Rates and consequences of recombination between rRNA operons

A mutant strain of Escherichia coli was created by inserting a cassette encoding sucrose sensitivity and neomycin resistance (sacB-neo) into the small-subunit rRNA-encoding gene rrs in the rrnB operon. During growth in a complex medium, the cassette was lost from the population, and a complete rrs gene was restored at a rate of 5 x 10(-9) per cell division. Repair of this lesion required flanking regions of DNA that were similar to the six remaining intact rRNA operons and reestablished the full complement of seven rRNA operons. The relative fitness of strains with restored rrnB operons was 1 to 2% higher than that of the mutant strain. The rrnB operon normally contains a spacer region between the 16S and 23S rRNA-encoding genes that is similar in length and tRNA gene content to the spacer in rrnC, -E, and -G. In 2 of the 14 strains in which rrnB was restored, the spacer region had the same length as the spacer region in rrnA, -D, and -H. The requirement for flanking regions of nearly identical DNA and the replication of the spacer region from other rRNA operons during the repair of rrnB suggest that the restoration was accomplished via gene conversion. The rate of gene conversion was 10-fold less than the fixation of point mutations in the same region of the chromosome but was apparently sufficient to homogenize the sequences of rRNA genes in E. coli. These findings are discussed in the context of a conceptual model describing the presence of sequence heterogeneity in coevolving rRNA genes.     

Formation of delta tra F' plasmids: specific recombination at oriT

Delta tra F' plasmids can be isolated from matings between Hfr donors and recA- recipients, with selection for transfer of proximal chromosomal genes. Previous experiments indicate that F DNA from the neighborhood of the transfer origin up to the proximal junction with the chromosomal DNA is present on these plasmids, together with chromosomal segments, some of which belong to distinct size classes. We have sequenced across the novel joints contained in five delta tra FproA+ plasmids and in five delta tra FpurE+ plasmids, and we have compared these with the F sequence near oriT and with a chromosomal site near purE. The previously reported specificity in formation of some of these classes is confirmed at the nucleotide sequence level. The F DNA in nine of these novel joints extended beyond the nicking sites identified by others in lambda oriT+ bacteriophages up to a position between two sequenced oriT- mutations. Small plasmids containing these novel joints are mobilized in trans by pOX38 at frequencies less than 5 X 10(-7) times the mobilization frequencies for similar plasmids that contain oriT. The relations of these findings to the location of the nicking site at oriT are discussed.     

Redundant transfer of F' plasmids occurs between Escherichia coli cells during nonlethal selections.

Surface exclusion is the mechanism by which F plasmids prevent the redundant entry of additional F plasmids into the host cell during exponential growth. This mechanism is relaxed in cells that are in stationary phase. Using genetically marked F' plasmids and host strains, we extend this finding to Escherichia coli populations during extended nonlethal selection in bacterial lawns. We show that a high level of redundant transfer occurs between these nongrowing cells during the selection. This result has implications for the mechanism of adaptive mutagenesis.     

dnaB gene of Escherichia coli K-12 affects superinfection inhibition between F' plasmids.

F' Escherichia coli K-12 strains bearing the chromosomal mutation dnaB43 offer significantly less resistance to the conjugational introduction of a second F' plasmid than do nonmutant strains. Both the entry exclusion and incompatibility components of superinfection inhibition are altered. This action of dnaB43 occurs regardless of the presence of a recA-minus mutation in matings in liquid cultures and on membrane filters and is not limited to a particular set of F' plasmids. These effects are co-transducible by phage P1 with the temperature sensitivity conferred by dnaB43. The effects also occur with a strain carrying dnaB107. In the double F' strains that arise, the two plasmids exist as units autonomous of one another and the chromosome.     

Analysis of operons encoding 23S rRNA of Clostridium botulinum type A.

Southern hybridization analysis of Clostridium botulinum type A chromosomal DNA indicated the presence of six copies of the 23S rRNA gene. Fragments of DNA encoding 23S rRNA were amplified by polymerase chain reaction and cloned in Escherichia coli. Three clones examined by restriction enzyme and sequence analysis were found to be derived from different operons. Sequence determination of the entire insert of two clones revealed nine nucleotide changes in the genes coding for 23S rRNA (99.7% sequence identity) between operons encoded on the same chromosome, showing microheterogeneity in the rRNA operons of this organism.     

Mitotic recombination between dispersed but related rRNA genes of Schizosaccharomyces pombe generates a reciprocal translocation

Summary Recombination between dispersed yet related serine tRNA genes of Schizosaccharomyces pombe does occur during mitosis but it is approximately three orders of magnitude less frequent than in meiosis. Two mitotic events have been studied in detail. In the first, a sequence of at least 18 nucleotides has been transferred from the donor sup3 gene on the right arm of chromosome I to the related acceptor gene sup12 on the left arm of the same chromosome, thereby leading to the simultaneous change of 8 bp in the acceptor gene. This event must be explained in terms of recombination rather than mutation. It is assumed that it represents mitotic gene conversion, although it was not possible to demonstrate that the donor gene had emerged unchanged from the event. The second case reflects an interaction between sup9 on chromosome III and sup3 on chromosome I. Genetic and physical analysis allows this event to be described as mitotic gene conversion associated with crossingover. The result of this event is a reciprocal translocation. No further chromosomal aberrations were found among an additional 700 potential intergenic convertants tested. Thus intergenic conversion is much less frequently associated with crossingover than allelic conversion. However, the rare intergenic conversion events associated with crossingover provide a molecular mechanism for chromosomal rearrangements.     

Instability of rRNA operons in Bacillus subtilis.

Many laboratory strains of Bacillus subtilis contain 9 rather than 10 rRNA operons due to deletions occurring within the rrnJ-rrnW or rrnI-rrnH-rrnG gene cluster. These operons are members of two sets of closely spaced clusters located in the cysA-aroI region. Analysis of rescued DNA from integrants with insertions into rrnG and rrnH indicated that these tandemly arranged operons allowed frequent deletions of an rrn operon equivalent. These events may arise spontaneously by intrachromosomal recombination or by simultaneous double crossovers with a multimeric integrative plasmid.     

Transduction-related cointegrate formation between staphylococcal plasmids: A new type of site-specific recombination

Small R plasmids are frequently cotransduced by staphylococcal transducing phages. Most cotransductants contain independent plasmids indistinguishable from those of the donor strain; occasionally recombinational exchanges can be demonstrated including the formation of stable cointegrates that appear to contain all of the genomes of the two starting plasmids. These cointegrates do not dissociate at a detectable frequency upon subculture of the host strain or upon further transduction. Examination of a series of these cointegrates derived by recombination between various pairs of plasmids has revealed the existence of a new type of site-specific recombination. Nineteen different cointegrates involving five different plasmids were studied by restriction endonuclease analysis and electron microscopic examination of heteroduplexes. To the limits of resolution of these techniques, it can be concluded that each plasmid contains one or more specific sites that is used for the formation of cointegrates with the other plasmids. In addition, the cointegrates are orientation specific as well as site specific so that the recombination process resembles that of prophage integration more than that of transposon insertion. Strikingly, in cases where cointegrates between one plasmid, A, and two or more others (e.g., B and C) have been isolated, the same site on A is used for cointegrate formation with both B and C; moreover, B and C also form cointegrates with each other, using the same site and orientation with which both recombine with A. The formation of cointegrates probably involves identical sequences of ≤ 100 nucleotides and is not demonstrably related to overall homology.     

Site-specific recombination between plasmids of Staphylococcus aureus

Anomalous recombination between two similar but nonidentical, naturally occurring penicillinase plasmids, pI258 and pI524, leading to duplication and deletion of the beta-lactamase locus, is described. Physical mapping of these plasmids by heteroduplex and restriction analysis revealed that the beta-lactamase loci were homologous and in inverted orientation with respect to one another and that their respective locations were separated by a short region of homology. This intervening region of homology included one copy of a segment that was repeated on pI524 in inverted orientation at a distance of 2.2 kilobase pairs and contained a recognition sequence for a site-specific, rec-independent recombination function that caused reversible inversion of this segment on pI524. It is proposed that site-specific, intermolecular recombination involving this repeated sequence was responsible for the observed results.     

Specificity in formation of type II F'' plasmids.

Eight new F' plasmids derived from Hfr strains in which F is integrated at the chromosomal element alpha 3 beta 3 have been isolated and subjected to restriction enzyme, hybridization, and electron microscope heteroduplex analysis. Plasmids carrying extensive amounts of bacterial deoxyribonucleic acid were produced even though they were obtained by selection for transfer of lac, which is closely linked to F in the parental Hfr strains. Seven plasmids were type II Flac+ proC+ purE+ plasmids, and one was a type I Flac+ proC+ plasmid. Five of the Flac+ proC+ purE+ plasmids contain approximately 284 kilobases of bacterial deoxyribonucleic acid, which is identical for all five within the resolution of the restriction enzyme analysis. Theses results indicate that type II F' plasmids are the predominant tra+ F' type from this region of the Escherichia coli K-12 chromosome and that the recombination events leading to formation of these plasmids exhibit site specificity.     

An alternative pathway of recombination of chromosomal fragments precedes recA-dependent recombination in the radioresistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans R1 and other members of this genus are able to repair and survive extreme DNA damage induced by ionizing radiation and many other DNA-damaging agents. The ability of R1 to repair completely > 100 double-strand breaks in its chromosome without lethality or mutagenesis is recA dependent. However, during the first 1.5 h after irradiation, recA+ and recA cells show similar increases in the average size of chromosomal fragments. In recA+ cells, DNA continues to enlarge to wild-type size within 29 h. However, in recA cells, no DNA repair is observed following the first 1.5 h postirradiation. This recA-independent effect was studied further, using two slightly different Escherichia coli plasmids forming adjacent duplication insertions in the chromosome, providing repetitive sequences suitable for circularization by non-recA-dependent pathways following irradiation. After exposure to 1.75 Mrad (17,500 Gy), circular derivatives of the integration units were detected in both recA+ and recA cells. These DNA circles were formed in the first 1.5 h postirradiation, several hours before the onset of detectable recA-dependent homologous recombination. By comparison, D. radiodurans strains containing the same E. coli plasmids as nonrepetitive direct insertions did not form circular derivatives of the integration units before or after irradiation in recA+ or recA cells. The circular derivatives of the tandemly integrated plasmids were formed before the onset of recA-dependent repair and have structures consistent with the hypothesis that DNA repair occurring immediately postirradiation is by a recA-independent single-strand annealing reaction and may be a preparatory step for further DNA repair in wild-type D. radiodurans.     

Mapping and spacer identification of rRNA operons of Salmonella typhimurium.

The rRNA operons of Salmonella typhimurium have been characterized with respect to their map position, orientation, and type of tRNA spacer. One of the seven rrn operons was found to be linked to pheA and another was found to be linked to aroE. This information, together with published information about the other five rrn operons, shows that S. typhimurium and Escherichia coli are essentially identical in terms of the number, the map position, and the orientation of all seven operons. S. typhimurium and E. coli were also similar in that four of the rrn spacer regions code for tRNAGlu2 and three code for tRNAAla1B. However, the two species differed in that rrnD coded for tRNAGlu2 and rrnB coded for tRNAAla1B in S. typhimurium. This is the opposite of the arrangement in E. coli. We have tabulated the coordinates of the BamHI and PstI sites flanking six of the S. typhimurium rrn genes and present revisions for the coordinates of some of the E. coli sites.     

Analysis of genetic recombination between two partially deleted lactose operons of Escherichia coli K-12.

Genetic recombination between a nontandem duplication of two partially deleted lactose operons (lacMS286phi80dIIlacBK1) in Escherichia coli K-12 has been examined. Since the deletions were nonoverlapping, rare lactose-fermenting (Lac+) recombinants occurred and were detected qualitatively on lactose tetrazolium agar indicator plates as white papillae growing on the surface of red colonies or quantitively on lactose minimal agar plates. Formation of Lac+ recombinants required the recA, recB, and recC gene products. Indirect suppression of recB21 by sbcB15 led to an increase in the frequency of Lac+ recombinants over wild-type levels. recF143 did not appreciably alter the number of Lac+ progeny, whereas recL152 and sbcB15 strains yielded increased numbers of Lac+ recombinants. The nature and formation of Lac+ recombinants was also examined. Respreading analysis indicated that formation of recombinants occurred primarily as the cells entered early stationary phase on the surface of the minimal agar plates and that over 90% of the recombinants contained a phi80dIIlac+ prophage. Time-of-entry experiments suggested that the region of deoxyribonucleic acid between the two operons was not inverted as a result of the recombinational event.     

Isolation of F' plasmids carrying a portion of the Salmonella typhimurium histidine transport operon.

The transposable drug resistance element Tn10 was employed as a region of homology to direct the insertion of Tn10-containing derivatives of F'ts114 lac into the chromosome of a Salmonella typhimurium strain that carries a Tn10 insertion in the histidine transport operon. Based on the direction of transfer of the resulting Hfr strains, the chromosomal Tn10 insertion was determined to be in orientation "A." New F' plasmids were selectively generated from one of the Hfr strains. The F' factors carry an intact dhuA hisJ portion of the histidine transport operon. A Southern hybridization revealed that one of the F' plasmids was formed by a type II excision event.     

Formation of recombinants of F' plasmids in recombination-defective E. coli cells and their properties]

As a result of mating of cells carrying plasmid F' lac with cells carrying plasmid F'his merodiploids carrying plasmid complex F'lacF'his were isolated. The plasmid genes of the isolated merodiploids are donated together into the recipient cells and are eliminated jointly from host-cells both spontaneously and by acridine orange. As the formation of the plasmid complex took place in E. coli cells carrying mutation in rec A gene the recombination of plasmids F' is supposed to take place in the absence of the product of gene rec. A.     

recE4-Independent recombination between homologous deoxyribonucleic acid segments of Bacillus subtilis plasmids.

A plasmid (pLS104) carrying a tandem repetition of the leu region of the Bacillus subtilis chromosome arose spontaneously from pLS103, which carried a single copy of the leu region. Plasmid preparations from strains harboring pLS104 also contained the original plasmid, pLS103, and, in some preparations, plasmids carrying three or four repetitions of the leu region. These plasmids were shown to be generated by recombination between homologous deoxyribonucleic acid (DNA) segments in the tandemly repeated DNA regions on the plasmids, but not by recombinations between specific DNA sites. These phenomena were observed in a recE4-Independent background, showing that recombination of the homologous DNA sequences does not require the recE-Independent gene product(s).     

Replication origin region of Bacillus subtilis chromosome contains two rRNA operons.

The first replicating DNA fragment (BamHI-7) of the Bacillus subtilis chromosome contains two promoters for a rRNA operon. A map of restriction enzyme cleavage sites of the region of replication origin suggests the presence of a second rRNA operon in this region. Hybridization of rRNA genes (rDNA) with DNA fragments derived from the origin region by treatment with various enzymes clearly revealed two rRNA operons in this region, one at the B7-B3 junction and the other at the B5-B6 junction. The restriction enzyme cleavage sites surrounding the rRNA operons show that the operon at the B5-B6 junction corresponds to the rrnA operon. A novel operon at the B7-B3 junction was termed rrnO. Transformation by density-labeled fragments of the origin region showed that the first replicating marker, guaA, is located in the B3 fragment. From these results, a map was constructed for the first time to correlate the genetic markers with the physical structure of the replication origin region of the B. subtilis chromosome. The role of the rrnO operon in regulating the initiation of chromosomal replication is discussed, based on the fact that the promoter of the rrnO operon suppresses the replication of the plasmid carrying the promoter.