Transformation of Haemophilus influenzae by plasmid RSF0885 containing a cloned segment of chromosomal deoxyribonucleic acid.

A plasmid containing a single cloned insertion of Haemophilus influenzae chromosomal deoxyribonucleic acid that carried a novobiocin resistance marker was 2.6 times larger than the parent plasmid, RSF0885, which conferred ampicillin resistance. The most frequent type of transformation by this plasmid (designated pNov1) was the transfer of novobiocin resistance to the chromosome, with the loss of the plasmid from the recipient. In accord with this observation, after radioactively labeled pNov1 entered a competent cell, it lost acid-insoluble counts, as well as biological activity. The level of ampicillin transformation, which involved establishment of the plasmid, was almost two orders of magnitude lower than the level of novobiocin transformation. Both types of transformation were depressed profoundly in rec-1 and rec-2 mutants. Ampicillin transformants of wild-type cells always contained plasmids that were the same size as pNov1, although most of these transformants were not novobiocin resistant. Plasmid pNov1 in wild-type cells but not in rec-1 or rec-2 cells often recombined with the chromosome, causing a homologous region of the chromosome to be substituted for part of the plasmid, as shown by restriction and genetic analyses. Our data suggested that plasmid-chromosome recombination took place only around the time when the plasmid entered a cell, rather than after it became established.     

Loss of plasmids containing cloned inserts coding for novobiocin resistance or novobiocin sensitivity in Haemophilus influenzae.

Plasmids pNov1 and pNov1s , coding for resistance and sensitivity to novobiocin, respectively, were readily lost from wild-type Haemophilus influenzae but retained in a strain lacking an inducible defective prophage. The plasmid loss could be partly or wholly eliminated by a low-copy-number mutation in the plasmid or by the presence of certain antibiotic resistance markers in the host chromosome. Release of both phage HP1c1 , measured by plaque assay, and defective phage, measured by electron microscopy, was increased when the plasmids were present. The frequency of recombination between pNov1 and the chromosome, causing the plasmid to be converted to pNov1s , could under some circumstances be decreased from the normal 60 to 70% to below 10% by the presence of a kanamycin resistance marker in the chromosome. This suggested that a gene product coded for by the plasmid, the expression of which was affected by the kanamycin resistance marker, was responsible for the high recombination frequency. Evidence was obtained from in vitro experiments that the gene product was a gyrase.     

Mechanism of acquisition of chromosomal markers by plasmids in Haemophilus influenzae.

The hybrid plasmid pNov1 readily acquired genetic information from the chromosome of wild-type, but not rec-2, cells. Most of the recombination had taken place 1 h after entrance of the plasmid into the cell, as judged by transformation of rec-2 by lysates made from wild-type cells exposed to pNov1. Measurement of physical transfer from radioactively labeled cellular DNA to plasmids recombining in wild-type cells failed, since there was little more radioactivity in plasmids from such cells than from labeled rec-2 recipients, in which no recombination took place. EcoRI digestion of pNov1 divided the DNA into a 1.7-kilobase-pair fragment containing the novobiocin resistance marker and a 13-kilobase-pair fragment containing all of the original vector and considerable portions homologous to the chromosome. Transformation by the large fragment alone resulted in a plasmid the size of the original pNov1. Our hypothesis to explain the data is that genetic transfer from chromosome to plasmid took place by a copy choice mechanism.     

Mutations affecting gyrase in Haemophilus influenzae.

Mutants separately resistant to novobiocin, coumermycin, nalidixic acid, and oxolinic acid contained gyrase activity as measured in vitro that was resistant to the antibiotics, indicating that the mutations represented structural alterations of the enzyme. One Novr mutant contained an altered B subunit of the enzyme, as judged by the ability of a plasmid, pNov1, containing the mutation to complement a temperature-sensitive gyrase B mutation in Escherichia coli and to cause novobiocin resistance in that strain. Three other Novr mutations did not confer antibiotic resistance to the gyrase but appeared to increase the amount of active enzyme in the cell. One of these, novB1, could only act in cis, whereas a new mutation, novC, could act in trans. An RNA polymerase mutation partially substituted for the novB1 mutation, suggesting that novB1 may be a mutation in a promoter region for the B subunit gene. Growth responses of strains containing various combinations of mutations on plasmids or on the chromosome indicated that low-level resistance to novobiocin or coumermycin may have resulted from multiple copies of wild-type genes coding for the gyrase B subunit, whereas high-level resistance required a structural change in the gyrase B gene and was also dependent on alteration in a regulatory region. When there was mismatch at the novB locus, with the novB1 mutation either on a plasmid or the chromosome, and the corresponding wild-type gene present in trans, chromosome to plasmid recombination during transformation was much higher than when the genes matched, probably because plasmid to chromosome recombination, eliminating the plasmid, was inhibited by the mismatch.     

Differential behavior of plasmids containing chromosomal DNA insertions of various sizes during transformation and conjugation in Haemophilus influenzae.

Plasmids with chromosomal insertions were constructed by removal of a 1.1-kilobase-pair piece from the 9.8-kilobase-pair vector plasmid pDM2 by EcoRI digestion and inserting in its place various lengths of chromosomal DNA (1.7, 3.4, and 9.0 kilobase pairs) coding for resistance to novobiocin. A fourth plasmid was constructed by insertion of the largest piece of chromosomal DNA into the SmaI site of pDM2. The plasmids without inserts were taken up poorly by competent cells and thus were considered not to contain specific DNA uptake sites. The presence of even the smallest insert of chromosomal DNA caused a large increase in transformation of Rec+ and Rec- strains. The frequency of plasmid establishment in Rec+ cells by transformation increased exponentially with increasing insert size, but in Rec- cells there was less transformation by the larger plasmids. Conjugal transfer of these plasmids was carried out with the 35-kilobase-pair mobilizing plasmid pHD147. The frequency of establishment of plasmids by this method not only was not markedly affected by the presence of the insertions, but also decreased somewhat with increase in insert size and was independent of rec-1 and rec-2 genes. Recombination between plasmid and chromosome was readily detected after transformation, but could not be detected after transconjugation even when the recipient cells were Rec+ and made competent. These data suggested that there is a special processing of plasmid DNA that enters the competent cells in transformation that makes possible recombination of homologous regions of the plasmid with the chromosome and pairing with the chromosome that aids plasmid establishment.     

Homology and repair of UV-irradiated plasmid DNA in haemophilus influenzae

UV-irradiated plasmid pNov1 containing a cloned fragment of chromosomal DNA could be repaired by excision, but plasmid p2265 without homology to the chromosome could not. Establishment of pNov1 was more UV resistant in Rec⁻ than in Rec⁺ cells.     

Physical structure and genetic expression of the sulfonamide-resistance plasmid pLS80 and its derivatives in Streptococcus pneumoniae and Bacillus subtilis

The 10-kb chromosomal fragment of Streptococcus pneumoniae cloned in pLS80 contains the sul-d allele of the pneumococcal gene for dihydropteroate synthase. As a single copy in the chromosome this allele confers resistance to sulfanilamide at 0.2 mg/ml; in the multicopy plasmid it confers resistance to 2.0 mg/ml. The sul-d mutation was mapped by restriction analysis to a 0.4-kb region. By the mechanism of chromosomal facilitation, in which the chromosome restores information to an entering plasmid fragment, a BamHI fragment missing the sul-d region of pLS80 established the full-sized plasmid, but with the sul-s allele of the recipient chromosome. A spontaneous deletion beginning approximately 1.5 kb to the right of the sul-d mutation prevented gene function, possibly by removing a promoter. This region could be restored by chromosomal facilitation and be demonstrated in the plasmid by selection for sulfonamide resistance. Under selection for a vector marker, tetracycline resistance, only the deleted plasmid was detectable, apparently as a result of plasmid segregation and the advantageous growth rates of cells with smaller plasmids. When such cells were selected for sulfonamide resistance, the deleted region returned to the plasmid, presumably by equilibration between the chromosome and the plasmid pool, to give a low frequency (approximately 10(-3) of cells resistant to sulfanilamide at 2.0 mg/ml. Models for the mechanisms of chromosomal facilitation and equilibration are proposed. Several derivatives of pLS80 could be transferred to Bacillus subtilis, where they conferred resistance to sulfanilamide at 2 mg/ml, thereby demonstrating cross-species expression of the pneumococcal gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning of the purA16 locus in Rec+ cells of Bacillus subtilis

A portion of purA16 chromosomal locus of Bacillus subtilis was cloned into Rec+ cells of this microorganism with pBD12 plasmid (carrying chloramphenicol and kanamycin resistance determinants) serving as a vector. The hybrid plasmids were stably maintained in cells grown on media supplemented with antibiotics and were lost from cells in the absence of drugs. The cloned fragment could incorporate into the chromosome some with a frequency of 10(-2) per cell per generation. A clone carrying the hybrid plasmid inserted into the chromosome was detected.     

The plasmid carrying the temperature-sensitive mutation in the DNa-methylase gene of the PStI system: effect on host cells at nonpermissive temperature]

Temperature-sensitive (ts) derivatives of plasmid pRMP1, the derivative of PBR322 containing restriction and modification (RM) genes of the PstI system, were obtained using hydroxylamine mutagenesis. One of the isolated plasmids responsible for the inhibition of Escherichia coli cell growth at 42 degrees C, pRMPts, was analyzed in this work. Cells of Rec+ strains carrying this plasmid were unable to divide at 42 degrees C and formed long non-septated filaments that died upon prolonged cultivation. Cells of the RecA- strains carrying pRMPts did not form filaments at 42 degrees C and rapidly disappeared. On agar media with or without ampicillin, Rec+ and RecA- strains with this plasmid formed colonies of temperature-resistant (tr) derivatives with frequencies ranging from 1.5 x 10(-4) to 4 x 10(-6) in independent clones. The structure of plasmids from cells of tr-derivatives of Rec+ and RecA- strains carrying plasmid pRMPts was analyzed by the set of restriction enzymes. Reversions to the temperature-resistant phenotype were shown to result from the following events: (1) the insertional inactivation of the PstI restriction enzyme gene in pRMPts (the insertion of the IS1 element); (2) deletions in plasmid DNA fragments that partially or completely cover the restriction enzyme gene; (3) point mutations; and (4) others. The effect of the chromosomal sulA mutation on the maintenance of the ts-plasmid in bacterial cells was studied at 42 degrees C. High efficiency loss of the plasmid was detected in pRMPts-carrying Rec+ cells with the sulA::Tn5 mutation grown in liquid and solid nutrient media at this temperature. Under similar conditions, plasmid loss was not detected in SulA+ cells. On the basis of the data obtained, it is concluded that the ts-mutation is located in the DNA-methylase gene of plasmid pRMPts. Mutant DNA methylase was unable to methylate all sites in the chromosomal DNA at 42 degrees C. Some of the unmethylated sites can be digested with the PstI enzyme, which leads to the induction of SOS response in Rec+ cells or to total mortality in cells with the recA phenotype.     

Structural instability of IncP-1 plasmids in Pseudomonas aeruginosa PAT involves interaction with plasmid pVS1

The structural instability exhibited by IncP-1 plasmids in Pseudomonas aeruginosa strain PAT was shown to be Rec+ dependent and involved interaction with the resident plasmid pVS1. Structural instability resulted from deletion of plasmid deoxyribonucleic acid at a frequency of ca. 10(-2)/cell per generation. Deletants could be stabilized by transduction into P. aeruginosa strain PAO, but in strain PAT deletants had only a transient existence, as continued deletion led eventually to the loss of the entire plasmid. The patterns of markers lost in PAT were used to demonstrate a marker order for R68 similar to that published elsewhere for RP4 (Barth and Grinter, J. Mol. Biol. 113:455-474, 1977), except that only one Tra region was found. R68 also exhibited Rec+-dependent structural instability in PAO(pVS1) derivatives but, unlike the case in PAT, instability was not accompanied by chromosome mobilization. We isolated deletants of pVS1 which were unable to promote structural instability.     

A plasmid vector with a selectable marker for halophilic archaebacteria.

A mutant resistant to the gyrase inhibitor novobiocin was selected from a halophilic archaebacterium belonging to the genus Haloferax. Chromosomal DNA from this mutant was able to transform wild-type cells to novobiocin resistance, and these transformants formed visible colonies in 3 to 4 days on selective plates. The resistance gene was isolated on a 6.7-kilobase DNA KpnI fragment, which was inserted into a cryptic multicopy plasmid (pHK2) derived from the same host strain. The recombinant plasmid transformed wild-type cells at a high efficiency (greater than 10(6)/micrograms), was stably maintained, and could readily be reisolated from transformants. It could also transform Halobacterium volcanii and appears to be a useful system for genetic analysis in halophilic archaebacteria.     

Physical structure and genetic expression of the sulfonamide-resistance plasmid pLS80 and its derivatives in Streptococcus pneumoniae and Bacillus subtilis

Summary The 10-kb chromosomal fragment of Streptococus pneumoniae cloned in pLS80 contains the sul-d allele of the pneumococcal gene for dihydropteroate synthase. As a single copy in the chromosome this allele confers resistance to sulfanilamide at 0.2 mg/ml; in the multicopy plasmid it confers resistance to 2.0 mg/ml. The sul-d mutation was mapped by restriction analysis to a 0.4-kb region. By the mechanism of chromosomal facilitation, in which the chromosome restores information to an entering plasmid fragment, a BamHI fragment missing the sul-d region of pLS80 established the full-sized plasmid, but with the sul-s allele of the recipient chromosome.A spontaneous deletion beginning 1.5 kb to the right of the sul-d mutation prevented gene function, possibly by removing a promoter. This region could be restored by chromosomal facilitation and be demonstrated in the plasmid by selection for sulfonamide resistance. Under selection for a vector marker, tetracycline resistance, only the deleted plasmid was detectable, apparently as a result of plasmid segregation and the advantageous growth rates of cells with smaller plasmids. When such cells were selected for sulfonamide resistance, the deleted region returned to the plasmid, presumably by equilibration between the chromosome and the plasmid pool, to give a low frequency (10^-3) of cells resistant to sulfanilamide at 2.0 mg/ml. Models for the mechanisms of chromosomal facilitation and equilibration are proposed.Several derivatives of pLS80 could be transferred to Bacillus subtilis, where they conferred resistance to sulfanil-amide at 2 mg/ml, thereby demonstrating cross-species expression of the pneumococcal gene. Transfer of the plasmids to B. subtilis gave rise to large deletions to the left of the sul-d marker, but these deletions did not interfere with the sul-d gene function. Restriction maps of pLS80 and its variously deleted derivatives are presented.     

Cloning of the Bacillus subtilis sulfanilamide resistance gene in Bacillus subtilis

A recombinant plasmid was constructed by ligation of chromosomal DNA from a sulfanilamide-resistant strain of Bacillus subtilis to the plasmid vector pUB110 which specifies neomycin resistance. Recombinant molecules generated in vitro were introduced into a B. subtilis recipient strain which carried the recE4 mutation, and selection was for neomycin-sulfanilamide-resistant transformants. A single colony was isolated containing the recombinant plasmid pKO101. This 6.3-megadalton plasmid simultaneously conferred resistance to neomycin and sulfanilamide when transferred into sensitive Rec+ or Rec- cells by either transduction or transformation.     

Mutagenesis on cloned yeast genes. The mutation of the yeast gene comprising the plasmid and chromosome

The cells of Saccharomyces cerevisiae were transformed by plasmid pYG-007 treated in vitro with o-methylhydroxylamine. The plasmid consists of a portion of the bacterial plasmid with genes of resistance to ampicillin, chloramphenicol and tetracycline, 2 mkm yeast DNA and yeast genes ADE2 and LEU2. The collection of mutants containing a mutant allele of ADE2 gene within the plasmid was obtained. Interallelic complementation and that induced by suppression were studied in these ade 2 mutants. It was shown that all these induced ade 2 mutations were base-pair substitutions. Using the mechanism of conversion we managed to transfer the plasmid ade 2 mutations into the chromosome. Three pairs of strains carrying similar mutation in plasmid and chromosome were created. Analysis of frequency of reversions induced by UV-light and hydroxylaminopurine in the mutant ade2 locus comprised in the plasmid and chromosome showed that the former induced reversions in plasmid alleles less effectively than the latter.     

Characteristics of the het mutations in the Escherichia coli chromosome causing an increase in Tn1 transposition frequency

Four mutations were studied which lead to increasing the frequency of transposon Tn1 translocation into different replicons. These mutations (het1, het2, het3 and het4) increase the frequency of Tn1 translocation 10-20-fold. The het1 mutation is recessive and has been localized in the 90-94.5 min region of the bacterial chromosome. The mutation effects Tn1 transposition in the presence of F plasmid only. As we have demonstrated recently, F-plasmid inhibits Tn1 transposition in Escherichia coli cells. The het1 mutation eliminates this inhibition. Unlike het2, het3 and het4 mutations, het1 is responsible for resistance to male phages f1, f2, MS2 and inhibition of conjugative transfer in F+ bacteria.     

Two types of cold sensitivity associated with the A184-->V change in the DnaA protein

Multicopy dnaA(Ts) strains carrying the dnaA5 or dnaA46 allele are high-temperature resistant but are cold sensitive for colony formation. The DnaA5 and DnaA46 proteins both have an A184-->V change in the ATP binding motif of the protein, but they also have one additional mutation. The mutations were separated, and it was found that a plasmid carrying exclusively the A184-->V mutation conferred a phenotype virtually identical to that of the dnaA5 plasmid. Strains carrying plasmids with either of the additional mutations behaved like a strain carrying the dnaA+ plasmid. In temperature downshifts from 42 degrees C to 30 degrees C, chromosome replication was stimulated in the multicopy dnaA46 strain. The DNA per mass ratio increased threefold, and exponential growth was maintained for more than four mass doublings. Strains carrying plasmids with the dnaA(A184-->V) or the dnaA5 gene behaved differently. The temperature downshift resulted in run out of DNA synthesis and the strains eventually ceased growth. The arrest of DNA synthesis was not due to the inability to initiate chromosome replication because marker frequency analysis showed high initiation activity after temperature downshift. However, the marker frequencies indicated that most, if not all, of the newly initiated replication forks were stalled soon after the onset of chromosome replication. Thus, it appears that the multicopy dnaA(A184-->V) strains are cold sensitive because of an inability to elongate replication at low temperature. The multicopy dnaA46 strains, on the contrary, exhibit productive initiation and normal fork movement. In this case, the cold-sensitive phenotype may be due to DNA overproduction.     

High-level resistance to streptomycin inEscherichia coli with R1 plasmid and the analysis of genetic determinants

Some properties of streptomycin-resistant mutants of Escherichia coli were analyzed. In a R+ culture, the phenotype under study may be significantly selected at a frequency of 10(-5) on media with higher streptomycin level. The lrs mutation is present in the cells prior to the action of streptomycin and remains in the cells even after curing of the R1 plasmid. The mapping of the lrs gene by conjugation with a concomitant transfer of chromosome and the R1 plasmid in different Hfr strains of E. coli failed to establish the localization of this gene in the tested chromosome regions. The presence of a cryptic plasmid was detected in cells with the lrs mutation after curing of the R1 plasmid. This plasmid codes neither fertility functions nor chloramphenicol-acetyltransferase, streptomycin-adenyltransferase, or ampicillin-beta-lactamase.     

Chromosomal Assignment of Mutations by Specific Chromosome Loss in the Yeast Saccharomyces Cerevisiae

Yeast 2-microns plasmids were integrated near the centromere of a different chromosome in each of 16 cir0 mapping strains of Saccharomyces cerevisiae. The specific chromosomes containing the integrated 2-microns plasmid DNA were lost at a high frequency after crossing the cir0 strains to cir+ strains. A recessive mutation in a cir+ strain can then be easily assigned to its chromosome using this set of mapping strains, since the phenotype of the recessive mutation will be manifested only in diploids having the integrated 2-microns plasmid and the unmapped mutation on homologous chromosomes.     

Genetic recombination of bacterial plasmid DNA: effect of RecF pathway mutations on plasmid recombination in Escherichia coli.

Tn5 insertion mutations in the recN gene, and in what appears to be a new RecF pathway gene designated recO and mapping at approximately 55.4 min on the standard genetic map, were isolated by screening Tn5 insertion mutations that cotransduced with tyrA. The recO1504::Tn5 mutation decreased the frequency of recombination during Hfr-mediated crosses and increased the susceptibility to killing by UV irradiation and mitomycin C when present in a recB recC sbcB background, but only increased the sensitivity to killing by UV irradiation when present in an otherwise Rec+ background. The effects of these and other RecF pathway mutations on plasmid recombination were tested. Mutations in the recJ, recO, and ssb genes, when present in otherwise Rec+ E. coli strains, decreased the frequency of plasmid recombination, whereas the lexA3, recAo281, recN, and ruv mutations had no effect on plasmid recombination. Tn5 insertion mutations in the lexA gene increased the frequency of plasmid recombination. These data indicate that plasmid recombination events in wild-type Escherichia coli strains are catalyzed by a recombination pathway that is related to the RecF recombination pathway and that some component of this pathway besides the recA gene product is regulated by the lexA gene product.