Molecular genetic studies of the DNA promotor regions in Bacillus thuringiensis subsp. galleriae 69-6. II. Increased level of gene expression resulting from IS1 element integration

The 1.45 kb promoter containing HindIII fragment of Bacillus thuringiensis DNA promotes the expression of the tet gene of recombinant pPBT9 plasmid in Escherichia coli cells. Spontaneous mutants of this plasmid were isolated and analysed. They are responsible for an increase in the level of tetracycline resistance. This 3-fold increase resulted from integration of IS1 element into the bacillar promoter containing HindIII fragment, which led to formation of a mutant pPBT9::IS1 plasmid. The IS sequence integrated was defined as an IS1 element of the E. coli HB101 chromosomal DNA. The integration site of IS1 was localized.     

Cloning of Escherichia coli and Pseudomonas aeruginosa phosphomannose isomerase genes and their expression in alginate-negative mutants of Pseudomonas aeruginosa.

The phosphomannose isomerase (pmi) gene of Escherichia coli was cloned on a broad-host-range cosmid vector and expressed in Pseudomonas aeruginosa at a low level. Plasmid pAD3, which harbors the E. coli pmi gene, contains a 6.2-kilobase-pair HindIII fragment derived from the chromosome of E. coli. Subcloning produced plasmids carrying the 1.5-kilobase-pair HindIII-HpaI subfragment of pAD3 that restored alginic acid production in a nonmucoid, alginate-negative mutant of P. aeruginosa. This fragment also complemented mannose-negative, phosphomannose isomerase-negative mutants of E. coli and showed no homology by DNA-DNA hybridization to P. aeruginosa chromosomal DNA. By using a BamHI constructed cosmid clone bank of the stable alginate producing strain 8830, we have been able to isolate a recombinant plasmid of P. aeruginosa origin that also restores alginate production in the alginate-negative mutant. This new recombinant plasmid, designated pAD4, contained a 9.9-kilobase-pair EcoRI-BamHI fragment with the ability to restore alginate synthesis in the alginate-negative P. aeruginosa. This fragment showed no homology to E. coli chromosomal DNA or to plasmid pAD3. Both mucoid and nonmucoid strains of P. aeruginosa had no detectable levels of phosphomannose isomerase activity as measured by mannose 6-phosphate-to-fructose 6-phosphate conversion. However, P. aeruginosa strains harboring the cloned pmi gene of E. coli contained measurable levels of phosphomannose isomerase activity as evidenced by examining the conversion of mannose 6-phosphate to fructose 6-phosphate.     

Cloning and expression of a Streptomyces fradiae neomycin resistance gene in Escherichia coli

A Streptomyces fradiae DNA sequence, which codes for a neomycin phosphotransferase, has been subcloned from the Streptomyces recombinant plasmid pIJ2 [a chimera between the Streptomyces plasmid SLP1.2 and chromosomal DNA containing a neomycin (Nm) resistance gene] into the BamHI restriction enzyme site of pHV14. Three different recombinant plasmids (pWHR1, pWHR2, pWHR3) have been isolated which transform Escherichia coli to Nm resistance. Southern transfer hybridization experiments show that the recombinant plasmids contain the cloned Streptomyces Nm resistance gene, and lysates of E. coli containing the recombinant plasmids were shown to have Nm phosphotransferase activity, demonstrating that a gene from Streptomyces can be expressed in E. coli.     

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.     

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 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.     

Nucleotide sequence of a chromosomal mercury resistance determinant from a Bacillus sp. with broad-spectrum mercury resistance.

A 13.5-kilobase HindIII fragment, bearing an intact mercury resistance (mer) operon, was isolated from chromosomal DNA of broad-spectrum mercury-resistant Bacillus sp. strain RC607 by using as a probe a clone containing the mercury reductase (merA) gene. The new clone, pYW33, expressed broad-spectrum mercury resistance both in Escherichia coli and in Bacillus subtilis, but only in B. subtilis was the mercuric reductase activity inducible. Sequencing of a 1.8-kilobase mercury hypersensitivity-producing fragment revealed four open reading frames (ORFs). ORF1 may code for a regulatory protein (MerR). ORF2 and ORF4 were associated with cellular transport function and the hypersensitivity phenotype. DNA fragments encompassing the merA and the merB genes were sequenced. The predicted Bacillus sp. strain RC607 MerA (mercuric reductase) and MerB (organomercurial lyase) were similar to those predicted from Staphylococcus aureus plasmid pI258 (67 and 73% amino acid identities, respectively); however, only 40% of the amino acid residues of RC607 MerA were identical to those of the mercuric reductase from gram-negative bacteria. A 69-kilodalton polypeptide was isolated and identified as the merA gene product by examination of its amino-terminal sequence.     

A cloning vector for creation of Escherichia coli lacZ translational fusions and generation of linear template for chromosomal integration

A novel cloning vector to aid in the construction of single copy β-galactosidase reporter systems for gene expression studies in lactose metabolizing Escherichia coli strains, including STEC, is described. The plasmid allows construction of translational fusions of cloned gene promoters to a short segment of E. coli lacZ. A selectable spectinomycin resistance marker flanked by a short lacI segment is positioned 5' to the cloning site. PCR amplification using opposing primers complementary to the upstream lacI fragment and the downstream lacZ fragment generates a linear template suitable for integration using pRedET recombination. Integration of linear template derived from the recombinant plasmid into host strains replaces the entire native lacZ promoter and fuses the promoter of interest in-frame with the lacZ gene, thus simultaneously producing a single-copy, chromosomal reporter system and eliminating background lacZ expression. Studies comparing ahpC expression from a chromosomal fusion in the lac open with that on a plasmid in E. coli strain EDL933 are shown.     

Cloning and expression in Escherichia coli of a gene encoding proline oxidase of Serratia marcescens.

Proline plays a central role in the biosynthesis of prodigiosin by Serratia marcescens. Proline catabolism takes place by oxidation catalysed by the enzyme proline oxidase encoded by the gene putA. A gene bank of chromosomal DNA from S. marcescens was constructed using the plasmid vector pBR328, and then recombinant DNA was used in transformation experiments with Escherichia coli HB 101 as recipient strain. One of the recombinant plasmids, pSL001, was encoded for proline oxidase. Subcloning experiments led to a second plasmid pSL008 able to maintain proline oxidase activity.     

Isolation of an autonomously replicating DNA fragment from the region of defective bacteriophage PBSX of Bacillus subtilis

We have isolated a 5.4-kilobase fragment of Bacillus subtilis DNA that confers the ability to replicate upon a nonreplicative plasmid. The B. subtilis 168 EcoRI fragment was ligated into the chimeric plasmid pCs540, which contains a chloramphenicol resistance determinant from the Staphylococcus aureus plasmid pC194 and an HpaII fragment from the Escherichia coli plasmid, pSC101. A recE B. subtilis derivative, strain BD224, is capable of maintaining this DNA as an autonomously replicating plasmid. In rec+ recipients, chloramphenicol-resistant transformants do not contain free plasmid. The plasmid is integrated as demonstrated by alterations in the pattern of chromosomal restriction enzyme fragments to which the plasmid hybridizes. The site of plasmid integration was mapped by PBS1-mediated transduction to the metC-PBSX region. A strain was a deletion in the region of defective bacteriophage PBSX differs in the hybridization profile obtained by probing EcoRI digests with this cloned fragment. This same deletion mutant, though proficient in normal recombinational pathways, permits autonomous replication of the plasmid apparently owing to the lack of an homologous chromosomal region with which to recombine. We believe that, like E. coli. B. subtilis contains at least one DNA fragment capable of autonomous replication when liberated from its normally integrated chromosomal site and that this cloned DNA fragment comes from the region of defective bacteriophage PBSX.     

Molecular cloning and characterization of a genetic region from Serratia marcescens involved in DNA repair

We report here the molecular isolation of a DNA fragment which encodes Tag-like activity from the Gram-negative bacterium Serratia marcescens. A recombinant plasmid encoding Tag-like activity was isolated from a S. marcescens plasmid gene library by complementation of an Escherichia coli tag mutant, which is deficient in 3-methyladenine DNA glycosylase I. The clone complements E. coli tag, recA, alkA, but not alkB, mutants for resistance to the DNA-damaging agent methyl methanesulphonate (MMS). The coding region of the Tag activity, initially isolated on a 6.5kb BamHI fragment, was defined to a 1.8kb BglII-SmaI fragment. Labelling of plasmid-encoded proteins using maxicells revealed that the 1.8kb fragment encodes two proteins of molecular weights 42,000 and 16,000. Data presented here suggest that the cloned fragment encodes a DNA repair protein(s) that has similar activity to the 3-methyladenine DNA glycosylase I of E. coli.     

The cloning of chromosomal DNA associated with methicillin and other resistances in Staphylococcus aureus

Competitive hybridization was used to detect the deletion of chromosomal DNA accompanying the loss of resistance to methicillin (and concomitantly, to cadmium, mercury and tetracycline) from a clinical strain of methicillin-resistant Staphylococcus aureus (MRSA). The method was also used to screen a partial plasmid library of chromosomal HindIII fragments from the MRSA strain. Eight recombinant plasmid clones were identified as containing DNA included in the deletion. These clones were used as probes to screen a phage library of the total DNA of the same MRSA strain, resulting in the isolation of overlapping recombinant phage clones carrying 24 kb of the deleted DNA. Two of the cloned HindIII fragments were associated closely with methicillin resistance, as shown by probing DNA from an independent methicillin-sensitive/resistant transduced strain pair and from two MRSA strains following growth in the presence of high concentrations of methicillin. The endonuclease map of the cloned DNA indicates the presence of four copies of a direct repeat less than 1 kb in size. The map is also consistent with the presence in the chromosome of sequences for mercury resistance (mer A mer B) and for tetracycline-resistance plasmid pT181.     

Development of a method for heterologous gene expression in Enterobacter amnigenus, a potential host for the biological control of mosquito larvi

An integrative plasmid containing a 1.3 kb fragment of chromosomal DNA from Enterobacter amnigenus was constructed. The Omega fragment encoding spectinomycin/streptomycin resistance was cloned into the unique BglII site of the resulting plasmid, and the interrupted fragment was transferred via plasmid pMAK705 by electroporation into E. amnigenus with a selection for spectinomycin resistance. Cointegrants were resolved to generate an E. amnigenus strain that expressed spectinomycin resistance, but grew as rapidly as the parental strain. The cloned fragment encodes a putative homologue of the proW gene of Escherichia coli that is not essential for E. amnigenus growth. The integrative plasmid is now available to introduce any heterologous DNA into the E. amnigenus chromosome, for the construction of promoter-probe vectors for the studies of gene regulation, or to construct plasmids suitable for the isolation of secretion signals. Immediate applications of this system will include the expression and secretion of crystal toxins from bacilli for the biological control of mosquito larvae infected with the bacterial host.     

Analysis of the ruv locus of Escherichia coli K-12 and identification of the gene product.

The ruv gene of Escherichia coli, which is associated with inducible mechanisms of DNA repair and recombination, has been cloned into the low-copy plasmid vector pHSG415. The recombinant plasmid pPVA101 fully complements the DNA repair-deficient phenotype of ruv mutants. Restriction endonuclease analysis of this plasmid revealed a 10.6-kilobase (kb) HindIII DNA insert which contained a 7.7-kb PstI fragment identified as being from the chromosomal ruv region. Deletion analysis and Tn1000 insertional inactivation of ruv function located the ruv coding region to a 2.2-kb section of the cloned DNA fragment. A comparison of the proteins encoded by ruv wild-type and mutant plasmids identified the gene product as a protein of molecular weight 41,000.     

Cloning of the BamHI methyl transferase gene from Bacillus amyloliquefaciens

We wish to report the initial characterization of a recombinant clone containing the BamHI methylase gene. Genomic chromosomal DNA purified from Bacillus amyloliquefaciens was partially cleaved with HindIII, fractionated by size, and cloned into pSP64. Plasmid DNA from this library was challenged with BamHI endonuclease and transformed into Escherichia coli HB101. A recombinant plasmid pBamM6.5 and a subclone pBamM2.5 were shown to contain the BamHI methylase gene based on three independent observations. Both plasmids were found to be resistant to BamHI endonuclease cleavage, and chromosomal DNA isolated from E. coli HB101 cells harboring either of the plasmids pBamM6.5 or pBamM2.5 was resistant to cleavage by BamHI endonuclease. In addition, DNA isolated from lambda phage passaged through E. coli HB101 containing either plasmid was also resistant to BamHI cleavage. Expression of the BamHI methylase gene is dependent on orientation in pSP64. In these clones preliminary evidence indicates that methylase gene expression may be under the direction of the plasmid encoded LacZ promoter.     

The replication origin of Azotobacter vinelandii

The putative replication origin of Azotobacter vinelandii was cloned as an autonomously replicating fragment after ligation to an antibiotic resistance cartridge. The resulting plasmids could be isolated and labelled by Southern hybridisation with the antibiotic resistance cartridge as probe and also visualised by electron microscopy. These plasmids integrated into the chromosome after a few generations, even in the recA mutant of A. vinelandii. The integrated copy of the plasmid was re-isolated from the chromosome and the DNA and its subfragments were cloned in the plasmid vector pBR322. A 200-bp DNA fragment was sufficient to allow the replication of pBR322 in an Escherichia coli polA strain. Electron microscopic analysis of this plasmid showed that replication initiated mostly within the A. vinelandii DNA fragment. The nucleotide sequence of the putative replication origin and its flanking regions was determined. In the sequence of the 200-bp fragment many of the distinctive features found in other replication origins are lacking. A greater variation from the consensus DnaA binding sequence was observed in A. vinelandii. Direct sequencing of the relevant genomic fragment was also carried after amplifying it from A. vinelandii chromosomal DNA by PCR. This confirmed that no rearrangements had taken place while the cloned fragment was resident in E. coli. It was shown by hybridisation that the 200-bp chromosomal origin fragment of A. vinelandii was present in three other field strains of Azotobacter spp.