Activity of Chi Recombinational Hotspots in SALMONELLA TYPHIMURIUM

Chi sites have previously been shown to stimulate homologous recombination by the Escherichia coli RecBC pathway. To test the activity of Chi in another organism, bacteriophage lambda crosses were carried out in Salmonella typhimurium strains bearing the E. coli lambda receptor protein. Chi is active in these crosses in S. typhimurium, but is less active than in the same crosses carried out in E. coli. The lower Chi activity in S. typhimurium appears to be intrinsic to the S. typhimurium RecBC enzyme, since the Chi activity in E. coli-S. typhimurium hybrids depends on the species of origin of their RecBC enzyme. For these studies we constructed and F' factor and a pBR322-derived plasmid carrying the thyA+ recC+ recB+ argA+ region of the S. typhimurium chromosome.     

Incompatibility of Integrated Sex Factors in Double Male Strains of ESCHERICHIA COLI

Several strains of Escherichia coli K-12 harboring two F factors were isolated from Hfr x Hfr crosses. These strains were transiently capable of initiating chromosome transfer from two separate points of origin, and of transferring two different sex factors as integrated chromosomal markers. Each strain tested invariably reverted to a simple Hfr by loss of one of the inherited F factors. The F factor persisting in the revertant was, in nearly every case, that which had been inherited from the recipient Hfr parent.     

Fertility of Salmonella typhimurium Crosses with Escherichia coli

At least one factor that causes low fertility of Salmonella typhimurium LT2 strains in crosses with Escherichia coli K-12 Hfr's can be inhibited by growing the female strains in supplemented minimal salts medium rather than in nutrient broth and by incubating the female strains at 50 C immediately before mating with the Hfr. These pretreatments can enhance the recovery of prototrophic recombinants for markers injected early by the Hfr by a factor of as much as 10(4). The heat treatment is effective only on the female in intergeneric crosses and gradually loses (within 50 min) its effectiveness after return of heat-treated cells to 37 C. It is concluded that the restriction system of the female is heat-sensitive. Since markers injected late by the male enter females in which the heat-impaired restriction system has recovered, few recombinants for late markers are found. The presence of the leading end of an E. coli Hfr in an S. typhimurium-E. coli hybrid enhances by up to sevenfold the frequency of lac(+) recombinants in subsequent crosses with an E. coli Hfr if the E. coli segment is integrated into the chromosome of the hybrid; the effect is less marked if the E. coli segment is not integrated.     

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.     

Molecular evolution of the Escherichia coli chromosome. V. Recombination patterns among strains of diverse origin.

Incorporation patterns of donor DNA into recipient chromosomes following transduction or conjugation have been studied in the progeny of a variety of Escherichia coli crosses in which donor and recipient nucleotide sequences differ by 1-3%. Series of contiguous or variously spaced PCR fragments have been amplified from each recombinant chromosome and digested with a commercial restriction endonuclease previously shown to distinguish the respective parents in a given fragment. We conclude that entering donor DNA fragments are frequently abridged (cut and shortened) before incorporation, the cutting being due to restriction systems, and the shortening presumably due to exonuclease activity. Analysis of several backcrosses confirms, and extends to conjugation, the importance of restriction in E. coli recombination in nature. The transmission patterns in conjugation are similar to those of transduction, but (as expected) on a much larger scale. Asymmetric results of reciprocal crosses imply that mismatch frequency is not a major factor. Marked differences among the results of simple crosses according to parental strain combinations are consistent with observations that E. coli strains in nature vary dramatically in their restriction-modification systems.     

Mapping of two ugp genes coding for the pho regulon-dependent sn-glycerol-3-phosphate transport system of Escherichia coli.

Two genes, ugpA and ugpB, coding for a binding protein-dependent sn-glycerol-3-phosphate transport system, were mapped at 75.3 min on the Escherichia coli chromosome. A Tn10 insertion in ugpA resulted in loss of transport activity but still allowed the synthesis of the sn-glycerol-3-phosphate-binding protein. This Tn10 insertion was found to be linked by P1 transduction to pit, aroB, malA, asd, and livH with 2.5, 2.8, 25, 63.5, and 83% cotransduction frequency. An insertion of Mud (Ampr lac) in ugpB resulted in the loss of the binding protein. ugpB is closely linked to ugpA. It is either the structural gene for the binding protein or located proximal to it. The analysis of the crosses allowed the ordering of the markers in the clockwise direction as follows: aroB, malA, asd, ugpA, ugpB, livH, pit.     

Variants of the plasmid pAS8 delta with increased frequency of integration into Rhodopseudomonas sphaeroides chromosome--the result of IS8-element duplication

The possible participation of IS8 and IS elements of Rhodopseudomonas sphaeroides in cointegrate formation by chromosome of the purple bacterium and plasmid pAS8-121 delta has been studied. The plasmid derivatives having deleted Tn7 have been studied. Plasmid integration into the chromosome of the purple bacterium is shown to be mediated by IS8 element of the plasmid. Plasmid derivatives having the integration potential increased for two orders were isolated by a series of intergeneric conjugational crosses during which plasmid pAS8-121 delta was transferred from Rhodopseudomonas sphaeroides (cointegrate of plasmid and chromosome) to Escherichia coli (plasmid in an autonomous state) and back to Rhodopseudomonas sphaeroides. The restriction analysis of plasmid DNA digested by Hpal and Smal restriction endonucleases has revealed the tandem duplications of IS8 in plasmids capable of integration into the chromosome of the purple bacterium with a high frequency.     

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.     

Genetic Mapping of Loci for Glucose-6-Phosphate Dehydrogenase, Gluconate-6-Phosphate Dehydrogenase, and Gluconate-6-Phosphate Dehydrase in Escherichia coli

The loci on the Escherichia coli genome of mutations affecting the constitutive enzymes glucose-6-phosphate dehydrogenase (zwf) and gluconate-6-phosphate dehydrogenase (gnd), and the inducible enzyme gluconate-6-phosphate dehydrase (edd), were determined by conjugation and transduction experiments, chiefly by three-factor crosses. They are in the same region of the chromosome, and their order is gnd-his-(edd, zwf)-aroD; gnd and his are cotransduceable, as are zwf and edd. The position of gnd in Salmonella typhimurium was shown to be similar to that in E. coli.     

Enhancement of the viability of irradiated bacteria by chromosome transfer

The effect of x-irradiating recipient cells of Escherichia coli K-12 before mating on the formation of recombinants and on the distribution of parental genetic material among recombinants was investigated in both the wild-type (Rec+) and a recombination-deficient (Rec-) mutant. In crosses involving Rec- recipients, recombinants selected for a late donor marker were formed in almost normal numbers. Rec- cells exposed to otherwise lethal doses of x-rays were still able to form viable recombinants for a distal male marker. These recombinants had inherited almost all the transferred donor chromosome, as evidenced by the preponderance of male markers in the recombinants. In contrast, the recombinant-forming ability was about as x-ray-sensitive as the colony-forming ability in Rec+ cells. No preference for donor chromosomal material was observed in recombinants from crosses involving x-irradiated Rec+ cells.     

Genetic Mapping of Mutations Affecting Phosphoglucose Isomerase and Fructose Diphosphatase in Escherichia coli

The loci on the Escherichia coli genome of mutations affecting phosphoglucose isomerase (pgi) and fructose diphosphatase (fdp) have been determined by conjugation and transduction experiments, chiefly three-factor crosses. The loci for these two constitutive enzymes of central intermediary metabolism lie in the same region of the chromosome, but they are not cotransducible. The order of some markers in this region of the chromosome is arg(ACFH) metA pgi malB uvrA fdp pyrB thr.     

Mapping of sul, the suppressor of lon in Escherichia coli.

The suppressor sul, which is allele specific for the ultraviolet sensitivity gene lon, has been mapped by conjugation and transductional crosses in Escherichia coli K-12 and B/r. Previously, sul was reported to lie in the azi region of the Escherichia coli chromosome. Evidence is presented which positions sul close to and clockwise of fabA on the Escherichia coli map. Cotransductional frequencies of 31.3% were obtained between sul and pyrD, and frequencies of 82% were obtained between sul and fabA. Also, the mucoid phenotype of K-12 lon strains grown on minimal glucose agar plates at 37 C was not significantly effected in sul derivatives. No differences between the sul of Escherichia coli B/r and that of K-12 derivatives with regard to map location or effect on mucoid production were observed.     

Genetics of speciation in the Aedes (Stegomyia) scutellaris subgroup (Diptera:Culicidae). 4. Chromosomal relationships of Aedes cooki with four sibling species

Morphology and behavior of chromosomes and development of testes and sperm were examined in hybrids from interspecific crosses involving Aedes cooki and four sibling species of the Aedes (Stegomyia) scutellaris subgroup of mosquitoes. The degree of abnormality in hybrid spermatogenesis in interspecific crosses involving Aedes cooki males and females of four sibling species paralleled the geographic distributions of these species and the genetic divergence indicated by other genetic studies. Hybrids from crosses involving Aedes malayensis females and Aedes cooki males were characterized by atrophied testes and extensive chromosome breakage. Hybrids from crosses involving Aedes alcasidi females and Aedes cooki males suggested a possible pericentric inversion distinguishing the largest autosome of Aedes alcasidi from that of Aedes cooki. Hybrids from interspecific crosses involving females of Aedes polynesiensis and Aedes pseudoscutellaris and males of Aedes cooki showed high percentages of univalents of the smallest chromosome pair. Hybrid spermatogenesis in two interspecific crosses involving Aedes cooki females differed from results of reciprocal crosses. Data were scant, however, and interpretation was difficult in view of negligible hatch in all interspecific crosses involving Aedes cooki females.     

Mutation of msh-2 in Neurospora crassa does not reduce the incidence of recombinants with multiple patches of donor chromosome sequence

The Neurospora homologue msh-2 of the Escherichia coli mismatch repair gene mutS was mutated by repeat-induced point mutation (RIP) of a 1.9-kb duplication covering 1661bp of the coding sequence and 302 bp 5' of the gene. msh-2(RIP-LK1) exhibited a mutator phenotype conferring a 17-fold increase in the frequency of spontaneous mitotic reversion of his-3 allele K458. In msh-2(RIP-LK1) homozygotes, recombination frequency at the his-3 locus increased up to 2.9-fold over that in msh-2(+) diploids. Progeny of crosses homozygous msh-2(RIP-LK1), like those from crosses homozygous msh-2(+) frequently had multiple patches of donor chromosome sequence, suggesting that patchiness in msh-2(+) crosses is not explained by incomplete repair of heteroduplex DNA by MSH-2. These findings are consistent with data from the analysis of events in a Neurospora translocation heterozygote that suggested multiple patches of donor chromosome sequence arising during recombination reflect multiple template switches during DNA repair synthesis.     

Localization of the structural gene for threonine dehydrogenase in Escherichia coli.

The threonine dehydrogenase (tdh) gene of Escherichia coli, cloned within the plasmid pDR121, was inactivated in vitro by inserting a segment of DNA carrying the chloramphenicol acetyltransferase (cat) gene. The insertionally inactivated tdh gene was then transferred by homologous recombination into the E. coli chromosome by the procedure of Winans et al. (J. Bacteriol. 161:1219-1221, 1985). Mating experiments, followed by P1-mediated two- and three-point crosses, enabled us to localize tdh near min 81.2. The order with respect to known markers is mtl-cysE-tdh-pyrE.     

Genetic Location of a Mutant of Bacteriophage T4 Deficient in the Ability to Induce Endonuclease II

Reciprocal three-factor crosses and the use of a partial revertant of a putative ribonucleotide reductase mutant of Escherichia coli B/5 as indicator have made it possible to map denA (deficient in endonuclease II) between nrd-11 (ribonucleotide reductase gene B) and amM69 (gene 63) on the bacteriophage T4 chromosome.     

HrpA, a DEAH-box RNA helicase, is involved in mRNA processing of a fimbrial operon in Escherichia coli

Endonucleolytic cleavage of mRNA in the daa operon of Escherichia coli is responsible for co-ordinate regulation of genes involved in F1845 fimbrial biogenesis. Cleavage occurs by an unidentified endoribonuclease, is translation dependent and involves a unique recognition mechanism. Here, we present the results of a genetic strategy used to identify factors involved in daa mRNA processing. We used a reporter construct consisting of the daa mRNA processing region fused to the gene encoding green fluorescent protein (GFP). A mutant defective in daa mRNA processing and expressing high levels of GFP was isolated by flow cytometry. To determine the location of mutations, two different genetic approaches, Hfr crosses and P1 transductions, were used. The mutation responsible for the processing defect was subsequently mapped to the 32 min region of the E. coli chromosome. A putative DEAH-box RNA helicase-encoding gene at this position, hrpA, was able to restore the ability of the mutant to cleave daa mRNA. Site-directed mutagenesis of the hrpA regions predicted to encode nucleotide triphosphate binding and hydrolysis functions abolished the ability of the gene to restore the processing defect in the mutant. We propose that HrpA is a novel enzyme involved in mRNA processing in E. coli.     

Convenient construction of strains useful for transducing recA mutations with bacteriophage P1

Abstract The generalized transducing phage P1 grew well on heterozygous Escherichia coli carrying recA srlC 300::Tn 10 on the chromosome and recA ⁺ on a pBR322-derived plasmid. Because of the close linkage of Tn 10 to recA mutations, including recA 1, recA 13, recA 56, recA deletion and recA allele of E. coli BNG30, the latter can be moved to other strains in transductional crosses for selective resistance to tetracycline.     

Chromosomal location and expression of the structural gene for major outer membrane protein Ia of Escherichia coli K-12 and of the homologous gene of Salmonella typhimurium.

The gene determining the structure of a major outer membrane protein of Escherichia coli, protein Ia, has been located between serC and pyrD, at the min 21 region of the linkage map. This is based on the isolation and characterization of E. coli-Salmonella typhimurium intergeneric hybrids as well as analyses of a mutation (ompF2) affecting the formation of protein Ia. When the serC region of the S. typhimurium chromosome was transduced by phage P1 into E. coli, two classes of transductants were obtained; one produced protein Ia like the parental strain of E. coli, whereas the other produced not protein Ia but a pair of outer membrane proteins structurally related to 35K protein, one of the major outer membrane proteins of S. typhimurium. Furthermore, a strain of S. typhimurium harboring an F' plasmid which carries the ompF region of the E. coli chromosome was found to produce a protein indistinguishable from protein Ia, beside the outer membrane proteins characteristic to the parental Salmonella strain. These results suggest that the structural genes for protein Ia (E. coli) and for 35K protein (S. typhimurium) are homologous to each other and are located at the ompF region of the respective chromosome. The bearing of these findings on the genetic control of protein Ia formation is discussed.     

Cytological and breeding behavior of pentaploids derived from 3x × 4x crosses in potato

Cytology and breeding behavior of Solanum commersonii - S. tuberosum hybrids derived from 3 x x 4 x crosses was examined. The chromosome number of hybrids ranged from hypo-pentaploid (2 n=5 x - 8=52), to hyper-pentaploid (2 n=5 x + 7=67), with the euploid pentaploid 2 n=5 x=60 class predominant. The high variability in chromosome number of the 3 x x 4 x hybrids was attributed to the fact that meiotic restitution during megasporogenesis of the 3 x female may have involved poles with various chromosome numbers, resulting in 2 n eggs with 24-48 chromosomes. Microsporogenesis analyses provided evidence that chromosome pairing between S. commersonii and S. tuberosum genomes occurred. In addition, chromosome distribution at anaphase I and anaphase II revealed an average chromosome number of 29.5 and 29.1 per pole, respectively. To further study the extent of transmission of extra genome chromosomes from pentaploids, 5 x x 4 x and 4 x x 5 x crosses were performed, and the chromosome number of resulting progeny was determined. Ploidy ranged from 2 n=4 x=48 to 2 n=5 x=60 following 5 x x 4 x crosses, and from 2 n=4 x + 1=49 to 2 n=5 x=60 following 4 x x 5 x crosses. These results provided indirect evidence that the pentaploid hybrids produced viable aneuploid gametes with a chromosome number ranging from 24 to 36. They also demonstrated that gametes with large numbers of extra chromosomes can be functional, resulting in sporophytes between the 4 x and 5 x ploidy level. Fertility parameters of crosses involving various (aneuploid) pentaploid genotypes were not influenced by chromosome number, suggesting a buffering effect of polyploidy on aneuploidy. The possibility of successfully using (aneuploid) pentaploid genotypes for further breeding efforts is discussed.