Optimal conditions for genetic transformation of the cyanobacterium Anacystis nidulans R2

Under optimal conditions, the cyanobacterium Anacystis nidulans R2 was transformed to ampicillin resistance at frequencies of greater than 10(7) transformants per microgram of plasmid (pCH1) donor DNA. No stringent period of competency was detected, and high frequencies of transformation were achieved with cultures at various growth stages. Transformation increased with time after addition of donor DNA up to 15 to 18 h. The peak of transformation efficiency (transformants/donor molecule) occurred at plasmid concentrations of 125 to 325 ng/ml with an ampicillin resistance donor plasmid (pCH1) and 300 to 625 ng/ml for chloramphenicol resistance conferred by plasmid pSG111. The efficiency of transformation was enhanced by excluding light during the incubation or by blocking photosynthesis with the electron transport inhibitor 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU) or the uncoupler carbonyl cyanide-m-chlorophenyl hydrazone. Preincubation of cells in darkness for 15 to 18 h before addition of donor DNA significantly decreased transformation efficiency. Growth of cells in iron-deficient medium before transformation enhanced efficiency fourfold. These results were obtained with selection for ampicillin (pCH1 donor plasmid)- or chloramphenicol (pSG111 donor plasmid)-resistant transformants. Approximately 1,000 transformants per microgram were obtained when chromosomal DNA from an herbicide (DCMU)-resistant mutant was used as donor DNA. DCMU resistance was also transferred to recipient cells by using restriction fragments of chromosomal DNA from DCMU-resistant mutants. This procedure allowed size classes of fragments to be assayed for the presence of the DCMU resistance gene.     

Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences.

Use of the sacB gene (J. L. Ried and A. Collmer, Gene 57:239-246, 1987) provides a simple, effective, positive selection for double recombinants in Anabaena sp. strain PCC 7120, a filamentous cyanobacterium. This gene, which encodes the secretory levansucrase of Bacillus subtilis, was inserted into the vector portion of a suicide plasmid bearing a mutant version of a chromosomal gene. Cells of colonies in which such a plasmid had integrated into the Anabaena chromosome through single recombination were plated on solid medium containing 5% sucrose. Under this condition, the presence of the sacB gene is lethal. A small fraction of the cells from initially sucrose-sensitive colonies became sucrose resistant; the majority of these sucrose-resistant derivatives had undergone a second recombinational event in which the sacB-containing vector had been lost and the wild-type form of the chromosomal gene had been replaced by the mutant form. By the use of this technique, we mutated two selected genes in the chromosome of Anabaena sp. strain PCC 7120. The conditionally lethal nature of the sacB gene was also used to detect insertion sequences from this Anabaena strain. Sucrose-resistant colonies derived from cells bearing a sacB-containing autonomously replicating plasmid were analyzed. Five different, presumed insertion sequences were found to have inserted into the sacB gene of the plasmids in these colonies. One of them, denoted IS892, was characterized by physical mapping. It is 1.7 kilobases in size and is present in at least five copies in the genome of Anabaena sp. strain PCC 7120.     

Cloning the alpha-amylase gene of Bacillus amyloliquefaciens in cyanobacteria cells

The recombinant plasmids of pIAH4amy series were constructed containing the alpha-amylase gene of Bacillus amyloliquefaciens A50 with its own promoter and leading sequence within an integrative vector plasmid pIAH4 (CmR) for cyanobacterium Anacystis nidulans R2. At Anacystis nidulans transformation the hybrid plasmids integrate into cyanobacterium chromosome with high efficiency and all CmR transformants produce alpha-amylase. Expression of bacillar alpha-amylase gene in cyanobacterium cells is independent of the cloned gene orientation in the vector plasmid. Secretion of alpha-amylase into the cyanobacterial periplasm has been demonstrated.     

Direct selection for the exchange of alleles between a plasmid and the Escherichia coli chromosome

Recombination is extensively used in order to move alleles between replicons. The exchange of wild-type chromosomal and mutant plasmid-borne alleles is a two-step process entailing the formation of a cointegrate between the entire plasmid and the chromosome, followed by resolution of such cointegrates to give a mutant chromosome and a plasmid carrying the wild-type chromosomal sequence. Often the cointegrate and the resolved forms cannot be distinguished phenotypically. To enable the direct isolation of the resolved products we have developed a positive selection technique. Cells containing a cointegrated plasmid R1 were constructed by transduction using a P1 lysate prepared from cells harbouring a plasmid comprising a mutant chromosomal allele and the so-called omega fragment which carries an aad (aminoglycoside adenylyltransferase) gene. P1 transduction from the cointegrate strain into an SmD recipient allowed direct selection for the resolved complex, since transduction of the aad gene is lethal to an SmD strain.     

A new approach for molecular cloning in cyanobacteria: cloning of an Anacystis nidulans met gene using a Tn901-induced mutant

A new strategy for molecular cloning in the cyanobacterium Anacystis nidulans R-2 is described. This strategy involved the use of a transposon and was developed for the cloning of a gene encoding methionine biosynthesis. A met::Tn901 mutant was isolated. Chromosomal DNA fragments were cloned in the Escherichia coli plasmid vector pACYC184. A recombinant plasmid carrying the inactivated met::Tn901 gene was selected after transformation to E. coli. The cloned met::Tn901 DNA fragment was used as a probe to select the corresponding A. nidulans R-2 wild-type met gene from a gene library prepared in E. coli, using the newly constructed shuttle cosmid vector pPUC29. When transformed into A. nidulans Met- mutants, this cloned gene allowed the mutants to grow prototrophically.     

Transformation of Azotobacter vinelandii OP with a broad host range plasmid containing a cloned chromosomal nif-DNA marker

The non-nitrogen-fixing (Nif-) strain UW10 of Azotobacter vinelandii OP (UW) was naturally induced to competence and transformed with broad host range plasmid pKT210 containing the cloned wild-type nif-10 locus from A. vinelandii UW (Nif+); this marker was unable to complement the nif-10 mutation in trans, but could through recombination with the chromosome. The most frequent type of transformation event observed was recombination between the homologous regions of the plasmid and chromosome (producing Nif+ transformants) with loss of the plasmid vector. At a substantially lower frequency, transformants expressing the plasmid-encoded antibiotic resistance determinants were isolated which were phenotypically Nif-. Agarose gel electrophoresis showed that these transformants contained a plasmid migrating with the same mobility as the original donor plasmid. During culture these transformants acquired a Nif+ phenotype without the loss of the plasmid, as judged by the use of a hybridization probe specific for the cloned nif-DNA fragment. These data indicate that plasmids carrying sequences homologous to chromosomal sequences could be maintained in recombination-proficient A. vinelandii UW. The introduction of plasmids containing sequences homologous to chromosomal sequences was facilitated by prelinearization of the plasmid using a restriction endonuclease generating cohesive ends. Because the site of linearization could be chosen outside the region of shared homology, it was unlikely that the route of plasmid establishment occurred via a homology-facilitated transformation mechanism. The data also indicated that A. vinelandii UW could harbor broad host range cloning vectors based on plasmid RSF1010 without significant impairment of its nitrogen-fixation ability.     

Isolation and characterization of the Aspergillus niger trpC gene

The Aspergillus niger trpC gene was isolated by complementation experiments with an Escherichia coli trpC mutant. Plasmid DNA containing the A. niger trpC gene transforms an Aspergillus nidulans mutant strain, defective in all three enzymatic activities of the trpC gene, to Trp+, indicating the presence of a complete and functional trpC gene. Southern blot analysis of DNA from these Trp+ transformants showed that plasmid DNA was present but that this DNA was not integrated at the site of the chromosomal trpC locus. The A. niger trpC gene was localized on the cloned fragment by heterologous hybridization experiments and sequence analysis. These experiments suggest that the organization of the A. niger trpC gene is identical to that of the analogous A. nidulans trpC and the Neurospora crassa trp-1 genes.     

Gene disruption through homologous recombination in Spiroplasma citri: an scm1-disrupted motility mutant is pathogenic

To determine whether homologous recombination could be used to inactivate selected genes in Spiroplasma citri, plasmid constructs were designed to disrupt the motility gene scm1. An internal scm1 gene fragment was inserted into plasmid pKT1, which replicates in Escherichia coli but not in S. citri, and into the S. citri oriC plasmid pBOT1, which replicates in spiroplasma cells as well as in E. coli. Electrotransformation of S. citri with the nonreplicative, recombinant plasmid pKTM1 yielded no transformants. In contrast, spiroplasmal transformants were obtained with the replicative, pBOT1-derived plasmid pCJ32. During passaging of the transformants, the plasmid was found to integrate into the chromosome by homologous recombination either at the oriC region or at the scm1 gene. In the latter case, plasmid integration by a single crossover between the scm1 gene fragment carried by the plasmid and the full-length scm1 gene carried by the chromosome led to a nonmotile phenotype. Transmission of the scm1-disrupted mutant to periwinkle (Catharanthus roseus) plants through injection into the leafhopper vector (Circulifer haematoceps) showed that the motility mutant multiplied in the insects and was efficiently transmitted to plants, in which it induced symptoms similarly to the wild-type S. citri strain. These results suggest that the spiroplasmal motility may not be essential for pathogenicity and that, more broadly, the S. citri oriC plasmids can be considered promising tools for specific gene disruption by promoting homologous recombination in S. citri, a mollicute which probably lacks a functional RecA protein.     

Direct and indirect gene replacements in Aspergillus nidulans.

We performed three sets of experiments to determine whether cloned DNA fragments can be substituted for homologous regions of the Aspergillus nidulans genome by DNA-mediated transformation. A linear DNA fragment containing a heteromorphic trpC+ allele was used to transform a trpC- strain to trpC+. Blot analysis of DNA from the transformants showed that the heteromorphic allele had replaced the trpC- allele in a minority of the strains. An A. nidulans trpC+ gene was inserted into the argB+ gene, and a linear DNA fragment containing the resultant null argB allele was used to transform a trpC- argB+ strain to trpC+. Approximately 30% of the transformants were simultaneously argB-. The null argB allele had replaced the wild-type allele in a majority of these strains. The A. nidulans SpoC1 C1-C gene was modified by removal of an internal restriction fragment and introduced into a trpC- strain by transformation with a circular plasmid. A transformant containing a tandem duplication of the C1-C region separated by plasmid DNA was self-fertilized, and trpC- progeny were selected. All of these had lost the introduced plasmid DNA sequences, whereas about half had retained the modified C1-C gene and lost the wild-type copy. Thus, it is possible with A. nidulans to replace chromosomal DNA sequences with DNA fragments that have been cloned and modified in vitro by using either one- or two-step procedures similar to those developed for Saccharomyces cerevisiae.     

Hfr mapping of mutations in Bordetella pertussis that define a genetic locus involved in virulence gene regulation.

We report the development of techniques for the genetic mapping of point mutations in the bacterial pathogen Bordetella pertussis. A plasmid vector which is self-transmissible by conjugation and which, by insertion into the B. pertussis chromosome, can mobilize chromosomal sequences during conjugation with a recipient B. pertussis bacterium has been constructed. This vector is used in conjunction with a set of strains containing kanamycin resistance gene insertions at defined physical locations in the B. pertussis genome. In crosses between these donor strains and a mutant recipient strain, transfer of a chromosomal segment flanking the kanamycin resistance gene insertion is selected for, and the percentage of exconjugants which reacquire the wild-type trait is scored. In this way the linkage of the mutant allele to these markers, and thus the approximate chromosomal position of the mutant allele, is determined. We have used this genetic system to map a newly described locus in B. pertussis involved in the regulation of the virulence genes ptx (pertussis toxin) and cya (adenylate cyclase toxin).     

Transformation reveals a chromosomal locus of the gene(s) for methicillin resistance in Staphylococcus aureus.

The localization of the gene(s) mediating methicillin (mecr) in Staphylococcus aureus was determined by transformation with deoxyribonucleic acid (DNA) from a natural mecr strain (DU 4916) and transformation obtained with DNA from this strain. Streptomycin resistance genes (strr) and novobiocin resistance genes (novr) were used concurrently as representatives for chromosomal genes; penicillinase (PI254) and tetracycline plasmids were used as examples of medium- and small-size extrachromosomal genes, respectively. Superinfection of the lysogenic recipients with the competence-inducing phage phi11 or 83A enhanced transformation for all markers. Phenotypic expression of cadmium (cadr), tetracycline (tetr), or methicillin resistance (mecr) did not appear to require a host recombination system since a recA1 mutant could serve as the recipient provided it was superinfected with a competence-inducing phage. There was, furthermore, no requirement for preexisting plasmids for phenotypic expression. Ultraviolet irradiation of transforming DNA enhanced at low doses the transformation frequency for chromosomal genes strr and novr but not for mecr, cadr, or tetr. The gene(s) for mecr was transformed with chromosomal DNA after sodium dodecyl sulfate-sodium chloride extraction and after neutral sucrose gradient centrifugation of bulk DNA from wild-type strain DU 4916 and the transformats. No cavalently closed circular DNA or open circular DNA carrying the methicillin resistance gene(s) could be detected in the wild type or the transformants either by ethidium bromide-cesium chloride gradient centrifugation or by zonal rate centrifugation of cells directly lysed on top of the gradients. The mecr gene(s) is thus probably of chromosomal nature but possibly under recombinational control of phage genes, since transfer of mecr is independent of the recA1 gene(s) but can be accomplished in this strain after superinfection with a competence-inducing phage. Ultraviolet light inactivation of transforming DNA shows first-order kinetics for mecr transformability similar to that observed for both transfecting and plasmid DNA.     

Acquisition of a sucrose utilization system in Escherichia coli K-12 derivatives and its application to industry.

An Escherichia coli strain, B-62, that was isolated from a clinical source and was epidemiologically unrelated to E. coli K-12 was the source of chromosomal DNA for a sucrose utilization system (Scr+) in the construction of a plasmid, pST621. The cloned insert of a gene encoding Scr+ in pST621 conferred a sucrose-positive phenotype onto transformed cells of E. coli K-12 derivatives. Sucrase activity of the transformants was as high as that which would correspond to a "gene dosage effect" of a vector plasmid pBR322, whereas the transformants' sucrose uptake activity was always lower than that of E. coli B-62. A region within an XhoI-SacI fragment (3.2 kb) of pBR322-glyA was replaced in the construction of another plasmid, pST5R7, by a fragment (about 2.6 kb) of pST622 containing the gene encoding Scr+. A genetically stable Scr+ derivative of E. coli K-12 was obtained by introducing the gene encoding Scr+ onto E. coli chromosome via homologous recombination between pST5R7 and the chromosome and subsequent plasmid segregation. The use of low-copy-number plasmid RP4 as a cloning vector was also effective for enhancing the stability of Scr+. Tryptophan producers E. coli SGIII1032S, in which the gene encoding Scr+ was cloned onto the chromosome, and E. coli SGIII1032, which carried Scr+ plasmid RP4.5R7, produced from 6% sucrose in shake flasks (33 degrees C, 96 h) 2.3 and 5.7 g of tryptophan per liter, respectively.     

Acquisition of a sucrose utilization system in Escherichia coli K-12 derivatives and its application to industry.

An Escherichia coli strain, B-62, that was isolated from a clinical source and was epidemiologically unrelated to E. coli K-12 was the source of chromosomal DNA for a sucrose utilization system (Scr+) in the construction of a plasmid, pST621. The cloned insert of a gene encoding Scr+ in pST621 conferred a sucrose-positive phenotype onto transformed cells of E. coli K-12 derivatives. Sucrase activity of the transformants was as high as that which would correspond to a "gene dosage effect" of a vector plasmid pBR322, whereas the transformants' sucrose uptake activity was always lower than that of E. coli B-62. A region within an XhoI-SacI fragment (3.2 kb) of pBR322-glyA was replaced in the construction of another plasmid, pST5R7, by a fragment (about 2.6 kb) of pST622 containing the gene encoding Scr+. A genetically stable Scr+ derivative of E. coli K-12 was obtained by introducing the gene encoding Scr+ onto E. coli chromosome via homologous recombination between pST5R7 and the chromosome and subsequent plasmid segregation. The use of low-copy-number plasmid RP4 as a cloning vector was also effective for enhancing the stability of Scr+. Tryptophan producers E. coli SGIII1032S, in which the gene encoding Scr+ was cloned onto the chromosome, and E. coli SGIII1032, which carried Scr+ plasmid RP4.5R7, produced from 6% sucrose in shake flasks (33 degrees C, 96 h) 2.3 and 5.7 g of tryptophan per liter, respectively.     

Saccharomyces cerevisiae centromere CEN11 does not induce chromosome instability when integrated into the Aspergillus nidulans genome.

We constructed Aspergillus nidulans transformation plasmids containing the A. nidulans argB+ gene and either containing or lacking centromeric DNA from Saccharomyces cerevisiae chromosome XI (CEN11). The plasmids transformed an argB Aspergillus strain to arginine independence at indistinguishable frequencies. Stable haploid transformants were obtained with both plasmids, and strains were identified in which the plasmids had integrated into chromosome III by homologous recombination at the argB locus. Plasmid DNA was recovered from a transformant containing CEN11, and the sequence of the essential portion of CEN11 was determined to be unaltered. The transformants were further characterized by using them to construct heterozygous diploids and then testing the diploids for preferential loss of the plasmid-containing chromosomes. The CEN11 sequence had little or no effect on chromosome stability. Thus, CEN11 does not prevent chromosomal integration of plasmid DNA and probably lacks centromere activity in Aspergillus spp.     

The bacteriophage T4 insertion/substitution vector system. A method for introducing site-specific mutations into the virus chromosome

A bacteriophage T4 insertion/substitution vector system has been developed as a means of introducing in vitro generated mutations into the T4 chromosome. The insertion/substitution vector is a 2638-base pair plasmid containing the pBR322 origin of replication and ampicillin resistance determinant, a T4 gene 23 promoter/synthetic supF tRNA gene fusion, and a polylinker with eight unique restriction enzyme recognition sites. A T4 chromosomal "target" DNA sequence is cloned into this vector and mutated by standard recombinant DNA techniques. Escherichia coli cells containing this plasmid are then infected with T4 bacteriophage that carry amber mutations in two essential genes. The plasmid integrates into the T4 chromosome by recombination between the plasmid-borne T4 target sequence and its homologous chromosomal counterpart. The resulting phage, termed "integrants," are selectable by the supF-mediated suppression of their two amber mutations. Thus, although the integrants comprise 1-3% or less of the total phage progeny, growth on a nonsuppressing host permits their direct selection. The pure integrant phage can be either analyzed directly for a possible mutant phenotype or transferred to nonselective growth conditions. In the latter case, plasmid-free phage segregants rapidly accumulate due to homologous recombination between the duplicated target sequences surrounding the supF sequence in each integrant chromosome. A major fraction of these segregants will retain the in vitro generated mutation within their otherwise unchanged chromosomes and are isolated as stable mutant bacteriophage. The insertion/substitution vector system thereby allows any in vitro mutated gene to be readily substituted for its wild-type counterpart in the bacteriophage T4 genome.     

Expression of the trpC gene from Penicillium chrysogenum in Aspergillus nidulans

The heterologous expression in Aspergillus nidulans of a gene involved in tryptophan biosynthesis from Penicillium chrysogenum is described. With the chimeric plasmid pPC-31, which carries the cloned trpC gene, approximately 10-40 "stable" transformants per microgram of DNA were obtained, with selection for complementation of the mutant allele. This frequency was increased 10-fold by the insertion of the ans1 fragment into the transformation vector. Southern hybridization analysis revealed that transformation occurred as a consequence of the integration of vector sequences into the host chromosome at a variety of sites within the genome.     

Phenotypic expression of PCR-generated random mutations in a Pseudomonas putida gene after its introduction into an Acinetobacter chromosome by natural transformation

Localized sets of random point mutations generated by PCR amplification can be transferred efficiently to the chromosome of Acinetobacter ADP1 (also known as strain BD413) by natural transformation. The technique does not require cloning of PCR fragments in plasmids: PCR-amplified DNA fragments are internalized by cells and directly incorporated into their genomes by homologous recombination. Previously such procedures for random mutagenesis could be applied only to Acinetobacter genes affording the selection of mutant phenotypes. Here we describe the construction of a vector and recipient that allow for mutagenesis, recovery, and expression of heterologous genes that may lack a positive selection. The plasmid carries an Acinetobacter chromosomal segment interrupted by a multiple cloning site next to a kanamycin resistance marker. The insertion of heterologous DNA into the multiple cloning site prepares the insert as a target for PCR mutagenesis. PCR amplifies the kanamycin resistance marker and a flanking region of Acinetobacter DNA along with the insert of heterologous DNA. Nucleotide sequence identity between the flanking regions and corresponding chromosomal segments in an engineered Acinetobacter recipient allows homologous recombination of the PCR-amplified DNA fragments into a specific chromosomal docking site from which they can be expressed. The recipient strain contains only a portion of the kanamycin resistance gene, so donor DNA containing both this gene and the mutagenized insert can be selected by demanding growth of recombinants in the presence of kanamycin. The effectiveness of the technique was demonstrated with the relatively GC-rich Pseudomonas putida xylE gene. After only one round of PCR amplification (35 cycles), donor DNA produced transformants of which up to 30% carried a defective xylE gene after growth at 37 degrees C. Of recombinant clones that failed to express xylE at 37 degrees C, about 10% expressed the gene when grown at 22 degrees C. The techniques described here could be adapted to prepare colonies with an altered function in any gene for which either a selection or a suitable phenotypic screen exists.     

Transformation of the fungal maize pathogen Cochliobolus heterostrophus using the Aspergillus nidulans amdS gene

Summary Cochliobolus heterostrophus protoplasts transformed with a plasmid carrying the Aspergillus nidulans amdS gene (Hynes et al. 1983) gave rise to colonies on a selective medium that did not support significant growth of wild type cells. The plasmid integrated at a single chromosomal locus in each transformant analyzed and the site of integration differed among transformants. Some transformants had one copy of the plasmid, others had multiple copies tandemly arranged and oriented head-to-tail. Both single and multiple copies segregated meiotically as single genes and were mitotically stable on either selective or nonselective medium. The andS gene is advantageous for transformation of genetically undeveloped fungi because it is selectable in wild type cells in organisms that lack a functional amdS gene, thus eliminating the need for induced mutations in recipient strains. Moreover, there is no background due to reversion of a counter-selected mutant allele.     

A hybrid plasmid is a stable cloning vector for the cyanobacterium Anacystis nidulans R2.

Anacystis nidulans R2 is a highly transformable strain which is suitable as a recipient for molecular cloning in cyanobacteria. In an effort to produce an appropriate cloning vector, we constructed a hybrid plasmid molecule, pSG111, which contained pBR328 from Escherichia coli and the native pUH24 plasmid of A. nidulans. pSG111 replicated in and conferred ampicillin and chloramphenicol resistance to both hosts. It contained unique sites for the restriction enzymes EcoRI, SalI, SphI, and XhoI, which could be used for the insertion of exogenous DNA. To demonstrate that a molecule like pSG111 could serve as a shuttle vector for the cloning of A. nidulans genes, we constructed a hybrid plasmid, pRNA404, containing an A. nidulans rRNA operon. This recombinant molecule was genetically and structurally stable during passage through A. nidulans and E. coli. The stability of the hybrid plasmid and the inserted rRNA operon demonstrates the feasibility of cloning in A. nidulans with hybrid vectors, with the subsequent retrieval of cloned sequences.