Isolation and Characterization of a Recombination-Deficient Hfr Strain

The isolation of a rec(-) Hfr strain of Escherichia coli K-12 is described. The method used consisted of mating AB2463 F(-) Rec(-) His(-) Lac(-) with P4X6 Hfr Rec(+) His(+) Lac(+), selecting Rec(-) His(-) Lac(+) recombinants, and searching for Hfr strains. One Hfr rec(-) strain, no. 12, was used as donor in crosses with Rec(+) and Rec(-) recipients. Crosses with Rec(+) recipients are fertile, and those with Rec(-) recipients are almost infertile, the frequency of recombinants being 10(-2) to 10(-3) that found with Rec(+) recipients. The Rec(-) mutant marker is transfered to and integrated into Rec(+) recipients. Zygotic induction of prophage lambda is observed in crosses between two Rec(-) strains. In crosses of F(-) Rec(-) with Hfr Rec(-), the gradient of integration frequencies for markers progressively more distant from the origin is steeper than in the Rec(+) x Rec(-) or the Rec(-) x Rec(+) crosses.     

Lethal zygosis in recombination-deficient mutants of Escherichia coli.

Use of nonselective medium for plating cells following mating has revealed that Rec recipient strains of E. coli may be killed as a result of conjugation. Sensitivity of RecA-, RecB-, and RecC- recipients increases with ratio of donor: recipient cells in mating mixtures and with time of mating. A Rec+ recipient shows no lethal zygosis in these experiments performed without aeration. Cell contact does not seem to be responsible for the sensitivity of Rec- strains, since lethality is prevented when cell contact is permitted but DNA transfer is not. Thus, an event(s) occuring subsequent to entry of donor DNA appears to cause lethality in Rec- recipients.     

New indications for the participation of group P plasmids R in the transfer of chromosomal genes in intergenera crosses]

Use of E. coli strains with phenotypes Rec+ and Rec- asrecipients in intergenera crosses confirmed the supposition put forward by the authors formerly that new chromosomal markers in transconjugantes originated due to Psuedomonas aeruginosa. These chromosomal markers were transferred together with plasmid R conditioning the conjugation, and maintained without being built-into E. coli chromosome. Between the arg+ marker and the plasmid R18 there existed labile physical connection demonstrable only under definite conditions of recombinant selection.     

Mechanism of conjugation and recombination in bacteria. XXI. Role of F factor genes in post-conjugational recombination in Escherichia coli K-12

Temperature sensitive dnaA recipient crossed at the restrictive temperature with HfrH, free from contaminating F+ cells, forms recombinants almost as proficiently as at the permissive temperature. The merozygotes are able to synthesize DNA at 42 degrees C, although the recipient and donor cells do not incorporate 3H-thymine. A substantial fraction of Lac+ recombinants, irrespective of the mating temperature, is temperature resistant (42 C-R); 15% from among those mated at 35 C and 30% from those mated at 42 C. The presence of dnaA mutation in these Lac+ 42 C-R recombinants was ascertained by co-transduction with ilv. Cell division at 42 C is inhibited in the Lac 42 C-R recombinants by acridine orange. The presence of F factor DNA in these recombinants was demonstrated directly by DNA: DNA hybridization. Suppression of dnaA mutation in Lac+ 42 degrees C-R recombinants and their sensitivity to acridine orange at 42 degrees C suggests that at least part of the F factor is integrated into the recombinant chromosome. A large fraction of the Lac+ 42 degrees C-R recombinants (up to 80%) is sensitive to male phage R17 and fertile. In crosses with HfrC there is a marked decrease of recombination frequency at 42 degrees C in the dnaA recipient. The fraction of Lac+ 42 degrees C-R recombinants is low (up to 10%) and the 42 degrees C-R recombinants are neither sensitive to male phage nor fertile. The results are discussed on the basis of the previously proposed model of post-conjugational recombination.     

Characterization and genetic mapping of a mutation affecting apurinic endonuclease activity in Staphylococcus aureus.

Protoplast fusion between the Rec- mutant RN981 (L. Wyman, R. V. Goering, and R. P. Novick, Genetics 76:681-702, 1974) of Staphylococcus aureus NCTC 8325 and a Rec+ NCTC 8325 derivative yielded Rec+ recombinants that exhibited the increased sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine characteristic of RN981. Transformation analyses identified a specific mutation, designated ngr-374, that was responsible not only for N-methyl-N'-nitro-N-nitrosoguanidine sensitivity, but also sensitivity to methyl methanesulfonate, ethyl methanesulfonate, nitrous acid, and UV irradiation. However, ngr-374-carrying recombinants showed no significant increase in their sensitivity to mitomycin C or 4-nitroquinoline 1-oxide and were unaffected in recombination proficiency. In vitro assays showed that ngr-374-carrying strains had lower apurinic/apyrimidinic endonuclease activities than the wild type. The chromosomal locus occupied by ngr-374 was shown to exist in the gene order omega(Chr::Tn551)40-ngr-374-thrB106.     

Novobiocin resistance marker in Haemophilus influenzae that is not expressed on a plasmid.

The plasmid pNov2, carrying a cloned chromosomal marker conferring resistance to at least 2.5 micrograms of novobiocin per ml, was constructed with a new Haemophilus influenzae cloning vehicle, pDM2. The novobiocin marker of pNov2 was not normally expressed, but in Rec+ cells approximately one in 10(4) cells in a culture of a transformant became novobiocin resistant, a frequency about four orders of magnitude higher than the spontaneous mutation frequency. Variants of such cells that had lost the plasmid were also novobiocin resistant. Since Rec- cultures bearing pNov2 showed novobiocin resistance only at the normal mutation frequency, we concluded that the Rec+ novobiocin-resistant transformants arose because of a rare recombination between plasmid and chromosome in which the chromosome acquired the novobiocin marker from the plasmid. Evidence is presented that novobiocin sensitivity is dominant over this particular novobiocin resistance marker.     

Characterization of Pseudomonas aeruginosa mutants deficient in the establishment of lysogeny.

Mutants of Pseudomonas aeruginosa with impaired ability to establish a lysogenic relationship with temperate bacteriophage (Les-) have been isolated. These les mutations map to two areas of the P. aeruginosa chromosomal map as determined by conjugational and transductional analyses. Two phenotypic classes of Les- mutants were identified. One class of mutations has pleiotropic effects on DNA metabolism. These mutants are unable to recombine genetic material acquired as a result of either conjugation or transduction (Rec-). In addition, the ability of these Les- Rec- mutants to repair UV-induced damage to bacteriophage is reduced (host-cell reactivation deficient, Hcr-). Mutants of the second class are Les-, Rec+, and Hcr+.     

Transduction of fimbriation demonstrating common ancestry in FIRN strains of Salmonella typhimurium.

The production of fimbriate (Fim+) recombinants was observed in transductional crosses between different pairs of wild-type strains of different biotypes of Salmonella typhimurium. Fim+ recombinants were readily produced in transductions from Fim+ donor strains to Fim- recipient strains and, less frequently, between some pairs of Fim- strains, for example, between almost any strain of the firn biogroup (Fim- Inl- Rha- Bxyl-) and many strains of the non-FIRN Fim- biogroup. None of numerous crosses between different pairs of FIRN strains gave Fim+ recombinants, suggesting that the fim mutation was present at the same intragenic site in all FIRN strains. FIRN strains are thought to have descended from a single ancestral FIRN bacterium which originated by a series of mutations from a strain of the common biotype 1a (Fim+ Inl+ Rha+ Bxyl+). Two FIRN-like (Fim- Inl+ Rha- Bxyl-) strains that did not yield Fim+ recombinants in crosses with FIRN strains were probably wild-type Inl+ mutants from FIRN strains.     

Construction of a genetic map for Caulobacter crescentus.

RP4-mediated conjugation has been used to transfer large fragments of chromosomal material in Caulobacter crescentus. In this system, conjugation proceeds from multiple origins, and haploid recombinants are recovered at frequencies of 10(-6) and 10(-7) per donor cell. The data from five-factor crosses were subjected to computer-assisted crossover analyses as a rapid method to determine marker order. Using this information and data from additional two- and three-factor crosses mediated by RP4 or the generalized transducing bacteriophage phi Cr30, we constructed the first genetic map for C. crescentus.     

Role of Rec pathways on sensitivity of Escherichia coli to near-ultraviolet and visible light.

In Escherichia coli lack of the RecRC or RecF pathway is found to cause sensitivity to near-ultraviolet and visible light. Resistance to this light is restored in the RecBC-defective strain carrying either the sbcB (Rec+) or xonA (Rec-) mutation. The sensitivity, therefore, is not found to correlate with the degree of recombination proficiency as measured by genetic crosses.     

Conjugational Recombination in Escherichia Coli: Genetic Analysis of Recombinant Formation in Hfr X F(-) Crosses

The formation of recombinants during conjugation between Hfr and F(-) strains of Escherichia coli was investigated using unselected markers to monitor integration of Hfr DNA into the circular recipient chromosome. In crosses selecting a marker located ~500 kb from the Hfr origin, 60-70% of the recombinants appeared to inherit the Hfr DNA in a single segment, with the proximal exchange located >300 kb from the selected marker. The proportion of recombinants showing multiple exchanges increased in matings selecting more distal markers located 700-2200 kb from the origin, but they were always in the minority. This effect was associated with decreased linkage of unselected proximal markers. Mutation of recB, or recD plus recJ, in the recipient reduced the efficiency of recombination and shifted the location of the proximal exchange (s) closer to the selected marker. Mutation of recF, recO or recQ produced recombinants in which this exchange tended to be closer to the origin, though the effect observed was rather small. Up to 25% of recombinant colonies in rec(+) crosses showed segregation of both donor and recipient alleles at a proximal unselected locus. Their frequency varied with the distance between the selected and unselected markers and was also related directly to the efficiency of recombination. Mutation of recD increased their number by twofold in certain crosses to a value of 19%, a feature associated with an increase in the survival of linear DNA in the absence of RecBCD exonuclease. Mutation of recN reduced sectored recombinants in these crosses to ~1% in all the strains examined, including recD. A model for conjugational recombination is proposed in which recombinant chromosomes are formed initially by two exchanges that integrate a single piece of duplex Hfr DNA into the recipient chromosome. Additional pairs of exchanges involving the excised recipient DNA, RecBCD enzyme and RecN protein, can subsequently modify the initial product to generate the spectrum of recombinants normally observed.     

Conjugal transfer of genetic material by Lactococcus lactis subsp. lactis 11007

Conjugal transfer of genetic material by Lactococcus lactis subsp. lactis 11007 was examined. A plasmid of 88 MDa (pJS88) was identified in addition to the previously reported conjugally transferred plasmids of 32 (pKB32) and 4.8 MDa. Proteinase activity, reduced bacteriophage sensitivity, bacteriocin resistance, and conjugal transfer ability were encoded by pJS88. The ability to metabolize lactose (Lac+) was encoded by pKB32, and the 4.8-MDa plasmid was cryptic. When a strain containing both pKB32 and pJS88 was mated with a recipient deficient in host-mediated homologous recombination (Rec-), a plasmid of 40 MDa (pJS40) was observed in approximately 50% of the Lac+ transconjugants. DNA-DNA hybridization results indicated that pJS40 contained homology with both pKB32 and pJS88. These results indicated that pKB32 was conjugally transferred via conduction and suggested that pJS40 is a deletion derivative of a pKB32::pJS88 cointegrate. A Rec- strain containing pKB32 and pJS88 mediated Lac+ conjugal transfer, suggesting that the pKB32::pJS88 cointegrate could form via a rec-independent event. Resolution of the pKB32::pJS88 cointegrate was observed in both Rec- and Rec+ hosts. Cointegrate formation and resolution via rec-independent mechanisms suggest the involvement of a transposable element in the Tn3 family.     

Plaque color method for rapid isolation of novel recA mutants of Escherichia coli K-12: new classes of protease-constitutive recA mutants

As a prerequisite to mutational analysis of functional sites on the RecA protein of Escherichia coli, a method was developed for rapid isolation of recA mutants with altered RecA protease function. The method involves plating mutagenized lambda recA+ cI ind on strains deleted for recA and containing, as indicators of RecA protease activity, Mu d(Ap lac) fusions in RecA-inducible genes. The lambda recA phages were recognized by their altered plaque colors, and the RecA protease activity of the lambda recA mutant lysogens was measured by expression of beta-galactosidase from dinD::lac. One class of recA mutants had constitutive protease activity and was designated Prtc; in these cells the RecA protein was always in the protease form without the usual need for DNA damage to activate it. Some Prtc mutants were recombinase negative and were designated Prtc Rec-. Another class of 65 recA mutants isolated as being protease defective were all also recombinase defective. Unlike the original temperature-dependent Prtc Rec+ mutant (recA441), the new Prtc Rec+ mutants showed constitutive protease activity at any growth temperature, with some having considerably greater activity than the recA441 strain. Study of these strong Prtc Rec+ mutants revealed a new SOS phenomenon, increased permeability to drugs. Use of this new SOS phenomenon as an index of protease strength clearly distinguished 5 Prtc mutants as the strongest among 150. These five strongest Prtc mutants showed the greatest increase in spontaneous mutation frequency and were not inhibited by cytidine plus guanosine, which inhibited the constitutive protease activity of the recA441 strain and of all the other new Prtc mutants. Strong Prtc Rec+ mutants were more UV resistant than recA+ strains and showed indications of having RecA proteins whose specific activity of recombinase function was higher than that of wild-type RecA. A Prt+ Rec- mutant with an anomalous response to effectors is described.     

Linkage distortion following conjugational transfer of sbcC+ to recBC sbcBC strains of Escherichia coli.

Conjugational recombination in Escherichia coli depends normally on RecBCD enzyme, a multifunctional nuclease and DNA helicase produced by the recB, recC, and recD genes. However, recombination can proceed efficiently without RecBCD in recB or recC strains carrying additional mutations in both the sbcB and sbcC genes. Recombination in these strains, sometimes referred to as the RecF pathway, requires gene products that are not essential in the RecBCD-dependent process predominating in the wild type. It has also been reported to produce a different spectrum of recombinant genotypes in crosses with Hfr donors. However, the sbcC+ gene was unknowingly transferred to the recipient strain in some of these crosses, and this may have affected the outcome. This possibility was examined by conducting parallel crosses with Hfr donors that were either wild type or mutant for sbcC. Transfer of sbcC+ from an Hfr donor is shown to alter the frequency of recombinant genotypes recovered. There is a severe reduction in progeny that inherit donor markers linked to the sbcC+ allele and an increase in the incidence of multiple exchanges. Colonies of mixed genotype for one or more of the unselected proximal markers are also much more prevalent. Since the yield of recombinants is lower than normal, these changes are attributed to the reduced viability of recombinants that inherit sbcC+ from the Hfr donor. When the Hfr donor used is also mutant for sbcC, the yield of recombinants is greater and the frequencies of the different genotypes recovered are similar to those obtained in crosses with a rec+ sbc+ recipient, in which transfer of sbcC+ has no apparent effect. Earlier studies are re-examined in light of these findings. It is concluded that, while recombination in recBC sbcBC strains involves different enzymes, the underlying molecular mechanism is essentially the same as that in the wild type.     

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.     

Merodiploidy in ESCHERICHIA COLI-SALMONELLA TYPHIMURIUM Crosses: The Role of Unequal Recombination between Ribosomal RNA Genes

Previous workers have shown that intergeneric crosses between Salmonella typhimurium and Escherichia coli produce a high proportion of merodiploid recombinants among the viable progeny. We have examined the unequal cross-over event that was responsible for a number of intergeneric merodiploids. The merodiploids that we studied were all heterozygous for the metB-argH interval and were the products of intergeneric conjugal crosses. We found that when the S. typhimurium donor had its transfer origin closely linked to metB and argH, all recombinants examined were merodiploid, and they generally arose as F-prime factors. Many of these F-prime factors had been created by recombination between flanking rrn genes in the donor. When the S. typhimurium Hfr transfer origin was more distant from the selected markers, quite different results were obtained. Depending on the donor, 19-47% of the recombinants that acquired the donor argH+ or metB+ genes were merodiploid for these loci, but none of the recombinants were F-prime. A majority of the merodiploids had a novel (nonparental) rrn gene, indicating that unequal recombination between nonidentical rrn genes was a prevalent mechanism for establishing the merodiploidy. Both tandem and nontandem duplications were found. Some of the merodiploids duplicated E. coli genes in addition to acquiring S. typhimurium genes. Some merodiploids contained the oriC region from each parent. Of a total of 118 intergeneric merodiploids characterized from all donors, 48 different genotypes were observed, and 38 of the 48 had one or more nonparental rrn operons.     

Evolution of Pseudomonas R-plasmids: consequences of Tn1 insertion and resultant partial diploidy to chromosome and Tra- R-plasmid mobilization.

Tn1 transposes from pRO161, a Tra- derivative of RP1, to Pseudomonas aeruginosa sex factor FP2. The acquisition of Tn1 by FP2 results in its ability to mobilize pRO161 to other bacteria. Genetic evidence presented here suggests two sequential mechanisms. Initially, transposition of Tn1 results in trans-diploidy for the Tra+ and Tra- plasmids. This subsequently allows mobilization of the Tra- R-plasmid dependent on a host recombination mechanism. Transconjugants from this mating contain either stable cointegrate R-plasmids or aggregates resulting from dissociation of the cointegrates into a Tra+ and Tra- plasmid. These aggregates have lost at least part of Tn1 from their parent FP2:Tn1 component, but now they mobilize the tra- R-plasmid from a recombination-deficient (Rec-) genetic background as well as from Rec+ donor strains. Transconjugants from these retransfer matings are aggregates. These results suggest a contribution of transposons to R-plasmid evolution and dissemination beyond the mere acquisition of resistance to a given antibiotic.     

Rec effect of certain textile dyes in Bacillus subtilis

A large number of compounds are toxic, genotoxic, mutagenic, teratogenic and/or carcinogenic. The genotoxicity of four textile dyes commonly used in India namely Sulphur Red Brown 360 (SRB), Jade Green 2G (JG), Reactofix Turquoise Blue 5GFL (RTB) and Direct Scarlet 4BS (DS) was determined by Bacillus subtilis spore Rec assay, both in the presence and absence of metabolizing activation mixture (S9 mix). Each dye was toxic at higher dose levels. A dose-dependent increase in the depth of growth inhibition zones was observed for all dyes. Zones of inhibition were usually clearer at higher doses of the dyes and with Rec- bacteria, but were translucent with Rec+ bacteria. SRB and DS were toxic to Rec+ and Rec- bacteria. JG was less genotoxic in the absence of S9 mix, however, its genotoxic potential increased in the presence of S9 mix. Reactofix T blue was more genotoxic in the absence of S9 mixture.     

Heat and UV light resistance of vegetative cells and spores of Bacillus subtilis Rec-mutants.

The heat and UV light resistance of spores and vegetative cells of Bacillus subtilis BD170 (rec+) were greater than those of B. subtilis BD224 (recE4). Strain BD170 can repair DNA whereas BD224 is repair deficient due to the presence of the recE4 allele. Spores of a GSY Rec+ strain were more heat resistant than spores of GSY Rec- and Uvr- mutants. The overall level of heat and UV light resistance attained by spores may in part be determined by their ability to repair deoxyribonucleic acid after exposure to these two physical mutagens.     

Meiotic exchange within and between chromosomes requires a common Rec function in Saccharomyces cerevisiae.

We used haploid yeast cells that express both the MATa and MAT alpha mating-type alleles and contain the spo13-1 mutation to characterize meiotic recombination within single, unpaired chromosomes in Rec+ and Rec- Saccharomyces cerevisiae. In Rec+ haploids, as in diploids, intrachromosomal recombination in the ribosomal DNA was detected in 2 to 6% of meiotic divisions, and most events were unequal reciprocal sister chromatid exchange (SCE). By contrast, intrachromosomal recombination between duplicated copies of the his4 locus occurred in approximately 30% of haploid meiotic divisions, a frequency much higher than that reported in diploids; only about one-half of the events were unequal reciprocal SCE. The spo11-1 mutation, which virtually eliminates meiotic exchange between homologs in diploid meiosis, reduced the frequency of intrachromosomal recombination in both the ribosomal DNA and the his4 duplication during meiosis by 10- to greater than 50-fold. This Rec- mutation affected all forms of recombination within chromosomes: unequal reciprocal SCE, reciprocal intrachromatid exchange, and gene conversion. Intrachromosomal recombination in spo11-1 haploids was restored by transformation with a plasmid containing the wild-type SPO11 gene. Mitotic intrachromosomal recombination frequencies were unaffected by spo11-1. This is the first demonstration of a gene product required for recombination between homologs as well as recombination within chromosomes during meiosis.