded restriction fragments of DNA (srf DNA), carrying alkylating reagents. The gene coding for tetracycline resistance of plasmid pBR322 was used as a target. Srf DNA was prepared by a partial digestion of a double-stranded EcoRI-BamHI restriction fragment (377 base pairs) from Tcr by E. coli exonuclease III. The residues of an alkylating reagent N,N,N'-tri(beta-chlorethyl)-N'-(p-formylphenyl) propylenediamine 1,3 (TFP) were attached covalently to 4-5% of sfr DNA bases. The alkylating derivative of the sfr DNA was hybridized with supercoiled pBR322 plasmid DNA. The hybridization conditions (37 degrees C, 40% formamide, 0,2 M NaCl, 0,1 M Tris-HCl pH 7,5, 0,001 M EDTA) under which the bases carrying TFP residues are not eliminated from the sfr DNA, and transforming activity of pBR322 DNA does not decrease were established. It was shown that about 20% of plasmid pBR322 molecules form D-loops with alkylating sfr DNA under these conditions. It was shown that sfr DNA, carrying TFP can alkylate the complementary region of plasmid DNA, forming cross-linked D-loops. A method for the site-directed mutagenesis of switching off the preselected genes or non-transcribed DNA functional regions (promotors, introns etc) integrated into plasmids of other vectors is suggested.     

Transformation and expression of a staphylococcal plasmid in Escherichia coli

Abstract A multiple antibiotic-resistant Staphylococcus aureus , was found to possess three plasmid bands in agarose gel electrophoresis. A plasmid of approximately 4.3 kb (pMC790/2) was found to code for ampicillin and tetracycline resistance and to have one Eco RI site when transformed into S. aureus RN 4220. pMC790/2 in unmodified form was transformed into a recA⁻ E. coli at a frequency of 1.2×10⁴ transformants/μg of plasmid DNA. Plasmid (pMC790/2) replicated, maintained itself stably and expressed far better in the E. coli host than in S. aureus .