Chromosomal Gene Inactivation in the Green Sulfur Bacterium Chlorobium tepidum by Natural Transformation

Conditions for inactivating chromosomal genes of Chlorobium tepidum by natural transformation and homologous recombination were established. As a model, mutants unable to perform nitrogen fixation were constructed by interrupting nifD with various antibiotic resistance markers. Growth of wild-type C. tepidum at 40°C on agar plates could be completely inhibited by 100 μg of gentamicin ml⁻¹, 2 μg of erythromycin ml⁻¹, 30 μg of chloramphenicol ml⁻¹, or 1 μg of tetracycline ml⁻¹ or a combination of 300 μg of streptomycin ml⁻¹ and 150 μg of spectinomycin ml⁻¹. Transformation was performed by spotting cells and DNA on an agar plate for 10 to 20 h. Transformation frequencies on the order of 10⁻⁷ were observed with gentamicin and erythromycin markers, and transformation frequencies on the order of 10⁻³ were observed with a streptomycin-spectinomycin marker. The frequency of spontaneous mutants resistant to gentamicin, erythromycin, or spectinomycin-streptomycin was undetectable or significantly lower than the transformation frequency. Transformation with the gentamicin marker was observed when the transforming DNA contained 1 or 3 kb of total homologous flanking sequence but not when the transforming DNA contained only 0.3 kb of homologous sequence. Linearized plasmids transformed at least an order of magnitude better than circular plasmids. This work forms a foundation for the systematic targeted inactivation of genes in C. tepidum, whose 2.15-Mb genome has recently been completely sequenced.     

Genetic transfer by conjugation in the thermophilic green sulfur bacterium Chlorobium tepidum.

The broad-host-range IncQ group plasmids pDSK519 and pGSS33 were transferred by conjugation from Escherichia coli into the thermophilic green sulfur bacterium Chlorobium tepidum. C. tepidum exconjugants expressed the kanamycin and ampicillin-chloramphenicol resistances encoded by pDSK519 and pGSS33, respectively. Ampicillin resistance was a particularly good marker for selection in C. tepidum. Both pDSK519 and pGSS33 were stably maintained in C. tepidum at temperatures below 42 degrees C and could be transferred between C. tepidum and E. coli without modifications. Conjugation frequencies ranged from 10(-1) to 10(-4) exconjugants per donor cell, and frequencies of 10(-2) to 10(-3) were consistently obtained when ampicillin resistance was used as a selectable marker. Methods for growth of C. tepidum on agar, isolation of plating strains and antibiotic-resistant mutants of wild-type C. tepidum cells, and optimum conditions for conjugation were also investigated.     

Genetic transformation of Streptococcus pneumoniae by DNA cloned into the single-stranded bacteriophage f1.

A Staphylococcus aureus plasmid derivative, pFB9, coding for erythromycin and chloramphenicol resistance was cloned into the filamentous Escherichia coli phage f1. Recombinant phage-plasmid hybrids, designated plasmids, were isolated from E. coli and purified by transformation into Streptococcus pneumoniae. Single-stranded DNA was prepared from E. coli cells infected with two different plasmids, fBB101 and fBB103. Introduction of fully or partially single-stranded DNA into Streptococcus pneumoniae was studied, using a recipient strain containing an inducible resident plasmid. Such a strain could rescue the donor DNA marker. Under these marker rescue conditions, single-stranded fBB101 DNA gave a 1% transformation frequency, whereas the double-stranded form gave about a 31% frequency. Transformation of single-stranded fBB101 DNA was inhibited by competing double-stranded DNA and vice versa, indicating that single-stranded DNA interacts with the pneumococcus via the same binding site as used by double-stranded DNA. Heteroduplexed DNA containing the marker within a 70- or 800-base single-stranded region showed only slightly greater transforming activity than pure single-stranded DNA. In the absence of marker rescue, both strands of such imperfectly heteroduplexed DNA demonstrated transforming activity. Pure single-stranded DNA demonstrated low but significant transforming activity into a plasmid-free recipient pneumococcus.     

Genetic transformation of Streptococcus pneumoniae by heterologous plasmid deoxyribonucleic acid.

A number of heterologous plasmid deoxyribonucleic acids (DNAs) coding for erythromycin, tylosin, lincomycin, tetracycline, or chloramphenicol resistance have been introduced into Streptococcus pneumoniae via genetic transformation with frequencies that varied between 10(-5) to as high as 5 x 10(-1) per colony-forming unit. Transformation with plasmid DNA required pneumococcal competence, was competed by chromosomal DNA, and showed a saturation at about 0.5 micrograms/ml (with a recipient population of 3 x 10(7) colony-forming units of competent cells per ml). Plasmid transformation did not occur with a recipient strain, 410, defective in endonuclease I activity and in chromosomal genetic transformation. All erythromycin-resistant transformants examined contained covalently closed circular DNA with the same electrophoretic mobility on agarose gels as the donor DNAs, and when examined in detail the plasmid reisolated from the transformants had the same restriction patterns and the same specific transforming activity as the donor DNA. In the cases of two plasmids examined in detail--pAM77 and pSA5700 Lc9--most of the transforming activity was associated with DNA monomers; DNA multimers present in pSA5700 Lc9 also had biological activity. An unexpected finding was the demonstration of transformation (2 x 10(-5) per colony-forming unit) with plasmid DNAs linearized by treatment with S1 nuclease or with restriction endonucleases.     

Development of a transformation system for the flavinogenic yeast Candida famata

Riboflavin-overproducing mutants of the flavinogenic yeast Candida famata are used for industrial riboflavin production. This paper describes the development of an efficient transformation system for this species. Leucine-deficient mutants have been isolated from C. famata VKM Y-9 wild-type strain. Among them leu2 mutants were identified by transformation to leucine prototrophy with plasmids YEp13 and PRpL2 carrying the Saccharomyces cerevisiae LEU2 gene. DNA fragments (called CfARSs) conferring increased transformation frequencies and extrachromosomal replication were isolated from a C. famata gene library constructed on the integrative vector containing the S. cerevisiae LEU2 gene as a selective marker. The smallest cloned fragment (CfARS16) has been sequenced. This one had high adenine plus thymine (A+T) base pair content and a sequence homologous to the S. cerevisiae ARS Consensus Sequence. Methods for spheroplast transformation and electrotransformation of the yeast C. famata were optimized. They conferred high transformation frequencies (up to 10(5) transformants per microg DNA) with a C. famata leu2 mutant using replicative plasmids containing the S. cerevisiae LEU2 gene as a selective marker. Riboflavin-deficient mutants were isolated from the C. famata leu2 strain and their biochemical identification was carried out. Using the developed transformation system, several C. famata genomic fragments complementing mutations of structural genes for riboflavin biosynthesis (coding for GTP cyclohydrolase, reductase, dihydroxybutanone phosphate synthase and riboflavin synthase, respectively) have been cloned.     

Plasmid profiling and curing of Lactobacillus strains isolated from the gastrointestinal tract of chicken

In this study, we assessed the susceptibility of 12 Lactobacillus strains, all of which had been isolated from the gastrointestinal tracts of chicken, to three antibiotics (chloramphenicol, erythromycin and tetracycline) used commonly as selective markers in transformation studies of lactic acid bacteria. Among these strains, 17%, 58%, and 25% were found to exhibit a high degree of resistance to 200 microg/ml of tetracycline, erythromycin, and chloramphenicol, respectively. Seven of the 12 Lactobacillus strains exhibiting resistance to at least 50 microg/ml of chloramphenicol or erythromycin, and five strains exhibiting resistance to at least 50 microg/ml of tetracycline, were subsequently subjected to plasmid curing with chemical curing agents, such as novobiocin, acriflavin, SDS, and ethidium bromide. In no cases did the antibiotic resistance of these strains prove to be curable, with the exception of the erythromycin resistance exhibited by five Lactobacillus strains (L. acidophilus I16 and I26, L. fermentum I24 and C17, and L. brevis C10). Analysis of the plasmid profiles of these five cured derivatives revealed that all of the derivatives, except for L. acidophilus I16, possessed profiles similar to those of wild-type strains. The curing of L. acidophilus I16 was accompanied by the loss of 4.4 kb, 6.1 kb, and 11.5 kb plasmids.     

Transformation of Acidiphilium by electroporation and conjugation.

Two techniques, electroporation and conjugation, have been used to introduce the RK2-based broad-host-range plasmids pRK415 and pLAFR3 into strains of the bacterial genus Acidiphilium. Using electroporation, cells were also transformed with a series of chimeric plasmids constructed by cloning cryptic Acidiphilium plasmids into the Escherichia coli vector pBR328. Various parameters affecting electroporation were investigated. Transformation efficiency varied widely with different recipient strains. Growth at an elevated temperature (37 degrees C) prior to electroporation increased transformation efficiency 10-fold compared with growth at 32 degrees C. For three strains tested, optimum transformation efficiency was obtained with field strengths of 10-15 kV/cm. Transformation efficiency increased linearly with increasing DNA concentration up to 10 micrograms/mL. Transformation efficiencies in these experiments ranged up to 10(4) transformants/micrograms DNA. Mobilization of pRK415 and pLAFR3 from E. coli strain S17.1 into several Acidiphilium strains was achieved following incubation for 3 h on nutrient agar medium (pH 7.0). Conjugation frequencies in the range of 10(-5)-10(-9) per recipient cell were obtained. Conjugation frequency was also dependent on recipient strain.     

Natural transformation-mediated transfer of erythromycin resistance in Campylobacter coli strains from turkeys and swine

Erythromycin resistance in Campylobacter coli from meat animals is frequently encountered and could represent a substantial barrier to antibiotic treatment of human infections. Erythromycin resistance in this organism has been associated with a point mutation (A2075G) in the 23S rRNA gene. However, the mechanisms responsible for possible dissemination of erythromycin resistance in C. coli remain poorly understood. In this study, we investigated transformation-mediated acquisition of erythromycin resistance by genotypically diverse C. coli strains from turkeys and swine, with total genomic DNA from erythromycin-resistant C. coli of either turkey or swine origin used as a donor. Overall, transformation to erythromycin resistance was significantly more frequent in C. coli strains from turkeys than in swine-derived strains (P < 0.01). The frequency of transformation to erythromycin resistance was 10(-5) to 10(-6) for turkey-derived strains but 10(-7) or less for C. coli from swine. Transformants harbored the point mutation A2075G in the 23S rRNA gene, as did the erythromycin-resistant strains used as DNA donors. Erythromycin resistance was stable in transformants following serial transfers in the absence of the antibiotic, and most transformants had high MICs (>256 microg/ml), as did the C. coli donor strains. In contrast to the results obtained with transformation, spontaneous mutants had relatively low erythromycin MICs (32 to 64 microg/ml) and lacked the A2075G mutation in the 23S rRNA gene. These findings suggest that natural transformation has the potential to contribute to the dissemination of high-level resistance to erythromycin among C. coli strains colonizing meat animals.     

Plasmid transformation of Streptococcus sanguis (Challis) occurs by circular and linear molecules

Summary Transformation of Streptococcus sanguis (Challis) by antibiotic resistance plasmids has shown that (a) competente developed with identical kinetics for chromosomal and plasmid DNA; (b) dependence of transformant yield on plasmid DNA concentration was second order; (c) open circular plasmid DNA transformed Challis, although at reduced frequency; (d) linearization of plasmid DNA by restriction enzymes cutting at unique sites inactivated the transforming capacity; (e) transforming activity was restored when linear plasmid molecules generated by different restriction enzymes were mixed; (f) restoration of transforming activity depended on the distance between the linearizing cuts, i.e. on the presence of sufficiently long overlapping homologous sequences; (g) when linear deletion mutants were mixed with linear parental plasmids the smaller plasmid was restored with significantly higher frequency.Based on these data, a model for plasmid transformation of Challis is proposed according to which circular plasmid is linearized during binding and uptake. One DNA strand enters the cell and restoration of circular plasmids inside the cell occurs by annealing of complementary single strands from two different donor molecules. Implications of this model for recombinant DNA experiments in streptococci are discussed.     

Plasmid transfer from Streptomyces to Mycobacterium smegmatis by spontaneous transformation

Hybrids of the Streptomyces coelicolor conjugative plasmid SCP2* and the Mycobacterium plasmid pAL5000 were transferred from Streptomyces coelicolor or Streptomyces lividans to Mycobacterium smegmatis mc2155 in plate crosses. Inactivation of the SCP2* transfer function did not prevent or reduce plasmid transfer. This transfer was DNase I sensitive and thus involved release of DNA from Streptomyces, followed by transformation of M. smegmatis. M. smegmatis growing on specific solid media was also transformed by pure CCC and linear plasmid DNA. Small plasmids were taken up intact but large plasmids suffered deletions. Competence developed within 24 h of incubation at 30 degrees C or 37 degrees C, and up to 400 transformants were obtained per microg of CCC plasmid DNA. Transformation frequencies were higher when M. smegmatis was co-cultivated with plasmid-free Streptomyces, but unaffected by resident homologous sequences or inactivation of recA in M. smegmatis. Spontaneous transformation was also observed with a circular Streptomyces transposable element which inserted into chromosomal sites. Transformative plasmid transfer was also shown to occur between M. smegmatis strains. This is the first report of non-artificially induced, spontaneous plasmid transformation in Mycobacterium.     

Characterization of plasmid transformation in Bacillus subtilis: kinetic properties and the effect of DNA conformation.

Summary Transformation of competent cells of Bacillus subtilis with antibiotic resistance plasmid DNA has shown that (a) competence for plasmid and chromosomal DNA develops with similar kinetics; (b) DNA linearized with a variety of restriction endonucleases does not transform; (c) CCC plasmid DNA is inactivated for transformation by a single nick; (d) T4 ligase restores transforming activity to both nicked and linearized DNA; (e) CCC relaxed DNA is fully active in transformation; (f) the DNA concentration-dependence of plasmid transformation is first order; and (g) plasmid transformation proceeds with a low efficiency, requiring the uptake of 10³ to 10⁴ DNA molecules per transformant.Based on this information, a model for the processing of chromosomal, plasmid and transfecting DNA is proposed.Abbreviations Cm Chloramphenicol - Em erythromycin - Km kanamycin - Sm streptomycin - CCC covalently closed circular - TBAB tryptose blood agar base - NCE nicking and closing enzyme In partial fulfillment of the requirements for the doctoral degree in the Department of Microbiology at the New York University School of Medicine, for S.C.     

Transformation of the fungusPyrenopeziza brassicae,cause of light leaf spot of brassicas,and complementation of mutants using a genomic library

An efficient gene transfer system is a prerequisite for the molecular genetic analysis of pathogenicity and other genes of plant pathogens. A transformation procedure for the fungusPyrenopeziza brassicae was therefore devised. Three plasmids, encoding hygromycin resistance (pAN7-1, pAN7-2) or phleomycin resistance (pAN8-1), were used to transform conidial protoplasts ofP. brassicae in the presence of calcium chloride and polyethylene glycol. Transformation arose due to integration of transforming DNA, apparently at random sites, and multiple integration events were common. The frequency of transformation was variable but similar to that reported for other phytopathogenic fungi (up to 20 μg⁻¹ DNA) and was increased when homologous DNA was included in the vector. The pathogenicity of the transformants was unchanged by transformation and, when reisolated from inoculated host tissue, the transformants were found to have retained their antibiotic resistance. The transformation technique was used to complement adeninerequiring and extracellular enzyme-deficient, UV-induced mutants to prototrophy and extracellular protease production, respectively, with cosmids from a genomic library of the fungus.     

DNA repair and the evolution of transformation IV. DNA damage increases transformation

Natural genetic transformation in the bacterium Bacillus subtilis provides a model system to explore the evolutionary function of sexual recombination. In the present work, we study the response of transformation to UV irradiation using donor DNAs that differ in sequence homology to the recipient's chromosome and in the mechanism of transformation. The four donor DNAs used include homologous-chromosomal-DNA, two plasmids containing a fragment of B. subtilis trp⁺ operon DNA and a plasmid with no sequence homology to the recipient cell's DNA. Transformation frequencies for these DNA molecules increase with increasing levels of DNA damage (UV radiation) to recipient cells, only if their transformation requires homologous recombination (i.e. is recA⁺-dependent). Transformation with non-homologous DNA is independent of the recipient's recombination system and transformation frequencies for it do not respond to increases in UV radiation. The transformation frequency for a selectable marker increases in response to DNA damage more dramatically when the locus is present on small, plasmid-borne, homologous fragments than if it is carried on high molecular weight chromosomal fragments. We also study the kinetics of transformation for the different donor DNAs. Different kinetics are observed for homologous transformation depending on whether the homologous locus is carried on a plasmid or on chromosomal fragments. Chromosomal DNA- and non-homologous-plasmid-DNA-mediated transformation is complete (maximal) within several minutes, while transformation with a plasmid containing homologous DNA is still occurring after an hour. The results indicate that DNA damage directly increases rates of homologous recombination and transformation in B. subtilis. The relevance of these results and recent results of other labs to the evolution of transformation are discussed.     

Stable allele replacement and unstable non-homologous integration events during transformation of Ascobolus immersus

An homologous transformation system for the filamentous fungus Ascobolus immersus has been developed, based on the complementation of a met2 mutation by the wild-type (wt) allele gene encoding homoserine O-transacetylase. Transformation of A. immersus met2 mutants occurs with moderate frequencies (about 50 transformants per microgram input DNA). Analysis of the DNA of the met2+ transformants showed that transformation resulted either in a single integration of the donor DNA into the genome by many different nonhomologous recombination events or in the substitution of the endogenous met2 mutation by the wt transforming allele. The relative frequencies of both events depended on the vector sequences carrying the cloned met2 gene. Whereas the substitution event led, as expected, to genetically stable transformants, the non-homologous integration was always associated with a strong instability when transformants were crossed and underwent meiosis.     

Replicative transformation of the filamentous fungus Ashbya gossypii with plasmids containing Saccharomyces cerevisiae ARS elements.

We have developed a transformation system for the filamentous ascomycete fungus Ashbya gossypii. Mycelial protoplasts were transformed to geneticin-resistance with plasmids containing the Escherichia coli kanamycin-resistance gene as a selectable marker and autonomously replicating sequences (ARS) from Saccharomyces cerevisiae (ARS1, 2 mu ARS). Transformation frequencies of up to 63 transformants per microgram of plasmid DNA were obtained. The transformants were unstable under nonselective conditions. Southern analysis of DNA separated by conventional and pulsed-field-gel electrophoresis showed that the transforming DNA was present as autonomously replicating plasmid. Plasmid integration into chromosomal DNA was not detected. We concluded that the S. cerevisiae ARS elements are functional in A. gossypii, since vectors lacking such elements did not yield transformants.     

Natural transformation in Campylobacter species.

Growing cells of Campylobacter coli and C. jejuni were naturally transformed by naked DNA without the requirement for any special treatment. Transformation frequencies for homologous chromosomal DNA were approximately 10(-3) transformants per recipient cell in C. coli and 10(-4) in C. jejuni. Maximum competence was found in the early log phase of growth. Campylobacters preferentially took up their own DNA in comparison with Escherichia coli chromosomal DNA, which was taken up very poorly. Three new Campylobacter spp.-to-E. coli shuttle plasmids, which contained additional cloning sites and selectable markers, were constructed from the shuttle vector pILL550A. These plasmid DNAs were taken up by campylobacters much less efficiently than was homologous chromosomal DNA, and transformation into plasmid-free cells was very rare. However, with the use of recipients containing a homologous plasmid, approximately 10(-4) transformants per cell were obtained. The tetM determinant, originally obtained from Streptococcus spp. and not heretofore reported in Campylobacter spp., was isolated from an E. coli plasmid and was introduced, selecting for tetracycline resistance, by natural transformation into C. coli.     

Efficient plasmid transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts.

A procedure for efficient transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts with plasmid DNA was developed. Transformation frequencies with S. fradiae protoplasts were strongly influenced by the temperatures for cell growth, protoplast formation, and protoplast regeneration. Transformation frequencies for both species were also influenced by the culture age before protoplast formation, the source and concentration of polyethylene glycol, the transformation-inducing agent, the concentration of protoplasts used in the transformation procedure, and the number of protoplasts added to regeneration plates. Transformation frequencies were substantially higher for both species when calf thymus DNA and protamine sulfate were added to the transformation mix. With S. fradiae, transformation frequencies were much lower with plasmid DNA prepared from other species than with the same plasmids prepared from S. fradiae, suggesting that S. fradiae expresses restriction and modification. With the modified transformation procedures using DNA prepared from homologous hosts, S. ambofaciens and S. fradiae are now transformed routinely at frequencies of 10(6) to 10(7) transformants per micrograms of plasmid DNA.     

Construction of a bacterial artificial chromosome library of Phytophthora infestans and transformation of clones into P. infestans

A bacterial artificial chromosome (BAC) library of Phytophthora infestans was constructed in a derivative of pBELOBACII that had been modified by adding a npt selectable marker gene for transforming P. infestans. A total library of 8 genome equivalents was generated and 16,128 clones with inserts averaging 75 kb (4.9 genome equivalents) were individually picked and stored as an arrayed library in microtiter plates. This coverage was confirmed by screening the library for 11 DNA loci by colony hybridization and by polymerase chain reaction of DNA pools. Transformation of P. infestans with BAC clones containing inserts of 93 to 135 kb was demonstrated. The efficiency of transformation with most BACs was noticeably higher than that with smaller plasmids. Detailed analyses of transformants obtained with a 102-kb BAC indicated that entire inserts were present in about one-quarter of the transformants.     

Inefficient replication reduces RecA-mediated repair of UV-damaged plasmids introduced into competent Escherichia coli

Transformation of Escherichia coli with purified plasmids containing DNA damage is frequently used as a tool to characterize repair pathways that operate on chromosomes. In this study, we used an assay that allowed us to quantify plasmid survival and to compare how efficiently various repair pathways operate on plasmid DNA introduced into cells relative to their efficiency on chromosomal DNA. We observed distinct differences between the mechanisms operating on the transforming plasmid DNA and the chromosome. An average of one UV-induced lesion was sufficient to inactivate ColE1-based plasmids introduced into nucleotide excision repair mutants, suggesting an essential role for repair on newly introduced plasmid DNA. By contrast, the absence of RecA, RecF, RecBC, RecG, or RuvAB had a minimal effect on the survival of the transforming plasmid DNA containing UV-induced damage. Neither the presence of an endogenous homologous plasmid nor the induction of the SOS response enhanced the survival of transforming plasmids. Using two-dimensional agarose-gel analysis, both replication- and RecA-dependent structures that were observed on established, endogenous plasmids following UV-irradiation, failed to form on UV-irradiated plasmids introduced into E. coli. We interpret these observations to suggest that the lack of RecA-mediated survival is likely to be due to inefficient replication that occurs when plasmids are initially introduced into cells, rather than to the plasmid's size, the absence of homologous sequences, or levels of recA expression.     

Restriction and sequence alterations affect DNA uptake sequence-dependent transformation in Neisseria meningitidis

Transformation is a complex process that involves several interactions from the binding and uptake of naked DNA to homologous recombination. Some actions affect transformation favourably whereas others act to limit it. Here, meticulous manipulation of a single type of transforming DNA allowed for quantifying the impact of three different mediators of meningococcal transformation: NlaIV restriction, homologous recombination and the DNA Uptake Sequence (DUS). In the wildtype, an inverse relationship between the transformation frequency and the number of NlaIV restriction sites in DNA was observed when the transforming DNA harboured a heterologous region for selection (ermC) but not when the transforming DNA was homologous with only a single nucleotide heterology. The influence of homologous sequence in transforming DNA was further studied using plasmids with a small interruption or larger deletions in the recombinogenic region and these alterations were found to impair transformation frequency. In contrast, a particularly potent positive driver of DNA uptake in Neisseria sp. are short DUS in the transforming DNA. However, the molecular mechanism(s) responsible for DUS specificity remains unknown. Increasing the number of DUS in the transforming DNA was here shown to exert a positive effect on transformation. Furthermore, an influence of variable placement of DUS relative to the homologous region in the donor DNA was documented for the first time. No effect of altering the orientation of DUS was observed. These observations suggest that DUS is important at an early stage in the recognition of DNA, but does not exclude the existence of more than one level of DUS specificity in the sequence of events that constitute transformation. New knowledge on the positive and negative drivers of transformation may in a larger perspective illuminate both the mechanisms and the evolutionary role(s) of one of the most conserved mechanisms in nature: homologous recombination.