Effect of adenosine 5''-triphosphate-dependent deoxyribonuclease deficiency on properties and transformation of Haemophilus influenzae strains.

A transformation-deficient strain of Haemophilus influenzae, lacking adenosine 5'-triphosphate-dependent deoxyribonuclease activity, was isolated by selection for sensitivity to mitomycin. The mutant, designated JK57, possibily showed a moderate sensitivity to ultraviolet (UV) irradiation and treatment with methyl methane sulfonate. Contrary to the wild type, the mutant degraded chromosomal deoxyribonucleic acid (DNA) to some extent. However, after UV irradiation to the mutant degraded considerably less DNA than the wild type and the TD24 mutant of H. influenzae, the latter being equivalent to a recA mutant of Escherichia coli. A TD2457 double mutant, constructed by transferring the TD24 mutation into the JK57 strain, was as sensitive to deleterious agents and as deficient in transformation as the TD24 single mutant; in the double mutant, however, after UV irradiation chromosomal DNA was degraded to the same extent as in the JK57 mutant. The number of transformants per unit of radioactive donor DNA taken up by JK57 recipient cells was approximately 10-fold smaller than in the wild type. Presynaptically, the fate of donor DNA in the adenosine 5'-triphosphate-dependent deoxyribonuclease-deficient mutants was not different from that in the wild type. In contrast to TD24 and the TD2457 double mutant, in the JK57 mutant, recombinant-type activities (molecules carrying both the donor and recipient markers) were formed almost as well as in the wild type. After integration into the JK57 recipient genome, the rate of replication of the donor marker was equal to that of the recipient marker during a number of generations, which suggests that the donor DNA is normally integrated into the JK57 chromosome. It is suggested that transformed JK57 cells pass with a high frequency into a type of cells that can replicate their chromosomes many times but have lost the ability to form visible colonies after plating.     

Chromosomal Basis of the Merozygosity in a Partially Diploid Mutant of Pneumococcus

A sulfonamide-resistant mutant of pneumococcus, sulr-c, displays a genetic instability, regularly segregating to wild type. DNA extracts of derivatives of the strain possess transforming activities for both the mutant and wild-type alleles, establishing that the strain is a partial diploid. The linkage of sulr-c to strr-61, a stable chromosomal marker, was established, thus defining a chromosomal locus for sulr-c. DNA isolated from sulr-c cells transforms two mutant recipient strains at the same low efficiency as it does a wild-type recipient, although the mutant property of these strains makes them capable of integrating classical "low-efficiency" donor markers equally as efficiently as "high efficiency" markers. Hence sulr-c must have a different basis for its low efficiency than do classical low efficiency point mutations. We suggest that the DNA in the region of the sulr-c mutation has a structural abnormality which leads both to its frequent segregation during growth and its difficulty in efficiently mediating genetic transformation.     

Induction and Transmission of a Merodiploid Condition near the Terminal Area of the Chromosome of BACILLUS SUBTILIS

A small fraction (about 0.5%) of the transformants for a particular marker of B. subtilis (ilvA4; most probably a deletion) were found to be relatively unstable merodiploids. They possess a redundancy of the metB–ilvA chromosome segment. When their DNA is used as donor in transformation a merodiploid condition for the whole of this segment is created in all ilvA4⁺ transformants. For several of the duplicated loci both copies often are of recipient strain origin. Markers originally belonging to different copies of the diploidized region can be contransferred in PBS1-mediated transduction. The data are well in agreement with the hypothesis that the merodiploids carry a tandem duplication. An alternative hypothesis which does not call for integration of the exogenote within the recipient chromosome was also considered. Models are proposed for interpreting the segregation of the merodiploids, the transmission of the diploid state and its generation during transformation of the ilvA4 marker by wild-type DNA.     

Repair of ultraviolet-irradiated transforming deoxyribonucleic acid in Haemophilus influenzae

Ultraviolet-sensitive and wild-type Haemophilus influenzae cells were exposed to irradiated and unirradiated transforming deoxyribonucleic acid (DNA) containing a marker which can be linked to another marker in the cells. Lysates were made after various times of incubation and assayed for transforming activity on an excisionless recipient. Repair can be noted as an increase in activity from the irradiated donor DNA after its linkage to the recipient DNA. No repair can be observed in a mutant which is unable to integrate transforming DNA. There is a little repair in another mutant which is unable to excise pyrimidine dimers. H. influenzae cells also repair nondimer damage, as judged by the increase in activity observed in lysates made with irradiated and maximally photoreactivated DNA.     

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°C. Of recombinant clones that failed to express xylE at 37°C, about 10% expressed the gene when grown at 22°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.     

Formation of recombinants inEscherichia coli and a gene controlling the expression of a donor marker

In our experiments we tried to explain some anomalies in the formation of recombinants in anEscherichia coli mutant strain which, compared with the control, yields a lower number of recombinants. Following the transfer of C¹⁴-thymidine-labelled donor chromosome it was found that the low recombinant frequency is not due to a lower effectiveness of DNA transfer into the recipient cell; similarly, using the technique of interrupted mating between the cells we were able to detect that the rate of chromosome transfer is the same as in the control. The low frequency of recombinants may be explained by some restriction processes which take place in the recipient cell following the chromosome transfer. In addition, a relation between the localization of a marker and its subsequent expression in the recombinants was observed. Analyzing this phenomenon we were able to find a gene located near thethr marker which might modify the expression of integrated donor marker in the zygote. The probable mode of action of this gene is discussed.     

Ectopic integration of chromosomal genes in Streptococcus pneumoniae.

When a DNA fragment containing a marker gene was ligated to random chromosomal fragments of Streptococcus pneumoniae and used to transform a recipient strain lacking that gene, the gene was integrated at various locations in the chromosome. Such ectopic integration was demonstrated for the malM gene, and its molecular basis was analyzed with defined donor molecules consisting of ligated fragments containing the malM and sul genes of S. pneumoniae. In a recipient strain deleted in the mal region of its chromosome, these constructs gave Mal+ transformants in which the malM and sul genes were now linked, with malM located between duplicate sul segments. Ectopic integration was unstable under nonselective conditions; mal(sul) ectopic insertions were lost at a rate of 0.05% per generation. Several possible mechanisms of ectopic integration were examined. The donor molecule is most likely to be a circular form of ligated homologous and nonhomologous fragments that, after entry into the cell, undergoes circular synapsis with the recipient chromosome at the site of homology, followed by repair and additive integration.     

Ultraviolet inactivation and excision-repair in Bacillus subtilis. II. Differential inactivation and differential repair of transforming markers

Ultraviolet inactivation of transforming Bacillus subtilis markers was studied with the aid of an eightfold auxotrophic recipient and its excision-repair-deficient derivative. The results allow the following conclusions. (i) Wild-type B. subtilis cells are able to repair approx. 80% of the UV-induced lesions causing inactivation of transforming activity in UV-sensitive recipients; (ii) Saturating amounts of donor DNA increase the apparent marker sensitivities. This phenomenon is most pronounced in transformation of UV-sensitive recipients; (iii) various markers are inactivated to different degrees, both when assayed on the wild-type as well as on the UV-sensitive strain; (iv) Various markers are repaired to different degrees in the wild-type recipient.     

Molecular Basis for the Transformation Defects in Mutants of Haemophilus influenzae

To determine the molecular basis of transformation defects in Haemophilus influenzae, the fate of genetically marked, (32)P-labeled, heavy deoxyribonucleic acid (DNA) was examined in three mutant strains (rec(1) (-), rec(2) (-), and KB6) and in wild type having (3)H-labeled DNA and a second genetic marker. Transforming cells upon lysis with digitonin followed by low-speed centrifugation are separable into the supernatant fraction, containing mainly the unintegrated donor DNA, and the pellet, containing most of the resident DNA along with integrated donor DNA. Electron micrographs of digitonin-treated cells also indicate that the resident DNA is trapped inside a cellular structure but that cytoplasmic elements such as ribosomes are extensively released. DNA synthesis in digitonin-treated cells is immediately blocked, as is any further integration of donor DNA into the resident genome. Isopycnic and sedimentation analysis of supernatant fluids and pellets revealed that in strains rec(2) (-) and KB6 there is little or no association between donor and resident DNA, and thus there is negligible transfer of donor DNA genetic information. In these strains, the donor DNA is not broken into pieces of lower molecular weight as it is in strain rec(1) (-) and in the wild type, both of which show association between donor and recipient DNA. In strain rec(1) (-), although some donor DNA atoms become covalently linked to resident DNA, the incorporated material does not have the donor DNA transforming activity.     

Characterization of rpo-B mutant of a strain of Escherichia coli to confirm its suitability for routine work related to recombinant DNA technology

Rifampicin resistant spontaneous mutant of a popular laboratory strain of Escherichia coli (DH5 alpha) was isolated and found to resist high level of the drug in growth medium. The growth of the isolate was found to be slower than its wild-type counterpart. Its ability to get transformed into drug-resistant state through transformation by chemical means as tested using plasmid DNA from three different size categories, was found to be at par with the wild type. Other properties, viz., alpha-complementation and ability to express foreign gene remained unaltered. The utility of the rifampicin-resistant phenotype as a potential chromosomal genetic marker was demonstrated in a typical conjugation experiment to establish the ability of the mutant to act as recipient strain for a recombinant, mobilizable plasmid DNA molecule with the advantage of drug-mediated, high efficiency selection. Substitution of the wild strain with the mutant for routine experimentations related to recombinant DNA technology was concluded to be appropriate and of advantage.     

Genome-wide prediction methods for detecting genetic effects of donor chromosome segments in introgression populations

Background

Introgression populations are used to make the genetic variation of unadapted germplasm or wild relatives of crops available for plant breeding. They consist of introgression lines that carry small chromosome segments from an exotic donor in the genetic background of an elite line. The goal of our study was to investigate the detection of favorable donor chromosome segments in introgression lines with statistical methods developed for genome-wide prediction.

Results

Computer simulations showed that genome-wide prediction employing heteroscedastic marker variances had a greater power and a lower false positive rate compared with homoscedastic marker variances when the phenotypic difference between the donor and recipient lines was controlled by few genes. The simulations helped to interpret the analyses of glycosinolate and linolenic acid content in a rapeseed introgression population and plant height in a rye introgression population. These analyses support the superiority of genome-wide prediction approaches that use heteroscedastic marker variances.

Conclusions

We conclude that genome-wide prediction methods in combination with permutation tests can be employed for analysis of introgression populations. They are particularly useful when introgression lines carry several donor segments or when the donor segments of different introgression lines are overlapping.     

The Size and Continuity of DNA Segments Integrated in Bacillus Transformation

We investigated the size and continuity of DNA segments integrated in Bacillus subtilis transformation. We transformed B. subtilis strain 1A2 toward rifampicin resistance (coded by rpoB) with genomic DNA and with a PCR-amplified 3.4-kb segment of the rpoB gene from several donors. Restriction analysis showed that smaller lengths of donor DNA integrated into the chromosome with transformation by PCR-amplified DNA than by genomic DNA. Nevertheless, integration of very short segments (<2 kb) from large, genomic donor molecules was not a rare event. With PCR-amplified segments as donor DNA, smaller fragments were integrated when there was greater sequence divergence between donor and recipient. There was a large stochastic component to the pattern of recombination. We detected discontinuity in the integration of donor segments within the rpoB gene, probably due to multiple integration events involving a single donor molecule. The transfer of adaptations across Bacillus species may be facilitated by the small sizes of DNA segments integrated in transformation.     

Molecular mechanism of genetic recombination in bacterial transformation

Summary B. subtilis cells auxotrophic for two linked markers (ind-his, ind-tyr, his-tyr) have been transformed by means of DNA preparations obtained by hybridization of wild type DNA with the DNA of a strain auxotrophic for one of the linked markers. It was established that hybridization does not increase the transforming activity of DNA for the heterozygous marker. A genetic analysis of the progeny of cells transformed by hybrid or wild type DNA was performed. On the basis of the data obtained a model of genetic recombination in transformation is proved. According to this model both strands of the donor DNA interact independently with the chromosome, and either strand can be incorporated into the cell genome with equal probability. According to the estimate made on the basis of this hypothesis, the probability of integration of a single DNA strand carrying a particular genetic marker is 8%.With 3 Figures in the Text     

A very poorly transformable mutation at the threonine deaminase locus in Bacillu subtilis

Summary An isoleucine requiring mutant of Bacillus subtilis which displays some novel properties has been studied. The strain lacks threonine deaminase activity and is also more sensitive than the wild type to the action of mitomycin C without being more sensitive to UV light. The two features (isoleucine requirement and mitomycin sensitivity) are transferred simultaneously in transformation. The isoleucine marker of this strain is situated at the right end in the map of the threonine deaminase locus. It is at present the nearest known marker to the terminus of the B. subtilis chromosome. Relative transformation frequency for this marker is very low suggesting a low integration efficiency. This is true for both alleles (wild type and mutant) of the marker. SP 10 transduction frequency is almost nil whereas PBS-1 transduction is as effective as for any other marker in that region. Any treatment which causes breakage of the strands in wild type DNA (dilution, shearing, deoxyribonuclease I action) brings about a very important decrease in the relative frequency of transformation for this marker. The marker is more sensitive to these treatments than the weak linkage of two very distant markers. Results agree with the hypothesis that the mutation in this strain corresponds to a deletion in the terminal region of the chromosome. The deletion (suggested also by the absence of reversion) should nevertheless be very long, probably exceeding the average length of a transforming DNA segment. However, an alternative hypothesis has been considered and discussed.     

L-O-Methylthreonine-Resistant Mutant of Arabidopsis Defective in Isoleucine Feedback Regulation

Threonine dehydratase/deaminase (TD), the first enzyme in the isoleucine biosynthetic pathway, is feedback inhibited by isoleucine. By screening M2 populations of ethyl methane sulfonate-treated Arabidopsis thaliana Columbia wild-type seeds, we isolated five independent mutants that were resistant to L-O-methylthreonine, an isoleucine structural analog. Growth in the mutants was 50- to 600-fold more resistant to L-O-methylthreonine than in the wild type. The resistance was due to a single, dominant nuclear gene that was denoted omr1 and was mapped to chromosome 3 in GM11b, the mutant line exhibiting the highest level of resistance. Biochemical characteristics (specific activities, Km, Vmax, and pH optimum) of TD in extracts from the wild type and GM11b were similar except for the inhibition constant of isoleucine, which was 50-fold higher in GM11b than in the wild type. Levels of free isoleucine were 20-fold higher in extracts from GM11b than in extracts from wild type. Therefore, isoleucine feedback insensitivity in GM11b is due to a mutant form of the TD enzyme encoded by omr1. The mutant allele omr1 of the line GM11b could provide a new selectable marker for plant genetic transformation.     

Intergeneric Conjugation in Streptomyces peucetius and Streptomyces sp. Strain C5: Chromosomal Integration and Expression of Recombinant Plasmids Carrying the chiC Gene

Intergeneric conjugal transfer of plasmid DNA from Escherichia coli to Streptomyces circumvents problems such as host-controlled restriction and instability of foreign DNA during the transformation of Streptomyces protoplasts. The anthracycline antibiotic-producing strains Streptomyces peucetius and Streptomyces sp. strain C5 were transformed using E. coli ET12567(pUZ8002) as a conjugal donor. When this donor species, carrying pSET152, was mated with Streptomyces strains, the resident plasmid was mobilized to the recipient and the transferred DNA was also integrated into the recipient chromosome. Analysis of the exconjugants showed stable integration of the plasmid at a single chromosomal site (attB) of the Streptomyces genome. The DNA sequence of the chromosomal integration site was determined and shown to be conserved. However, the core sequence, where the crossover presumably occurred in C5 and S. peucetius, is TTC. These results also showed that the C31 integrative recombination is active and the phage attP site is functional in S. peucetius as well as in C5. The efficiency and specificity of C31-mediated site-specific integration of the plasmid in the presence of a 3.7-kb homologous DNA sequence indicates that integrative recombination is preferred under these conditions. The integration of plasmid DNA did not affect antibiotic biosynthesis or biosynthesis of essential amino acids. Integration of a single copy of a mutant chiC into the wild-type S. peucetius chromosome led to the production of 30-fold more chitinase.     

Involvement of the Reserve Material Poly-β-Hydroxybutyrate in Azospirillum brasilense Stress Endurance and Root Colonization

When grown under suboptimal conditions, rhizobacteria of the genus Azospirillum produce high levels of poly-β-hydroxybutyrate (PHB). Azospirillum brasilense strain Sp7 and a phbC (PHB synthase) mutant strain in which PHB production is impaired were evaluated for metabolic versatility, for the ability to endure various stress conditions, for survival in soil inoculants, and for the potential to promote plant growth. The carbon source utilization data were similar for the wild-type and mutant strains, but the generation time of the wild-type strain was shorter than that of the mutant strain with all carbon sources tested. The ability of the wild type to endure UV irradiation, heat, osmotic pressure, osmotic shock, and desiccation and to grow in the presence of hydrogen peroxide was greater than that of the mutant strain. The motility and cell aggregation of the mutant strain were greater than the motility and cell aggregation of the wild type. However, the wild type exhibited greater chemotactic responses towards attractants than the mutant strain exhibited. The wild-type strain exhibited better survival than the mutant strain in carrier materials used for soil inoculants, but no difference in the ability to promote plant growth was detected between the strains. In soil, the two strains colonized roots to the same extent. It appears that synthesis and utilization of PHB as a carbon and energy source by A. brasilense under stress conditions favor establishment of this bacterium and its survival in competitive environments. However, in A. brasilense, PHB production does not seem to provide an advantage in root colonization under the conditions tested.     

The structural gene for the ribosomal protein S18 in Escherichia coli. II. Chemical studies on the protein S18 having an altered electrophoretic mobility

A mutant of Escherichia coli strain CR341 has an altered 30 S ribosomal protein S18. The alteration involves a change in the electrophoretic mobility of S18. S18 proteins were purified from the mutant and the parent strain, respectively, and their amino acid composition and tryptic peptides were compared. The results have shown that the mutational alteration involves substitution of cysteine for arginine. In addition, we determined the electrophoretic mobility of S18 proteins modified by ethyleneimine. The modification, which involves conversion of cysteine residues to S-(2-aminoethyl)cysteine, causes a greater electrophoretic mobility increase in the mutant protein than in the wild type protein, resulting in identical mobilities for the aminoethylated proteins. This experiment gives further support to the conclusion that the original mobility difference between mutant and wild type proteins is due to the mutational substitution of cysteine for arginine. The S18 obtained from a recombinant was also studied. The recombinant protein was found to have the mobility of the wild type protein and the wild type primary structure, as judged by amino acid composition and tryptic peptide analysis. This recombinant was obtained from the mutant by introducing Hfr strain G10 chromosome segments in the region between 70 and 10 minutes, and not in the str-spc region at 64 minutes, as described in the preceding paper. These results, together with those in the preceding paper, show that the mutation studied here is in the structural gene for S18, and that it maps outside the str-spc region.     

Integration of donor DNA in bacterial conjugation

Conjugation between ¹³C¹⁵N- and ³H-labelled hybrid donors and ¹³C¹⁵N-labelled hybrid recipients of Escherichia coli gives rise to recombinant radioactive DNA of density greater than labelled hybrid. The donor radioactivity is present, in these molecules, in discrete heavy segments covalently attached to the light strand. When light radioactive Hfr cells are mated to heavy F^? cells in light medium, the donor label appears, in DNA extracted from the F^? cells, in labelled hybrid molecules. The radioactivity in these molecules is exclusively in the light strand. The insertion of donor material is thus restricted to a single newly formed strand of the recipient DNA and double-strand integrations do not occur. A temperature-sensitive recipient containing the dna B mutation ts₄₃ accumulates single-stranded Hfr DNA if mating is carried out at the nonpermissive temperature. The formation of a complementary strand in the recipient does not, therefore, appear to be necessary for continued transfer of Hfr DNA.     

Mapmi gene contributes to stress tolerance and virulence of the entomopathogenic fungus, Metarhizium acridum

Phosphomannose isomerase (PMI) catalyzes the reversible interconversion of fructose 6-phosphate (Fru-6-P) and mannose 6-phosphate (Man-6-P), providing a link between glycolysis and the mannose metabolic pathway. In this study, we identified pmi gene (Mapmi) from the entomopathogenic fungus, Metarhizium acridum, and analyzed its functions using RNA interference (RNAi). Amending the growth medium with cell stress chemicals significantly reduced growth, conidial production and percent germination in Mapmi-RNAi mutant strain, compared to the wild-type strain. Growth of RNAi mutant was lower than the wild type strain with glucose or fructose as sole carbon source. RNAi mutant exhibited a normal growth phenotype with mannose at low concentrations, while trace or high concentration of mannose was more negatively impacted the growth of RNAi mutant than the wild type strain. Infection with Mapmi-RNAi mutant against Locusta migratoria manilensis (Meyen) led to a significantly reduced virulence compared to infection with the wild-type strain. These results suggest that Mapmi plays essential roles in stress tolerance and pathogenicity of M. acridum.