Evidence that F plasmid transfer replication underlies apparent adaptive mutation.

An Escherichia coli K12 strain, FC40, has been used extensively in the analysis of adaptive mutability. This strain carries a revertible mutant lac allele on an F plasmid and accumulates Lac+ (lactose utilizing) revertants, but not unselected mutants, when placed on selective medium. These adaptive mutations are a subset of spontaneous types and their formation depends on the RecABC functions. Data presented here suggest that this phenomenon depends on transfer functions of the F factor. Fertility inhibition eliminates RecA-dependent adaptive reversion. Thus, "adaptive" revertants may form during replication from the transfer origin, whereas loci in the nonreplicating chromosome show little mutation.

Rearrangement of the bacterial chromosome: forbidden inversions.

The order of genes in the chromosome of enteric bacteria has been evolutionarily conserved despite the existence of mechanisms for rearrangement. Homologous chromosomal sequences in the same orientation recombine to form deletions or duplications. When homologous sequences in inverse orientation recombine, one expects to form an inversion of the intervening chromosomal segment. This expectation was tested by placing pairs of homologous sequences in inverse order at various points in the chromosome. Sequences at many pairs of sites (permissive) do recombine to generate the expected inversion, while the same sequences placed at other pairs of sites (nonpermissive) do not form an inversion. For the one nonpermissive interval tested, the missing inversion type can be constructed by an alternative transductional method; strains with this inversion are viable. Thus mechanistic limitations must prevent sequences at particular sites from undergoing the recombination event required to form an inversion.

Evidence that gene amplification underlies adaptive mutability of the bacterial lac operon.

Adaptive mutability is the apparent alteration in specificity or rate of mutability seen in bacteria during stress. A model is proposed by which gene amplification during selective growth can give the appearance of adaptive mutability without requiring any change in mutability. The model is based on two assumptions, that a mutant lac locus with residual function allows growth if its copy number is increased, and that true reversion events are made more likely by replication of chromosomes with many copies of the locus. Apparent directed mutability, its recombination requirement, and its apparent independence of cell growth are all accounted for by the model. Evidence is provided for the required residual function and gene amplification.

Structural and functional studies of insertion element IS200.

The nucleotide sequence of the insertion element IS200 has been determined partially, including the junctions between the element and the host chromosome at the insertion site. At most, two bases (A-A) are found repeated at the junctions and could be duplications of host sequences generated by the insertion of the element. No obvious sequence repeats, either direct or inverted, have been detected between the sequences just within the two ends of the element. The element is an extremely strong block to host transcription across the insertion site. A sequence similar to known transcription termination signals was found just within the element near the right end. Removal of less than 50 base-pairs at the right end of the element abolishes the transcription block. The putative terminator sequence is located within this 50 base-pair region. Genetic studies suggest that the element contains a promoter located more than 93 base-pairs from its left end. The proposed promoter and terminator are in proper orientation to form an internal transcription unit.

Reciprocality of recombination events that rearrange the chromosome.

We describe a genetic system for studying the reciprocality of chromosomal recombination; all substrates and recombination functions involved are provided exclusively by the bacterial chromosome. The genetic system allows the recovery of both recombinant products from a single recombination event. The system was used to demonstrate the full reciprocality of three different types of recombination events: (1) intrachromosomal recombination between direct repeats, causing deletions; (2) intrachromosomal recombination between inverse homologies, causing inversion of a segment of the bacterial chromosome; and (3) circle to circle recombination (in the absence of any plasmid or phage functions). Results suggest that intrachromosomal recombination in bacteria is frequently fully reciprocal.

Recombination between homologies in direct and inverse orientation in the chromosome of Salmonella: intervals which are nonpermissive for inversion formation.

Sequences placed in inverse order at particular chromosome sites (permissive) recombine to generate an inversion; the same sequences, placed at other sites (nonpermissive) interact recombinationally but do not form the expected inversion recombinants. We have investigated the events that occur between sequences at nonpermissive sites. Genetically marked lac operons in inverse order were placed at nonpermissive sites in a single chromosome and Lac+ recombinants were selected. No inversions were formed. The Lac+ recombinants recovered include double-recombinant types in which information appears to have undergone a nonreciprocal information exchange; one mutant copy is repaired with no alteration of the other copy. Recombination within the lac operon is stimulated more than 100-fold by the presence of extensive homology (antenna sequences) outside of the region for which recombination is selected. Sequences placed in direct order at the ends of the same noninvertible chromosome segment recombine to form all the expected recombinant types including those in which a reciprocal exchange has generated a duplication. All the detected recombinant types can be accounted for by recombination between sister chromosomes. These results are discussed in terms of two alternative models. One explanation of the failure to detect inversion of some intervals is that particular inversions are lethal, despite the fact that no essential sequences are disrupted. Another explanation is that chromosome topology prevents sequences at nonpermissive sites in a single chromosome from engaging in the direct interaction required for inversion formation, but allows the sister strand exchanges that can generate the recombinant observed.

Polarity effects in the hisG gene of salmonella require a site within the coding sequence.

A single site in the middle of the coding sequence of the hisG gene of Salmonella is required for most of the polar effect of mutations in this gene. Nonsense and insertion mutations mapping upstream of this point in the hisG gene all have strong polar effects on expression of downstream genes in the operon; mutations mapping promotor distal to this site have little or no polar effect. Two previously known hisG mutations, mapping in the region of the polarity site, abolish the polarity effect of insertion mutations mapping upstream of this region. New polarity site mutations have been selected which have lost the polar effect of upstream nonsense mutations. All mutations abolishing the function of the site are small deletions; three are identical, 28-bp deletions which have arisen independently. A fourth mutation is a deletion of 16 base pairs internal to the larger deletion. Several point mutations within this 16-bp region have no effect on the function of the polarity site. We believe that a small number of polarity sites of this type are responsible for polarity in all genes. The site in the hisG gene is more easily detected than most because it appears to be the only such site in the hisG gene and because it maps in the center of the coding sequence.

Pathways for homologous recombination between chromosomal direct repeats in Salmonella typhimurium.

Homologous recombination pathways probably evolved primarily to accomplish chromosomal repair and the formation of and resolution of duplications by sister-chromosome exchanges. Various DNA lesions initiate these events. Classical recombination assays, involving bacterial sex, focus attention on double-strand ends of DNA. Sexual exchanges, initiated at these ends, depend on the RecBCD pathway. In the absence of RecBCD function, mutation of the sbcB and sbcC genes activates the apparently cryptic RecF pathway. To provide a more general view of recombination, we describe an assay in which endogenous DNA damage initiates recombination between chromosomal direct repeats. The repeats flank markers conferring lactose utilization (Lac+) and ampicillin resistance (ApR); recombination generates Lac-ApS segregants. In this assay, the RecF pathway is not cryptic; it plays a major role without sbcBC mutations. Others have proposed that single-strand gaps are the natural substrate for RecF-dependent recombination. Supporting this view, recombination stimulated by a double-strand break (DSB) in a chromosomal repeat depended on RecB function, not RecF function. Without RecBCD function, sbcBC mutations modified the RecF pathway and allowed it to catalyze DSB-stimulated recombination. Sexual recombination assays overestimate the importance of RecBCD and DSBs, and underestimate the importance of the RecF pathway.

Histidine mutants requiring adenine: selection of mutants with reduced hisG expression in Salmonella typhimurium.

A method is described for the selection of Salmonella typhimurium mutants with reduced levels of hisG enzyme activity. This method is based on the fact that the hisG enzyme catalyzes the consumption of ATP in the first step of histidine biosynthesis. Normally, this reaction is closely regulated, both by feedback inhibition and by repression of the operon. However, conditions can be set up that result in the uncontrolled use of adenine in histidine biosynthesis. Cells grown under these conditions become phenotypic adenine auxotrophs. Some revertant clones that no longer require adenine contain mutations in hisG, hisE, or the his-control region. The hisG mutations are of all types (nonsense, frameshift, missense, deletion and leady types), and they map throughout the hisG gene.

Circularization of transduced fragments: a mechanism for adding segments to the bacterial chromosome.

Generalized transducing fragments that have redundant sequences in direct order can circularize during transduction events. The length of the required redundant sequences can be at least as short as IS10 (1.4 kb) (Kleckner 1977). The circular transduced fragment is able to recombine with homologous sequences in the chromosome. Circularization and insertion of transduced fragments allow addition of segments to the bacterial chromosome rather than replacement of recipient segments as in a normal transductional cross. It also provides a method for translocation of bacterial genes to a variety of specific sites on the chromosome in either orientation. The significance of these events to bacterial evolution is discussed.

Transitory cis complementation: a method for providing transposition functions to defective transposons.

A genetic complementation system is described in which the complementing components are close together in a single linear DNA fragment; the complementation situation is temporary. This system is useful for providing transposition functions to transposition-defective transposons, since transposition functions act preferentially in cis. The basic procedure involves placing a transposition-defective transposon near the gene(s) for its transposition functions on a single DNA fragment. This fragment is introduced, here by general transduction, into a new host. The transposase acts in cis to permit the defective element to transpose from the introduced fragment into the recipient chromosome. The helper genes do not transpose and are lost by degradation and segregation. The method yields single insertion mutants that lack transposase and are not subject to further transposition or chromosome rearrangement. The general procedure is applicable to other sorts of transposable elements and could be modified for use in other genetic systems.

Activation of silent genes by transposons Tn5 and Tn10.

The presence of transposons Tn10 or Tn5 in the genome increases the frequency with which a silent (promoter-less) gene (hisD) is mutationally activated. The activation frequency is increased 5-25-fold by Tn10 and 30-90-fold by Tn5. Activation of the hisD gene is achieved by transposition of the entire transposon or one of its flanking insertion sequences to a region just upstream of the silent gene, between a Rho-dependent termination site in the adjacent hisG gene and the hisD gene. For both Tn5 and Tn10 the component insertion sequences were found to transpose much more frequently than the entire composite element. Transposons Tn5 and Tn10 have previously been shown to carry promoters which direct transcripts into sequences adjacent to their insertion sites.

A search for a general phenomenon of adaptive mutability.

The most prominent systems for the study of adaptive mutability depend on the specialized activities of genetic elements like bacteriophage Mu and the F plasmid. Searching for general adaptive mutability, we have investigated the behavior of Salmonella typhimurium strains with chromosomal lacZ mutations. We have studied 30 revertible nonsense, missense, frameshift, and insertion alleles. One-third of the mutants produced > or = 10 late revertant colonies (appearing three to seven days after plating on selective medium). For the prolific mutants, the number of late revertants showed rank correlation with the residual beta-galactosidase activity; for the same mutants, revertant number showed no correlation with the nonselective reversion rate (from fluctuation tests). Leaky mutants, which grew slowly on selective medium, produced late revertants whereas tight nongrowing mutants generally did not produce late revertants. However, the number of late revertants was not proportional to residual growth. Using total residual growth and the nonselective reversion rate, the expected number of late revertants was calculated. For several leaky mutants, the observed revertant number exceeded the expected number. We suggest that excess late revertants from these mutants arise from general adaptive mutability available to any chromosomal gene.

Ability of a bacterial chromosome segment to invert is dictated by included material rather than flanking sequence.

Homologous recombination between sequences present in inverse order within the same chromosome can result in inversion formation. We have previously shown that inverse order sequences at some sites (permissive) recombine to generate the expected inversion; no inversions are found when the same inverse order sequences flank other (nonpermissive) regions of the chromosome. In hopes of defining how permissive and nonpermissive intervals are determined, we have constructed a strain that carries a large chromosomal inversion. Using this inversion mutant as the parent strain, we have determined the "permissivity" of a series of chromosomal sites for secondary inversions. For the set of intervals tested, permissivity seems to be dictated by the nature of the genetic material present within the chromosomal interval being tested rather than the flanking sequences or orientation of this material in the chromosome. Almost all permissive intervals include the origin or terminus of replication. We suggest that the rules for recovery of inversions reflect mechanistic restrictions on the occurrence of inversions rather than lethal consequences of the completed rearrangement.

Directed formation of deletions and duplications using Mud(Ap, lac).

A genetic procedure is directed for the isolation of chromosomal deletions and duplications with predetermined endpoints. These rearrangements are generated in transduction crosses using a mixture of P22-transducing phage lysates grown on two strains, each carrying a Mud-lac insertion. The formation of duplications and deletions was demonstrated in the his operon using insertions of Mud 1-8 (a transposition-defective Mud-lac phage). This technique was also used to make larger chromosomal duplications between Mud 1-8 insertions in the thr and leu biosynthetic operons and between Mud insertions in the thr and pyrB operons. Genetic evidence is presented that strongly suggests that inheritance of a single Mud prophage by P22-mediated crosses requires two transduced fragments; each carrying part of the Mud prophage. The two fragments must be involved in three recombinational exchanges; one exchange joins the donor Mud fragments and two exchanges occur between the composite fragment and the recipient chromosome, one on either side of the complete donor Mud element. Since duplications only occur between Mud insertions in the same orientation on the chromosome, the method of duplication formation provides a simple means of determining the orientation of Mud 1-8 on the chromosome and, therefore, the direction of transcription of the gene into which Mud is inserted. This method was also used to construct recombinants between a Mud 1-8 prophage and Casadaban's protein fusion vector Mud2 and, thereby, isolate Mud2-8, a Mud derivative containing the protein fusion ability of Mud2 and the defective transposition functions of Mud1-8.

A novel P22 prophage in Salmonella typhimurium.

Under several sets of conditions, all of which seem to perturb purine metabolism, Salmonella typhimurium releases a variety of phages which were not known to be present in the strain. These cryptic phages are not induced by UV irradiation. Furthermore, the induction process does not require a functional recA gene product. While phages of several phenotypic classes have been recovered, including both turbid and clear plaque formers, all appear to be variants of P22 because all show DNA restriction patterns indistinguishable from that of P22. The variety of types suggests that the cryptic prophage is mutagenized as a consequence of the induction process. All the temperature phages tested are capable of transducing a variety of chromosomal markers with high efficiency. The phages induced in this novel way are capable of forming plaques on the strains that gave rise to them. Since the strains releasing phage are not immune to P22, the parental lysogens must not express immunity and the phage must be held in a cryptic state by a novel mechanism. The released phage possess an intact P22 immunity system because many can form standard immune lysogens after reinfection of Salmonella. These results raise the possibility that Salmonella typhimurium harbors cryptic phages that are subject to a novel system of global control related to purine metabolism. Preliminary evidence suggests that the regulation system may involve DNA modification.

Role of recBC function in formation of chromosomal rearrangements: a two-step model for recombination.

The role of recBC functions has been tested for three types of chromosomal recombination events: (1) recombination between direct repeats to generate a deletion, (2) recombination between a small circular fragment and the chromosome, and (3) recombination between inversely oriented repeats to form an inversion. Deletion formation by recombination between direct repeats, which does not require a fully reciprocal exchange, is independent of recBC function. Circle integration and inversion formation are both stimulated by the recBC function; these events require full reciprocality. The results suggest that half-reciprocal exchanges can occur without recBC, but recBC functions greatly stimulate completion of a fully reciprocal exchange. We propose that chromosomal recombination is a two-step process, and recBC functions are primarily required for the second step.

Role of gene duplications in the adaptation of Salmonella typhimurium to growth on limiting carbon sources.

Duplication-containing cells are selected when growth of Salmonella typhimurium is limited by the availability of any one of several carbon and energy sources. Under conditions of extreme starvation, growth occurs almost exclusively in the duplication-containing fraction of the population. Cells with duplications of one large segment of the chromosome are repeatedly selected regardless of which of these carbon sources limits growth. The duplicated chromosomal segment encodes the transport systems for all of these carbon sources. This duplication is not selected during growth on a carbon source for which the permease is not included within the duplication segment. This suggests that the growth advantage conferred by the duplication may be due to increased transport of the limiting carbon source. Inclusion of the permease alone is not sufficient to explain the growth advantage of the duplications, since other common duplications that include the permease are not selected.

Approaches to half-tetrad analysis in bacteria: recombination between repeated, inverse-order chromosomal sequences.

In standard bacterial recombination assays, a linear fragment of DNA is transferred to a recipient cell and, at most, a single selected recombinant type is recovered from each merozygote. This contrasts with fungal systems, for which tetrads allow recovery of all meiotic products, including both ultimate recombinant products of an apparent single act of recombination. We have developed a bacterial recombination system in which two recombining sequences are placed in inverse order at widely separated sites in the circular chromosome of Salmonella typhimurium. Recombination can reassort markers between these repeated sequences (double recombination and apparent gene conversion), or can exchange flanking sequences, leading to inversion of the chromosome segment between the recombining sequences. Since two recombinant products remain in the chromosome of a recombinant with an inversion, one can, in principle, approach the capability of tetrad analysis. Using this system, the following observations have been made. (a) When long sequences (40 kb) recombine, conversion frequently accompanies exchange of flanking sequences. (b) When short sequences (5 kb) recombine, conversion rarely accompanies exchange of flanks. (c) Both recA and recB mutations eliminate inversion formation. (d) The frequency of exchanges between short repeats is more sensitive to the distance separating the recombining sequences in the chromosome. The results are presented with the assumption that inversions occur by simple interaction of two sequences in the same circular chromosome. In an appendix we discuss mechanistically more complex possibilities, some of which could also apply to standard fungal systems.

Selfish operons: horizontal transfer may drive the evolution of gene clusters.

A model is presented whereby the formation of gene clusters in bacteria is mediated by transfer of DNA within and among taxa. Bacterial operons are typically composed of genes whose products contribute to a single function. If this function is subject to weak selection or to long periods with no selection, the contributing genes may accumulate mutations and be lost by genetic drift. From a cell's perspective, once several genes are lost, the function can be restored only if all missing genes were acquired simultaneously by lateral transfer. The probability of transfer of multiple genes increases when genes are physically proximate. From a gene's perspective horizontal transfer provides a way to escape evolutionary loss by allowing colonization of organisms lacking the encoded functions. Since organism bearing clustered genes are more likely to act as successful donors, clustered genes would spread among bacterial genomes. The physical proximity of genes may be considered a selfish property of the operon since it affects the probability of successful horizontal transfer but may provide no physiological benefit to the host. This process predicts a mosaic structure of modern genomes in which ancestral chromosomal material is interspersed with novel, horizontally transferred operons providing peripheral metabolic functions.