Characterization of a conditionally transformation-deficient mutant of Haemophilus influenzae that carries a mutation in the rec-1 gene region.

A mutant of Haemophilus influenzae, designated HM5, carrying a mutation in the rec-1 gene region, is described. This mutant transformed approximately 100-fold less well than does the wild type, but approximately 100-fold better than rec1 mutants. The mutant was less sensitive to UV irradiation and less "reckless" than rec1 mutants. In contrast to rec1 lysogens, HP1c1 lysogens of the mutant were inducible, and during transformation, recombinant-type activity was formed to the same extent as in the wild type. Although the integration of donor DNA was complete, the integrated DNA was not replicated at 36 degrees C. Both the inhibition of replication of the donor-recipient DNA complex and the transformation deficiency could be suppressed when, after DNA entry, the cells were incubated under suboptimal conditions. The loss of colony formation after UV irradiation was suppressible by the same conditions.     

Recombination deficient mutant of Caulobacter crescentus

Summary A recombination-deficient (Rec^-) strain of Caulobacter crescentus has been isolated from a collection of mutants sensitive to ultraviolet irradiation. The Rec^- mutant fails to give recombinants following Cr30-mediated generalized transduction or following RP4-mediated conjugation. The recombination frequency in the Rec^- strain is at least 5000-fold lower than in the wild type strains. The Rec^- mutant is indistinguishable from wild type in terms of morphology, growth rate, viability, and phage sensitivities, differing only in properties known to be associated with recA-type mutations in other organisms: recombination frequency, ultraviolet sensitivity, and Weigle reactivation. The map location of the rec-526 allele has not been identified, but rec-526 can be cotransferred with the fla-169 mutation by RP4-mediated conjugation at low frequency. This apparent linkage has been used to move the rec mutation to other strains. The Rec^- mutant resembles recA strains of other organisms and provides a healthy strain severely deficient in recombination for use in complementation and cloning studies involving C. crescentus.     

Isolation of the rec Mutants in Staphylococcus aureus

A histidine auxotroph (his-) of Staphylococcus aureus MS3937 and mutants sensitive to ultraviolet (UV) irradiation were obtained. The UV-sensitive mutants were found also to be sensitive to N-methyl-N'-nitro-N-nitrosoguanidine and mitomycin C, and their sensitivity was accounted for by a defect in deoxyribonucleic acid repair. Transduction of either chromosomal or plasmid markers to UV-sensitive mutants showed that these staphylococcus mutants are of the recA (reckless) type mutants as reported in Escherichia coli and Salmonella typhimurium; therefore the UV-sensitive mutants (uvr-) were renamed recombination-deficient mutants (rec-). The biochemical and genetic properties of these mutants are described, and their usefulness for studies of staphylococcal plasmids is discussed.     

rpe, a cis-acting element from the strA region of the Haemophilus influenzae chromosome that makes plasmid establishment independent of recombination.

Plasmids that share homology with the Haemophilus influenzae chromosome transform wild-type cells more efficiently than they transform recombination-defective mutants. A 5.2-kilobase-pair chromosomal fragment containing the strA gene of H. influenzae was found to promote efficient plasmid establishment in recombination-defective mutants. A cis-acting element in the insert, called rpe for rec-less plasmid establishment, promoted plasmid transformation in rec-1 and rec-2 mutants without suppressing the recombination defects of these strains. The rpe locus increased plasmid transformation in wild-type cells without interfering with the pathway of plasmid establishment that is dependent on recombination functions.     

Recombination-deficient mutants of Bacillus subtilis.

Two mutant strains of Bacillus subtilis Marburg, NIG43 and NIG45, were isolated. They showed high sensitivities to gamma rays, ultraviolet light (UV), and chemicals. Deficiencies in genetic recombination of these two mutants were shown by the experiments on their capacity in transformation. SPO2 transfection, and PBS1 phage transduction, as well as on their radiation and drug sensitivities and their Hcr+ capacity for UV-exposed phage M2. Some of these characteristics were compared with those of the known strains possessing the recA1 or recB2 alleles. Mapping studies revealed that the mutation rec-43 of strain NIG43 lies in the region of chromosome replication origin. The order was purA dna-8132 rec-43. Another mutation, rec-45, of strain NIG45 was found to be tightly linked to recA1. The mutation rec-43 reduced mainly the frequency of PBS1 transduction. On the other hand, the mutation rec-45 reduced the frequency of recombination involved both in transformation and PBS1 transduction. The mutation rec-43 of strain NIG43 is conditional, but rec-45 of strain NIG45 is not. The UV impairment in cellular survival of strain NIG43 was gradually reverted at higher salt or sucrose concentrations, suggesting cellular possession of a mutated gene produce whose function is conditional. In contrast to several other recombination-deficient strains, SPO2 lysogens of strain NIG43 and NIG45 were not inducible, indicating involvement of rec-43+ or rec-45+ gene product in the development of SPO2 prophage to a vegetative form. The UV-induced deoxyribonucleic acid degradation in vegetative cells was higher in rec-43 and rec-45 strains.     

Genetic control of prophage induction in haemophilus influenzae after exposure to psoralen plus near-UV light.

Prophage S2 could be induced by psoralen plus near-UV light (PNUV) from a wild-type strain of Haemophilus influenzae, from UV light-sensitive strains uvr-1 and uvr-2 and PNUV-sensitive strains PSO1 amd PSO7, but not from a recombination-deficient strain, rec-1. The levels of prophage induction were comparable in the wild type and an ATP-dependent DNase-deficient strain, KW31, even though the PNUV-induced degradation in the latter strain was considerably lower. Prophage induction could be observed even with chloramphenicol present before, during, and 30 min after PNUV treatment.     

Isolation and characterization of mutants of Haemophilus influenzae deficient in an adenosine 5''-triphosphate-dependent deoxyribonuclease activity.

By a direct assay approach, mutants of Haemophilus influenzae Rd that are deficient in adenosine 5'-triphosphate-dependent deoxyribonuclease activity (add-) were isolated and characterized. A large proportion (50 to 90%) of the cells in cultures of these mutants failed to produce visible colonies when plated. An extensive analysis of the recombination proficiency of these strains revealed that the transformation frequency (transformants per competent cell) in the mutants was similar to that found in the wild type, but that the transformation efficiency (transformants per microgram of irreversibly bound deoxyribonucleic acid [DNA]) was reduced approximately fourfold. Sensitivities of the mutants to gamma rays, ultraviolet radiation, and methyl methane sulfonate were only slightly greater than wild-type levels. The rate of degradation of host DNA after ultraviolet irradiation was significantly reduced in the mutants. It is suggested that the adenosine 5'-triphosphate-dependent deoxyribonuclease in H. influenzae plays a nonessential role in DNA recombination and repair.     

The effect of radiation sensitivity and cell stage on liquid holding response in Schizosaccharomyces pombe

Summary The response of the wild type strain and 20 different radiation sensitive mutants of S. pombe to liquid holding after ultraviolet irradiation was ivestigated. Three of the sensitive mutants tested showed appreciable liquid holding recovery, as opposed to the negative liquid holding effect observed in the wild type cells. One of these mutants is reported to be recombination-deficient while the other two have a normal recombination capability. Further experiments were carried out by using G₁ cells and ascospores to test the possible role of a recombinational type of repair pathway in the failure of wild type S. pombe to show liquid holding recovery. Data from such studies indicated that the negative liquid holding effect observed in the wild type cannot be ascribed to this particular pathway. This conclusion is further supported by the observation that caffeine which is believed to inhibit mainly the recombinational repair in this yeast, did not alter the negative liquid holding effect in the wild type. This observation implies that the caffeine-sensitive repair process occurs only in a rich medium and not in the non-nutrient solution. Data have been discussed as these relate to possible cause(s) of negative liquid holding effect in this organism.     

Cloning and characterization of the Haemophilus influenzae Rd rec-1+ gene.

The Haemophilus influenzae Rd rec-1+ gene was cloned from a partial chromosomal digest into a plasmid vector as a 20-kilobase-pair (kbp) BstEII fragment and then subcloned. The smallest subclone with rec-1+ activity carried a 3.1-kbp EcoRI fragment. The identity of the rec-I gene in these clones was confirmed by transforming an Rd strain carrying a leaky rec-1 mutation (recA4) to resistance to methyl methanesulfonate (MMS) by using whole or digested plasmids. It was demonstrated that the Rec+ phenotype of the MMSr transformants was linked to the strA, novAB, and mmsA loci, as expected if the recA4 allele had been replaced by rec-1+. In growing cultures (rec-1 or rec+), all rec-1+-carrying plasmids induced near-maximal levels of transformability when their hosts reached stationary phase; these levels are 100 to 1,000 times higher than the values seen with strains not carrying a Rec plasmid. Transfer of the 3.1-kbp subclone was greatly reduced compared with transfer of similarly sized vector plasmids, and the resulting transformants grew slowly; this suggests an explanation of my failure to directly clone this fragment from chromosomal DNA digests. Transfer of a rec-1+ plasmid to a very poorly genetically transformable H. influenzae Rb strain resulted in greatly increased transformability. Transfer of such plasmids to a noncompetent H. influenzae Rc strain did not render this strain competent. It is suggested that transformability of Rd and Rb strains is limited by rec-1 expression but that the noncompetence of Rc has some other basis.     

A plasmid carrying mucA and mucB genes from pKM101 in Haemophilus influenzae and Escherichia coli.

The plasmid pMucAMucB, constructed from the Haemophilus influenzae vector pDM2, and a similar plasmid, constructed from pBR322, increased the survival after UV irradiation of Escherichia coli AB1157 with the umu-36 mutation and also caused UV-induced mutation in the E. coli strain. In H. influenzae, pMucAMucB caused a small but reproducible increase in survival after UV irradiation in wild-type cells and in a rec-1 mutant, but there was no increase in spontaneous mutation in the wild type or in the rec-1 mutant and no UV-induced mutation.     

Mechanism of gap-filling during postreplication repair of ultraviolet damage in Haemophilus influenzae.

Deoxyribonucleic acid (DNA), pulse labeled after ultraviolet irradiation of excision-defective mutants of Haemophilus influenzae, is of lower single strand molecular weight than that of unirradiated cells but approaches the size of DNA from unirradiated cells upon further incubation in growth medium. This gap-filling process is controlled by the rec-1 gene. Gap-filling occurs normally in a temperature-sensitive DNA synthesis mutant at the restrictive temperature showing that normal semiconservative DNA synthesis is not necessary for gap-filling. To test for recombinational events after irradiation, the DNA synthesized after irradiation was radioactively labeled for a short time in medium containing 5-bromodeoxyuridine followed by incubation for various times in non-radioactive, 5-bromodeoxyuridine-containing medium. The DNA was denatured and analyzed isopycnically. The labeled DNA was initially "heavy," but later shifted toward lighter densities. This shift occurred in the temperature-sensitive DNA synthesis mutant at the restrictive temperature and in the recombination-defective mutant rec-2, but was not seen in the rec-1 mutant. The density shift can be interpreted as evidence that rather extensive exchanges occurred between parental DNA and the DNA made after irradiation. These results suggest that such exchanges are important for gap-filling in H. influenzae.     

Sensitivity of wild type and recombination-deficient strains of Drosophila melanogaster to ultraviolet radiation.

The sensitivity of Drosophila melanogaster to ultraviolet light has been studied in wild type and recombination-deficient strains. Survival was measured as the proportion of irradiated embryos or larvae which developed to adult flies. In view of the fact that males of this species do not participate in meiotic recombination, emphasis was placed on the relative sensitivity of males and females. The results show that young wild type male larvae are more sensitive to UV radiation than are young female larvae. This difference in sensitivity, however, is not apparent in some recombination-deficient strains. In addition, young embryos of the recombination-deficient strain Df(3)sbd105/T(2;E)Xa are exceptionally sensitive to UV radiation.     

Isolation and properties of a recombination-deficient mutant of Micrococcus radiodurans.

A mutant of micrococcus radiodurans which is deficient in recombination has been isolated after treatment of the wild type with N-methyl-N'-nitro-N-nitrosoguanidine. We have called this mutant Micrococcus radiodurans rec30. The efficiency of recombination in this mutant, as measured by transformation, is less than 0.01% that of the wild type. It is 15 times more sensitive to the lethal action of ultraviolet radiation, 120 times more sensitive to ionizing radiation, and 300 times more sensitive to mitomycin C (MMC) than the wild type. It is probably inactivated by a single MMC-induced deoxyribonucleic acid cross-link per genome. The excision of ultraviolet-induced pyrimidine dimers is normal. There is no radiation-induced degradation of deoxyribonucleic acid. All spontaneous revertants selected for resistance to low levels of MMC had wild-type resistance to radiation and MMC, and the same efficiency of recombination as the wild type, suggesting that the recombination deficiency of the strain is due to a single mutation. Deoxyribonucleic acid from this mutant can transform M. radiodurans UV17 presumed deficient in an exr type gene to wild type.     

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.     

Characterization of the rec-1 gene of Haemophilus influenzae and behavior of the gene in Escherichia coli.

The rec-1 gene of Haemophilus influenzae was cloned into a shuttle vector that replicates in Escherichia coli as well as in H. influenzae. The plasmid, called pRec1, complemented the defects of a rec-1 mutant in repair of UV damage, transformation, and ability of prophage to be induced by UV radiation. Although UV resistance and recombination were caused by pRec1 in E. coli recA mutants, UV induction of lambda and UV mutagenesis were not. We suggest that the ability of the H. influenzae Rec-1 protein to cause cleavage of repressors but not the recombinase function differs from that of the E. coli RecA protein.     

The effect of three rad genes on survival, inter- and intragenic mitotic recombination in Saccharomyces. I. UV irradiation without photoreactivation or liquid-holding post-treatment

The effect of UV irradiation on the survival, inter- and intragenic mitotic recombination of 3 diploid UV sensitive Saccharomyces mutants was studied and compared with the wild type RAD. These strains, homozygous for either the RAD, r1s rad 9-4, or rad 2-20 gene, have DRF values for survival of 1:1.6:3:20.6 respectively, at LD1. Their recombination behaviour is not correlated to their survival characteristics. The RAD, r1s, and rad 2-20 strains showed UV induced mitotic inter- and intragenic recombinants; the induction in the r1s diploid is ca. 100 times greater for both the inter- and intragenic recombinants than in the RAD strain. The rad 9-4 diploid produced no UV induced mitotic recombinants whatsoever, and is therefore considered to be a rec- mutation.     

Two Haemophilus influenzae Rd genes that complement the recA-like mutation rec-1.

Two Haemophilus influenzae Rd genes each complemented the pleiotropic defects of the recA-like mutation rec-1. One gene, fec, was isolated on a 3.6-kilobase-pair EcoRI restriction fragment by complementation of the Fec- phenotype of bacteriophage lambda. The other gene, rec, was identified on a 3.1-kilobase-pair EcoRI fragment by Southern hybridization by using recA-like gene probes from Erwinia carotovora and Pseudomonas aeruginosa PAO. In a rec-1 strain of H. influenzae, the cloned genes restored resistance to UV irradiation, transformation by chromosomal DNA, and spontaneous release of HP1 prophage to wild-type levels. The fec and rec genes were located on the cloned segments by insertion and deletion mutagenesis and subcloning. The restriction endonuclease cleavage maps of the two DNAs were similar but not identical. Southern hybridization demonstrated that the two EcoRI restriction fragments contained homologous DNA sequences, but a fec gene-specific probe was prepared. Each gene encoded a 38,000-dalton polypeptide.     

Cloning of the rec-2 locus of Haemophilus influenzae

A collection of transposon mutants of Haemophilus influenzae was constructed by additive transformation with mutagenized chromosomal DNA. A rec-2::miniTn10 km mutation was cloned from a transformation-defective member of the mutant collection, followed by the reconstruction of the wild-type rec-2 locus by recombination to create pDM62. Southern blots showed that the commonly studied Rec-2 mutant, Rd(DB117)rec-, contained either a large deletion or a substitution that removed part of rec-2 locus. A collection of transposon mutations in pDM62 was used to characterize the rec-2 locus by complementation. A corresponding collection of mutants was also constructed. A single segment was required to complement the transformation defect in Rd(DB117)rec-. All of the transformation-defective transposon mutants failed to translocate donor DNA into then cell, in agreement with previous studies of Rd(DB117)rec-.     

Repair of Deoxyribonucleic Acid in Haemophilus influenzae I. X-Ray Sensitivity of Ultraviolet-sensitive Mutants and Their Behavior as Hosts to Ultraviolet-irradiated Bacteriophage and Transforming Deoxyribonucleic Acid

Seven mutants of Haemophilus influenzae were isolated by the criterion of sensitivity to ultraviolet (UV) inactivation of colony formation. These mutants and the wild type were characterized with regard to X-ray inactivation of colony formation, UV induction of division inhibition, the ability of the eight strains to act as recipients to UV-irradiated H. influenzae phage and transforming deoxyribonucleic acid (DNA), and the influence of acriflavine on the survival of UV-irradiated transforming DNA with these strains as recipients. The photoreactivable sector of transforming DNA with yeast photoreactivating enzyme was measured for the most UV-sensitive mutant and was found to be greater than that of wild type. Judged by the above criteria, the order of the strains' sensitivities shows some, but by no means complete, correlation from one type of sensitivity characterization to another, indicating that a minimum of two variables is needed to explain the differences in the strains. Acriflavine increases the UV sensitivity of transforming DNA except in the most sensitive mutant. This effect is usually, but not always, more pronounced in the case of the more UV-resistant marker. The acriflavine effect is postulated to be the result of at least two factors: (i) interference with repair of transforming DNA in the host cell, and (ii) interference with the probability of recombination between transforming DNA and host DNA.     

Construction and analysis of a recombination-deficient (radA) mutant of Haloferax volcanii

By deleting the radA open reading frame of an extreme halophile, Haloferax volcanii , we created and characterized a recombination-deficient archaeon. This strain, Hf. volcanii DS52, has no detectable DNA recombination, is more sensitive to DNA damage by UV light and ethylmethane sulfonate, and has a slower growth rate than the wild type. These characteristics are similar to those observed in recombination mutants of Eukarya and Bacteria, and show that the radA gene belongs in the recA / RAD51 family by function as well as sequence homology. In addition, strain DS52 was not transformable by plasmids pWL102 or pUBP2 (which contain pHV2 and pHH1 replicons, respectively), although it was readily transformed by plasmids containing a pHK2 replicon, indicating a role for radA in the maintenance or replication of some halobacterial plasmids. Despite its slower growth rate, Hf. volcanii DS52 was still easy to culture and transform, and should be suitable for use in studies where a recombination-deficient background is desired.