Genetic analysis of rec E activities in Bacillus subtilis

Summary ArecE mutant (recE6) ofBacillus subtilis was constructed by insertion of a selectable marker into therecE coding region. The insertional inactivation of therecE gene renders cells very sensitive to DNA damaging agents and severely impairs intermolecular recombination, but does not markedly affect plasmid interstrand annealing and intramolecular recombination. TherecE6 allele was then introduced into a set of DNA repair-deficient strains ofB. subtilis. The removal of DNA damage by therecF,addAaddB,recH,recL andrecP gene products is strictly dependent on an activerecE gene product (recE-dependent pathway). On the other hand, the increased sensitization to purine adducts in theuvrA42recE6 andpolA5recE6 strains suggests that such lethal lesions may be removed either by therecE-dependent or by therecE-independent pathway.     

Apparent gene conversion in an Escherichia coli rec+ strain is explained by multiple rounds of reciprocal crossing-over

Summary Gene conversion, the non-reciprocal transfer of sequence information between homologous DNA sequences, has been reported in lower eukaryotes, mammals and in Escherichia coli. In an E. coli rec ⁺ strain, we established a plasmid carrying two different deleted neo genes (neoDL and neoDR) in an inverted orientation and then selected for homologous recombination events that had reconstructed an intact neo ⁺ gene. We found some plasmids that had apparently experienced intramolecular gene conversion. Further evidence, however, suggests that they are products of multiple rounds of reciprocal crossing-over,apparently involving two plasmid molecules. First, most of the Neo⁺ clones contained multiple types of Neo⁺ plasmids, although the frequency of producing the neo ⁺ clones was low. Second, all the neo ⁺ clones also contained, as a minority, one particular form of dimer, which can be formed by reciprocal crossing-over between neoDL of one plasmid molecule and neoDR of another plasmid molecule. Third, in reconstruction experiments, we cloned and purified this dimer and transferred it back into the rec ⁺ cells. The dimer gave rise to clones containing multiple types of neo ⁺ recombinant monomers, including those apparent gene conversion types, and containing only few molecules of this dimer plasmid.     

ThenadB gene ofSalmonella typhimurium complements the nicotinic acid auxotrophy ofShigella flexneri

Shigella species are characteristically nicotinic acid (NA) auxotrophs. The invasiveS. flexneri strain M90T, transformed with the multicopy plasmid pZT349 encoding thenadB gene ofSalmonella typhimurium, can grow in minimal glucose medium without exogenous NA, whereas, M90T containing the control vector, pUC18 does not, suggesting that this species lacksl-aspartic acid oxidase, the first enzyme in the de novo NAD biosynthetic pathway. The estimated growth rate of strain M90T (pZT349) in HeLa cells was identical to that of M90T (pUC18), indicating the available intracellular concentration of NA is not limiting for bacterial growth.     

Evidence of abortive recombination in ruv mutants of Escherichia coli K12

Summary Genetic recombination in Escherichia coli was investigated by measuring the effect of mutations in ruv and rec genes on F-prime transfer and mobilization of nonconjugative plasmids. Mutation of ruv was found to reduce the recovery of F-prime transconjugants in crosses with recB recC sbcA strains by about 30-fold and with recB recC sbcB sbcC strains by more than 300-fold. Conjugative plasmids lacking any significant homology with the chromosome were transferred normally to these ruv mutants. Mobilization of the plasmid cloning vectors pHSG415, pBR322, pACYC184 and pUC18 were reduced by 20- to 100-fold in crosses with ruv rec ⁺ sbc ⁺ strains, depending on the plasmid used. Recombinant plasmids carrying ruv ⁺ were transferred efficiently. With both F-prime transfer and F-prime cointegrate mobilization, the effect of ruv was suppressed by inactivating recA. It is proposed that the failure to recover transconjugants in ruv recA ⁺strains is due to abortive recombination and that the ruv genes define activities which function late in recombination to help convert recombination intermediates into viable products.     

Transfer of an insecticidal protein gene ofBacillus thuringiensis into plant-colonizingAzospirillum

A fusion plasmid, pRKC, was constructed, using pACYC184, RSF1010 and a kanamycin-resistance cartridge from pUC4K, to convey thecryIA(a) gene intoAzospirillum spp. With the pRKC plasmid, the number of putative transconjugants obtained inA. lipoferum was about 300-fold higher than inA. brasilense. Conjugation frequency and plasmid stability inA. lipoferum were less for pBTF8, which carries thecryIA(a) gene in the correct orientation for a constitutive promoter, than for pBTF9, which carries the gene in the opposite orientation. Expression of thecryIA(a) gene was not apparent in SDS-PAGE analysis ofA. lipoferum transconjugants harbouring pBTF8. However,Escherichia coli transformants with the pBTF8 rescued fromA. lipoferum transconjugants produced an approximately 135 kDa Cry protein, indicating that thecry gene is intact in the transconjugants.V. Udayasuriyan was and A. Nakamura, H. Masaki and T. Uozumi are with the Department of Biotechnology, Faculty of Agriculture, The University of Tokyo, Yayoi 1-1-1, Bunkyo-Ku, Tokyo 113, Japan; V. Udayasuriyan is now with the Department of Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultral University, Coimbatore-641 003, India.     

Isolation of the RAD18 gene of Saccharomyces cerevisiae and construction of rad18 deletion mutants

Summary The RAD18 gene of Saccharomyces cerevisiae is involved in mutagenic DNA repair. We describe its isolation from a yeast library introduced into the centromeric YCp50 vector, a low copy number plasmid. The insert was sublconed into YCp50 and into the multicopy YRp7 plasmid. RAD18 is not toxic when present in multiple copies but the UV survival response indicates an heterogeneity in the cell population, a fraction of it being more sensitive. A DNA segment, close to RAD18, is toxic on the multicopy plasmid and may correspond to the tRAN sup61 known to be tightly linked to RAD18. Chromosomal deletions of RAD18 were constructed. The gene is not essential and the deleted strains have the properties of single site mutants. Thus, RAD18 appears to be essentially involved in DNA repair metabolism.     

Excision of pyrimidine dimers from the DNA of Neurospora

Summary Germinated conidia of Neurospora have been monitored for their ability to excise pyrimidine dimers. Dimer concentration was measured in DNA extracted immediately after UV treatment, and it was compared to that of DNA from cells which had a post-UV incubation before extraction. Two methods were used to assay dimer level in DNA: 1) measurement of the number of single-strand breaks (as revealed in alkaline sucrose gradients) produced by a dimer-specific endonuclease; 2) monitoring the ability to compete for binding to dimer-specific antibodies in a radioimmuno assay. Both methods showed efficient excision of dimers by wild-type and by uvs-2, even though an earlier study had reported that uvs-2 was unable to excise dimers.UV-induced mutation shows a dose-rate effect: acute UV yields several times as many mutations as does the same dose of chronic UV. There is a parallel effect on dimer accumulation. The concentration of dimers at the conclusion of the UV treatment shows a strong correlation with the resultant mutation frequency.     

Genetic interaction between the Ras-CAMP pathway and the Dis2s1/Glc7 protein phosphatase in Saccharomyces cerevisiae

The Saccharomyces cerevisiae DIS2S1/GLC7 gene encodes a type 1 protein phosphatase indispensable for cell proliferation. We found that introduction of a multicopy DIS2S1 plasmid impaired growth of cells with reduced activity of the cAMP-dependent protein kinase. In order to understand further the interaction between the two enzymes, a temperature-sensitive mutation in the DIS2S1 gene was isolated. The mutant accumulated less glycogen than wild type at the permissive temperature, indicating that activity of the Dis2s1 protein phosphatase is attenuated by the mutation. Furthermore, the dis2s1 ^ts mutation was shown to be suppressed by a multicopy plasmid harboring PDE2, a gene for cAMP phosphodiesterase. These results indicate that the Ras-cAMP pathway interacts genetically with the DIS2S1/GLC7 gene.     

A long region upstream of the IME1 gene regulates meiosis in yeast

Summary Meiosis and sporulation in yeast are subject to two types of regulation. The first depends on environmental conditions. The second depends on a genetic pathway which involves the control of the positive regulatory gene IME1 by RME1, which is in turn controlled by the MAT locus. The presence of IME1 on a multicopy plasmid enables cells to undergo meiosis regardless of their genotype at MAT or RME1. We show here that a multicopy plasmid carrying IME1 also enables meiosis, regardless of the environment. Therefore, both kinds of regulation appear to act through IME1. Furthermore, the behavior of multicopy plasmids carrying various segments from the IME1 region suggests that the region upstream of IME1 contains both positive and negative regulatory sites. Control of IME1 by the environment and by the MAT pathway both act through negative regulatory sites.     

TheCBP2 gene fromSaccharomyces douglasii is a functional homologue of theSaccharomyces cerevisiae gene and is essential for respiratory growth in the presence of a wild-type (intron-containing) mitochondrial genome

InSaccharomyces cerevisiae the only known role of theCBP2 gene is the excision of the fifth intron of the mitochondrialcyt b gene (bI5). We have cloned theCBP2 gene fromSaccharomyces douglasii (a close relative ofS. cerevisiae). A comparison of theS. douglasii andS. cerevisiae sequences shows that there are 14% nucleotide substitutions in the coding region, with transitions being three times more frequent than transversions. At the protein level sequence identity is 87%. We have demonstrated that theS. douglasii CBP2 gene is essential for respiratory growth in the presence of a wild-typeS. douglasii mitochondrial genome, but not in the presence of an intronlessS. cerevisiae mitochondrial genome. Also theS. douglasii andS. cerevisiae CBP2 genes are completely interchangeable, even though the intron bI5 is absent from theS. douglasii mitochondrial genome.     

Highly efficient integration of foreign DNA into the genome of the green sulfur bacterium,Chlorobium vibrioforme by homologous recombination

Highly efficient and reproducible transformation ofChlorobium vibrioforme with plasmid DNA has been achieved by electroporation. Specific parameters have been optimized for the electrotransformation procedure. The method was developed using a construct containing a full copy of thepscC gene encoding the cytochromec ₅₅₁ subunit of the photosynthetic reaction center complex and theaadA gene encoding streptomycin resistance as selectable marker. Southern blotting analysis showed that the tested colonies were true transformants with the plasmid integrated into the genome by single homologous recombination. No transformants were obtained using the vector without thepscC gene showing that this vector does not replicate inC. vibrioforme. Thus transformation is possible only by homologous recombination. When using constructs designed to inactivate thepscC gene by insertion no transformants were obtained, indicating that the gene is indispensable for growth. The vector pVS2 carrying genes for erythromycin and chloramphenicol resistance was shown to replicate inC. vibrioforme. The two transformations shown here, provide an important genetical tool in the further analysis of structure and function of the photosynthetic apparatus in green sulfur bacteria.     

Tagging of Bacillus subtilis soil isolates through illegitimate recombination for PCR detection

Although there are numerous bacteria of the genus Bacillus of great importance for biological control, little is known about their ecology in the soil. We wanted to test illegitimate recombination as a tagging system that would allow us to study selected or genetically engineered Bacillus soil isolates. Strains carrying the plasmid integrated into the chromosome were obtained by growing at a non-permissive temperature after transformation with a plasmid carrying a thermo-sensitive replication origin with selection for erythromycin. A laboratory strain, a commercial strain (Kodiak), and four other soil isolates were generated through this procedure and analysed. In all of these strains the integrated plasmid was maintained in multicopy. The erythromycin resistance gene (ermB) placed on the plasmid was used as a target for polymerase chain reaction (PCR). The tagged strains could be then detected when inoculated into microcosms prepared with non-sterile soil.     

The nucleotide sequence of the lipo-penicillinase gene of alkalophilic Bacillus sp. strain 170

The lipo-penicillinase (LIPEN) gene from an alkalophilic Bacillus sp. strain 170 was cloned in Escherichia coli using the vector pHSG399. A plasmid, pFAP121, was isolated from an ampicillin resistant transformant and the cloned LIPEN gene was found to be in a 2.2 kb DNA fragment. The nucleotide sequence of a 1.9 kb segment encoding the LIPEN was determined. This segment showed an open reading frame which would encode a polypeptide of 310 amino acids. The amino acid sequence of this LIPEN gene product has strong homology with those of the Bacillus cereus -lactamase III and Bacillus licheniformis penicillinase.     

DNA damage induced nucleotide excision repair in Saccharomyces cerevisiae

Nucleotide excision repair (NER) is the most versatile and universal pathway of DNA repair that is capable of repairing virtually any damages other than a double strand break (DSB). This pathway has been shown to be inducible in several systems. However, question of a threshold and the nature of the damage that can signal induction of this pathway remain poorly understood. In this study it has been shown that prior exposure to very low doses of osmium tetroxide enhanced the survival of wild type Saccharomyces cerevisiae when the cells were challenged with UV light. Moreover, it was also found that osmium tetroxide treated rad3 mutants did not show enhanced survival indicating an involvement of nucleotide excision repair in the enhanced survival. To probe this further the actual removal of pyrimidine dimers by the treated and control cells was studied. Osmium tetroxide treated cells removed pyrimidine dimers more efficiently as compared to control cells. This was confirmed by measuring the in vitro repair synthesis in cell free extracts prepared from control and primed cells. It was found that the uptake of active ³²P was significantly higher in the plasmid substrates incubated with extracts of primed cells. This induction is dependent on de novo synthesis of proteins as cycloheximide treatment abrogated this response. The nature of induced repair was found to be essentially error free. Study conclusively shows that NER is an inducible pathway in Saccharomyces cerevisiae and its induction is dependent on exposure to a threshold of a genotoxic stress.     

Heterogeneity in oligosaccharides from theO-polysaccharide chain of the lipopolysaccharide fromSalmonella typhi 253Ty determined by fast atom bombardment mass spectrometry

Oligosaccharides from theO-polysaccharide chain of theSalmonella typhi 253Ty lipopolysaccharide (LPS) were prepared from delipidated LPS by digestion with bacteriophage P22. The oligosaccharides were separated by gel chromatography into fractions representing monomers to polymers of the basic tetrasaccharide repeating unit. Fractions containing the dimer and the trimer were analysed by fast atom bombardment-mass spectrometry as methylated alditols. The mass spectra clearly showed heterogeneity in terms ofd-glucose substitution of thed-galactose residue in the tetrasaccharide repeating unit: dimers with none, one, or twod-glucosyl branches and trimers with none, one, two, or threed-glucosyl branches were found. This suggests a randomd-glucosylation of theO-polysaccharide chain ofS. typhi 253Ty.     

Selectable marker recycling in the chloroplast

The bacterial geneaadA is an important and widely used selectable marker for manipulation of the chloroplast genome through biolistic transformation. Because no other such marker is available, two strategies for recycling of theaadA cassette have been developed. One utilizes homologous recombination between two direct repeats flanking theaadA cassette to allow its loss under non-selective growth conditions. A second strategy is to perform co-transformation with a plasmid containing a modified, non-essential chloroplast gene and another plasmid in which theaadA cassette disrupts a chloroplast gene known to be essential for survival. Under selective growth conditions the first mutation can be transferred to all chloroplast DNA copies whereas theaadA insertion remains heteroplasmic. Loss of the selectable marker can be achieved subsequently by growing the cells on non-selective media. In both cases it is possible to reuse theaadA cassette for the stepwise disruption or mutagenesis of any gene in the same strain.     

IS21 insertion in the trfA replication control gene of chromosomally integrated plasmid RP1: a property of stable Pseudomonas aeruginosa Hfr strains

Summary Broad host range IncP-1 plasmids are able to integrate into the chromosome of gram-negative bacteria. Strains carrying an integrated plasmid can be obtained when the markers of a temperature-sensitive (ts) plasmid derivative are selected at non-permissive temperature; in this way Hfr (high frequency) donor strains can be formed. The integrated plasmids, however, tend to be unstable in the absence of continuous selective pressure. In order to obtain stable Hfr donor strains of Pseudomonas aeruginosa PAO, we constructed a derivative of an RP1 (ts) plasmid, pME134, which was defective in the resolvase gene (tnpR) of transposon Tn801. Chromosomal integration of pME134 was selected in a recombination-deficient (rec-102) PAO strain at 43°C. Plasmid integration occurred at different sites resulting in a useful set of Hfr strains that transferred chromosomal markers unidirectionally. The tnpR and rec-102 mutations prevented plasmid excision from the chromosome. In several (but not all) Hfr strains that grew well and retained the integrated plasmid at temperatures below 43°C, the insertion element IS21 of RP1 was found to be inserted into the trfA locus (specifying an essential trans-acting replication funtion) of the integrated plasmid. One such Hfr strain was rendered rec ⁺; from its chromosome the pME134::IS21 plasmid (=pME14) was excised and transferred by conjugation to Escherichia coli where pME14 could replicate autonomously only when a helper plasmid provided the trfA ⁺ function in trans. Thus, it appears that trfA inactivation favours the stability of chromosomally integrated RP1 in P. aeruginosa.     

Stable transformation and regulated expression of an inducible reporter construct inCandida albicans using restriction enzyme-mediated integration

To allow the regulated expression of cloned genes inCandida albicans, a plasmid was constructed using the inducible promoter of theC. albicans MAL2 gene. To demonstrate that theMAL2 promoter could regulate cloned genes placed under its control, a fusion construct was made with the coding sequence of theC. albicans URA3 gene. This plasmid was introduced into a Ura^– strain ofC. albicans using the process of restriction enzyme-mediated integration (REMI). This procedure involves the transformation of theBamHI-linearized plasmid in the presence ofBamHI enzyme. The majority of transformants generated contained insertions of the plasmid at chromosomalBamHI sites. All transformants examined were inducible forURA3 expression, which was determined by growth analysis and by measuring the level ofURA3 gene product activity. The Ura⁺ phenotype of the transformants was stable during growth under nonselective conditions. This system offers the advantages of stable transformation, easy recovery of integrated DNA, and inducible expression of genes inC. albicans.Deceased, December 15, 1995     

In vivo cloning ad characterization of mutations of the regulatory locus ompR of Escherichia coli K12

Summary The product of the ompR gene of E. coli K12 is a positive regulatory protein, which is needed for the expression of the major outer membrane proteins OmpC and OmpF in E. coli K12. A simple in vivo technique was used to transfer three ompR mutations (ompR101, ompR472, ompR4) onto a multicopy plasmid carrying the wild-type ompR gene. The resulting clones were transformed into wild type and corresponding mutant back-grounds to analyze their effects on ompC and ompF expression. All of the cloned ompR mutant alleles exhibited a dominant OmpC^- phenotype in an ompR ⁺background. In addition negative complementation of ompF expression was observed between chromosomal ompR4 and multicopy ompR101 alleles. The results suggest an interaction between different OmpR molecules, and thereby support the idea that OmpR can exist as a multimeric protein.