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EVER SINCE DARWIN proposed his theory of natural selection, biological evolution has been viewed as a process with two distinct stages. First, variation accumulates in a population of related organisms. This is now thought to occur through random mutation and recombination of DNA sequences. Second, competition among members of the variant population leads the bestadapted individuals to thrive and displace less well-adapted individuals. This principle, that evolutionary processes have a basal rate of variation and selection, is not disputed and, in fact, underpins many genetic procedures in bacteria, yeast and higher eukaryotes. However, debate about rates of variation and mechanisms for generating mutants continuesL Darwin suggested that evolution works mainly through gradual diversification, which allows useful adaptations to be incorporated slowly but ceaselessly over long time-frames. Darwin recognized that evolution accelerates when organisms move into novel environments, but there was no visible mechanism. In 1972, Eldredge and Gould proposed an alternative to evolutionary gradualism called punctuated equilibrium 2, which accounted for two observations in paleontology. First, many species have been remarkably stable - species life spans of more than ten million years are not unusual in the geological record. However, there is also evidence of the appearance of many new species during a very short time span, with no sign of a gradual series of intermediaries. The proposal of Eldredge and Gould that the rate of evolution may be highly variable stimulated new ideas about the role of catastrophic extinctions in evolution. It also suggested that evidence for both punctuation and equilibrium in variation could be found in present-day biological systems. Most molecular theories about variation assume that evolution is driven
Death and transfiguration among bacteria
When bacteria are placed in sub-optimal environments, they can respond by increasing the frequency of mutants created by base substitution, frame-shift and transposition mutations. Also, during periods of restrictive growth, 'dead' bacterial cells may transfer genetic material to neighboring colony-forming cells. This can be beneficial, resulting in a heterogeneous population that may exhibit differentiation and even produce killer cells. These discoveries reveal several conundrums about the control of an organism over mutations and the supposed randomness of genetic variation.
by random changes occurring in reproductive populations. Few, if any, scientists challenge the fact that there is a basal rate of mutation. However, in addition to constant slow change, mechanisms for dramatic changes have been uncovered. For example, moveable segments of DNA known as transposons influence chromosome structure and function through transposition between chromosomes in all cellular organisms. They alter gene activity by modifying transcriptional regulation, by disrupting active genes and by creating new genes with novel activities. Gilbert suggests that re-assorting as few as 2000 small genes of 40-60 amino acids (exons) might be sufficient to generate the complete repertoire of modern proteins 3. Transposition provides a mechanism for this DNA shuffling.
Detectingtransposons
When do transposons act, and do they move at fixed rates or with punctuated rhythms? The first indication that transposition rates fluctuate was in the 1940s. McClintock discovered transposons while examining maize plants with special chromosomes that often break during the cell divisions that follow meiosis. In several cases, a transposon changed anthocyanin pigment N. P. Higgins is at the Department of production only in the aleurone layer of Biochemistry, University of Alabama at a corn kernel, which restricted the Birmingham, 464 Basic Health Sciences transposition to a period covering only Building, 1918 University Blvd, UAB Station, Birmingham, AL 35294-0005, USA. two cell mitoses after introduction of a © 1992,ElsevierSciencePublishers, (UK) 0376-5067/92/$05.00
chromatid break 4. Thus, one response of maize to a chromosome break was an increased transposition rate. This revolutionary discovery was not widely appreciated until transposons were found years later in bacteria. However, McClintock recognized two important properties of this unexpected genetic behavior: genes in chromosomes can be surprisingly mobile and they can be induced to move with a genomic shock or challenge. Obtaining temporal information about transposon movement is difficult in most traditional genetic systems. However, novel properties of phage Mu permit transposition events to be monitored in living bacterial populations. Mu is both a transposon and a temperate virus. It is the most efficient transposon known, capable of replicating itself to new chromosomal sites in a single cell 100 times an hour. Mu DNA is not cut out of the host chromosome during transposition, so it scrambles the host gene order by making deletions and inversions and by joining together distant segments of the bacterial chromosome 5 (see Fig. 1). However, Mu may also remain silent and at the same position for many generations. One way to examine the timing of Mu transposition is to track subsets of selectable Mu-driven rearrangements that benefit the host. Shapiro studied a notable case in E. coil6. A Mu prophage was located between two non-functional
2O7
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Mu scrambles the host chromosome. Bacteriophage Mu is depicted as a bold arrow along a circular chromosome with gene order A-J. The consequence of replicative Mu transposition to a second site within the bacterial chromosome is either formation of a deletion, if Mu inserts in one orientation (left arrow), or an inversion, ifMu inserts in the opposite orientation (right arrow). Subsequent Mu transposition events may form more circular deletions or reform the large circle. An example of one gene order after nine cycles of Mu replication is shown in the bottom circle. In the lytic cycle Mu transposes to 100 positions within the host chromosome in an hour.
operons. On one side was an inactive lactose operon (lac) that contained functional genes for transporting and hydrolysing lactose but lacked a transcription promoter. On the other side of Mu was an arabinose operon (ara) bearing functional arabinose-sensitive
transcription control elements and an enzyme defective in its ability to hydrolyse arabinose. Thus, the parent strain would not grow on plates containing both arabinose and lactose as the carbon sources. However, Mu can generate ara-lac fusions that allow fermentation
R
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i
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Mu g e n e s
Rgure 2 Genetic structure of Mudl1168I, a non-plaque-forming derivative of Mu carrying the Mu c repressor, the A transposase and B replication protein. A selectable neomycin phosphotransferase gene from Tn903 (Kan) is near the center of the element. Arrows below the genes indicate the direction of transcription. Transcription of lac genes must be initiated outside the transposon (hyphenated arrow) and production of functional ~-galactosidase requires fusion of lacZ in the proper reading frame to a protein outside the eJement.
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of lactose in the presence of inducing levels of arabinose. In these rearranged chromosomes, Mu transposition connected the lac genes to the ara regulatory sequences, so these strains grew only when both lactose and arabinose were present. Ara-lac fusions, did not appear on plates with an abundant carbon source. However, after starvation on minimal plates containing both arabinose and lactose, ara-lac-utilizing colonies were frequently detected. After several days, hundreds of independent colonies appeared on a single plate. Shapiro's results and careful analysis clearly showed that ara-lac fusion formation was caused by punctuated transposon-driven genetic rearrangements and that the carbon limitation history of the culture was also important. A more general way to detect transposition in colonies is with non-plaqueforming Mu derivatives like MudIl1681 (Ref. 7). MudII1681 contains the viral DNA sequences and proteins needed to regulate and mobilize a phage genome. but it lacks phage structural genes (A in Fig. 2). A lacZ gene that lacks both a transcription promoter and a ribosomebinding site is situated towards the left. Functional B-galactosidase (E.C. 3.2.1.23) can be made only after MudII1681 transposes into the correct reading frame of another gene and creates a protein chimera. The amino-terminal domain of the chimera comes from a host protein; the carboxy-terminal segment is an active domain of lacZ. Because Mu may land in either orientation in any of three reading frames, only one-sixth of all transposition events into transcribed genes produce a lacE, chimera. However, the random transposition products that generate active B-galactosidase can be visualized and quantitated due to the blue stain that results from cleavage of galactose residues (Fig. 3). Reproducible patterns, which are caused by temporal variation in MudII1681 transposition rates, have been observed in many bacterial strains. Some strains produce B-galactosidase fusions near the colony center, giving a blue bull's-eye pattern with white edges like those in Fig. 3b. However, most E. coil strains produce colonies with a white center and concentric blue rings and develop wedgeshaped blue sectors (Fig. 3c; Ref. 7). To date, the most significant determinants of the MudII1681 transposition rate have been found to be the nutritional conditions on the plate and regulatory
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Figure 3 Mudl11681 transposition in S. typhimurium and E. coil (a) This sectored colony pattern was caused by a duplication of the genes between minutes 45-80 of the standard S. typhimurium map11. Cells carrying the duplication have a stimulated Mu transposition (Smt) phenotype. (b) Cells streaked from the dark blue sector make two types of colonies. If the 45-80 minute duplication is present, the colonies spread rapidly, appear flattened, and have dark blue centers caused by frequent Mudl11681 transposition. The smaller, light-staining colonies have lost the duplicated region by homologous recombination. (Picture and information courtesy of R. Sonti and J. Roth, University of Utah.) (c) An E. coil colony producing sectors with new growth and enzyme phenotypes. This colony was grown from a spot of 105 cells containing a silent copy of Mudl11681. Dark rings and sectors have high levels of Mudl11681 replication. See Ref. 7 for details. (Picture courtesy of J. Shapiro, University of Chicago.)
genes in the host. Transposition rings and sectored patterns appear in colonies of most strains only after several days of growth on minimal plates with a single limited carbon source. Under these circumstances, Mu transposition can be exceedingly variable.
plication have a selective advantage on media containing low concentrations of a carbon source. Other examples of punctuated transposon activity are known. One case, reminiscent of McClintock's elements in maize, is activation of genes for degrading ~-glucosides in E. coll. No repressor Genetics of punctuated transposition or activator protein specific for the bgl A sector . signifies an inherited operon is known, but Reynolds et al. change, so the Mu phenotype provides found that transposon insertions near an opportunity to study genetic control the bgl promoter activate this cryptic of transposition. Mutants have been operon 12. The transposons, present at isolated from blue sectors that retain a other locations in the host chromohypertransposition phenotype and some, moved near bgl only after culform many new ~galactosidase fusions tures were placed under selective conduring growth. Genetic analyses are ditions and not during growth in rich just beginning, but two genetic changes media. Hall studied a case in which a increase the transposition frequency of disruptive copy of IS103 was present in Mu. First, mutants with an inactive hns one of the bgl operon genes. Efficient gene exhibit an increased Mudl11681 IS103 excision occurred after cells were transposition frequency during carbon incubated for several days under seleclimitation 8. The hns gene encodes an tion 13. These examples show that carintriguing chromosome-associated pro- bon starvation can modulate transpotein responsible for silencing numerous sition frequencies of elements unreunrelated operons in enteric bacteria 9,~°. lated to Mu. A second change that stimulates Mu transposition is duplication of the chro- Transposition in dead cells mosomal segment spanning minute 45 Many cells die when E. coil cultures to minute 80 of the S. typhimurium or enter stationary phase, and cells survivE. coil chromosome (Fig. 3a and b; R. V. ing in stationary phase were recently Sonti and J. R. Roth, pers. commun.). shown to kill log phase cells TM. A dead Duplication of this 35-minute chromo- cell is defined as one that has lost the some arc occurs repeatedly using a ability to form a new colony, although precise chromosomal joint point u. In biochemical activity may be retained. addition to having a high Mu trans- Surprisingly, studies of mini-MudlI1681 position rate, strains bearing this du- transposition revealed that the bulk of
gene-fusion products were in noncolony-forming cellsL Southern blots showed that hundreds of Mu-driven rearrangements were present in the blue sectors of a colony7. In contrast, MudI11681 was found only at the original position in white sectors. When cells from blue or white sectors were inoculated into rich medium, grown overnight and then examined by Southern blot analysisl only a single Mu located at the original position could be detected. Since there is no obvious way to delete Mu from a rearranged chromosome, the potential for hypertransposition must have been carried by colony-forming cells, and the bulk of chimeric proteins must have been made in dead cells that can synthesize RNA, protein and DNA (since Mu DNA is amplified). Can rearranged genes in dead cells affect the development of a colonyforming population? If true, this could modify the darwinian emphasis on survival of the fittest. There is currently no clear answer to this question with regard to cells that have undergone hypertransposition with Mu. However, there are examples of dead cells that benefit adapting bacterial cultures. In cyanobacteria, a terminally differentiated heterocyst is produced when cells become starved of nitrogen ~s. The heterocyst is a permissive anaerobic compartment for redox enzymes that are poisoned by oxygen. In Anabaena it is
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estimated that 1000 genes are differentially expressed in the non-dividing heterocyst. Another notable example is Bacillus subtilis, where starvation induces a differentiation leading to a heat-resistant spore. During sporulation, unequal cytokinesis leads to formation of two compartments - one containing the forespore and the other housing a dead mother cell. In both Anabaena heterocysts 15 and the noncolony-forming B. subtilis mother celP 6, chromosomal deletions occurring during the developmental program lead to formation of proteins needed for specific differentiated functions. Thus, genes rearranged within dead cells can have significant impact on their sibling neighbors.
P22, ~., P1 and Mu. An interesting example in photosynthetic bacteria is the genetic transfer factor (GTF) TM,which is a phage-like agent of Rhodobacter capsulatus that cuts the host chromosome to uniform size pieces and packages the DNA into a tiny phage-like particle. The head is too small to package DNA of the virus-like element, but cultures harboring GTF are continually infected by DNA from dead sister cells. Conjugation is the third mechanism for transferring DNA in bacteria harboring plasmids. Many plasmids, like F, make a special pilus for attaching to its mating partner. However, DNA transfer is not limited purely to exchanges involving bacteria; F+ strains of E. coil also transfer DNA to yeast cells ~.
Transfer of dead-cell genes
Do dead-cell mutations count?
If transposition rates vary in response to external or internal genomic shocks and cultures harboring Mu make cells with extensively rearranged chromosomes, can bacteria inherit rearranged dead-cell genes? If so, what are the consequences of hypermutation in dead cells? In bacteria, three mechanisms facilitate intercellular DNA transfer: transformation, transduction and conjugation. In transformation and transduction, DNA moves from dead cells to colonyforming cells. Some striking examples illustrate these phenomena. Neisseria gonorrhoeae changes its cell-surface antigens to escape an immune response of the host. One of the dominant surface antigens is pilin. Multiple pilin genes have different antigenic character, but only one type of pilin is made by a single cell because only one gene resides in the expression locus on the bacterial chromosome. Pilin types change over time because alternative genes present at silent loci will recombine non-reciprocally at the expression site. One path of recombination leading to pilin switching is via DNA transformation ~7. When gonococci die and lyse, colony-forming cells take DNA from the dead bacteria and insert alternative alleles into the expression locus, converting the cell from one antigen type to another. This transfer reactio.n is facilitated by an extremely efficient DNA transformation system. In this case, cell death precedes. (triggers?) one type of antigen switching. A second mechanism for transferring DNA from terminal cells is transduction. Phages capable of transducing E. coli or S. typhimurium genes include
What could be the consequence of transferring hypermutated DNA from dead cells to colony-forming bacteria? There has been recent interest in a process called 'directed' mutation. Directed mutations occur as punctuated events during complex physiological selections. Results from two types of experiments suggest that environmental conditions can direct the recovery of certain mutants. Cairns et al. 2°, Cairns and Foster21and Hall22, find that bacteria bearing nonsense or frameshift mutations in different lacZ genes show mutation rates to lactose-utilization that depend on growth conditions. One explanation is hypermutation, which is feasible since inducible mutator systems exist in E. coli. However, the mutation rate in non-selected genes was not highly elevated as a consequence of a lactose selection. Thus, recovery of mutants that restore the lac operon function seemed to be directed by the selection. Shapiro's ara-lac fusion experiment 6 described above is similar. Cairns et aL 2° repeated this selection and found that Mu-driven rearrangements allowing fermentation of lactose in the presence of arabinose were not recovered if either sugar was missing from the medium. Again, hypermutation by Mu induction does not explain the result because Mu transposition should not fuse the lac and ara operons only when both substrates are present. Several theories have been proposed to explain the apparent direction of mutant formation in these systems 23-2s. These explanations assume that mutations are generated in colony-forming units that eventually exhibit the trait. However, if mutations arise through
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hypermutation in dead cells and can be transferred to neighboring cells, there would be less mystery surrounding directed mutagenesis. According to this alternative scenario, starvation induces hypermutation of a fraction of the deadcell population. Mutagenesia occurs randomly but with frequencies at least 1000-fold higher than the spontaneous mutation rate in colony-forming cells. Once a beneficial mutation is fixed, the dead cell acquires a new energy source, which allows it to activate DNA transfer machinery and mobilize the active genes to a neighbor that can form colonies. Thus, after fixation, the population appears to have undergone a directed mutation. In this case, only DNA containing the adaptive mutation would have to be placed into the colony-forming recombinant. DNA transfer could occur by any mechanism. Strains used by Cairns et al. ~° have an F plasmid and Shapiro's strain 6 has Mu. What mechanisms for responding to environmental challenges are hidden within the complex evolutionary history of a modern organism? The ability to punctuate periods of genetic equilibrium with episodes of highly active variation may be a great advantage for organisms. Temporally modulated transposition and directed mutagenesis suggest that organisms have a more active role in creating diversity than previously thought. And if adaptive mutations pass from hypermutated dead cells to colony-forming cells, then many assumptions about genetic selection warrant re-evaluation. While the theory of natural selection is holding up amazingly well, it was proposed 100 years before the structure of DNA was understood. New discoveries mean that darwinian theory itself must evolve. This does not denigrate Darwin's original thinking, but it is a natural outgrowth of the explosion of new information about regulatory mechanisms, where no important biological pathway seems to be unregulated or completely random. In this regard, the words of another British author seem relevant: 'There are more things in heaven and earth, Horatio, than are dreamt of in your philosophy' 26.
Acknowledgements I thank J. Shapiro, R. Sonti and J. Roth for photographs and for sharing information about strains. Thanks also to J. Shapiro, T. Elliott and V. McGovern
TIBS 1 7 - J U N E 1 9 9 2
for constructive c o m m e n t s on t h e manuscript. Work in the Higgins laborat o r y is s u p p o r t e d by NSF grant DMB 9122048.
References 1 Drake, J. W. (1991) Annu. Rev. Genet. 25, 125-146 2 EIdredge, N. and Gould, S. J. (1972) in Models in Paleobiology(Schopf, T. J. M., ed.), pp. 88-115, Freeman, Cooper and Co. 3 Dorit, R. L., Schoenbach, L. and Gilbert, W. (1990) Science 250, 1377-1382 4 McClintock, B. (1984) Science 226, 792-801 5 Pato, M. L. (1989) in Mobile DNA (Berg, D. and Howe, M. M., eds), pp. 23-52, American Society for Microbiology
6 Shapiro, J. A. (1984) Mol. Gen. Genet. 194, 79-90 7 Shapiro, J. A. and Higgins, N. P. (1989) J. Bacteriol. 171, 5975-5986 8 Falconi, M. et al. (1991) New Biol. 3, 615-625 9 Hulton, C. S. J. et al. (1990) Cell 63, 631-642 10 May, G. et al. (1990) Mol. Gen. Genet. 224, 81-90 11 Sonti, R. V. and Roth, J. R. (1989) Genetics 123, 19-28 12 Reynolds, A. E., Mahadevan, S., LeGrice, S. F. and Wright, A. (1986) J. Mol. Biol. 191, 85-95 13 Hall, B. G. (1988) Genetics 120, 887-897 14 Kolter, R. (1992) Life and Death in Stationary Phase, Am. Soc. Microbiol. News 58, 75-79 15 Haselkorn, R. (1989) in Mobile DNA (Berg, D. and Howe, M. M., eds), pp. 735-742, American Society for Microbiology 16 Stragier, P., Kunkel, B., Kroos, L. and Losick, R.
(1989) Science 243, 507-512 17 Seifert, H. S. et al. (1988) Nature 336, 392-395 18 Scolnik, P. A. and Marrs, B. L. (1987) Annu. Rev. Microbiol. 41, 703-726 19 Heineman, J. A. and Sprague, G. F. (1989) Nature 340, 205-209 20 Cairns, J., Overbaugh, J. and Miller, S. (1988) Nature 335, 142-145 21 Cairns, J. and Foster, P. (1991) Genetics 128, 695-701 22 Hall, B. (1991) Proc. Natl Acad. Sci. USA 88,
5882-5886 23 Davis, B. D. (1989) Proc. Natl Acad. Sci. USA
86, 5005-5009 24 Stahl, F. W. (1988) Nature 335, 112-113 25 Shapiro, J. A. and Leach, D. (1990) Genetics
126, 293-299 26 Shakespeare, W. (1954) The Tragedy of Hamlet, Prince of Denmark I,v, p. 167, Folio Society
LETTERS Are molecular filters really necessary? The article by Hopkins 1 places great emphasis on the role of molecular filters in membrane protein trafficking. The molecular filters he refers to are supposed to be those originally proposed and documented by Brets'cher 2'3. However, the concept of the molecular filter as defined by Bretscher differs in at least one fundamental respect from that invoked by Hopkins. Bretscher proposed that the molecular filter was a device for preventing plasma membrane proteins from entering domains such as the coated pit~ so that these structures could preferentially internalize lipid for recycling and maintaining a directed lipid flow on the plasma membrane. This extreme concept was moderated to the idea that the coated pit operated to exclude most membrane proteins from the coated regions 3. Even in this attenuated form, however, the molecular filter was essentially a negative device for excluding membrane proteins from specific domains. In contrast, Hopkins employs the term almost entirely in the positive sense, that is, as a device for concentrating membrane proteins at specific sites, although this was not the sense in which it was 'originally proposed and documented by Bretscher'. Is our understanding of membrane protein sorting enhanced by the above concept, given the confusion surrounding the use of the term 'molecular filter'? In the positive filtering idea, it is unclear whether there is a special process involved or whether it is simply a different name for a well-known process,
in which case introducing a new term is confusing and unhelpful. As described by Hopkins, the coated-pit positive filter is thought to operate through a shell of adaptor complexes between the lipid bilayer of the membrane and the clathrin lattice, and that these complexes bind trafficking proteins. The underlying physicochemical principle leading to this chain of events is the well-known one of preferential affinity between macromolecules, which operates in a host of familiar processes such as the assembly of budding viruses, secretory granules and even organelles. It involves complex simultaneous interaction between arrays of macromolecules in a manner analogous to crystallization in solution. These latter processes exhibit the same selectivity in their assembly in that they focus specific molecular species (and exclude others) at a particular site, but it has never been necessary to refer to them as possessing a special filtering mechanism. Thus the use of the term filter in this context is unnecessary and confusing. This then leaves the question of the advantage of using the term in the context of the apparent exclusion of some components from the coated regions, i.e. the negative filter, as originally proposed. The evidence for the existence of selective exclusion is at best controversial 4 and could reasonably be treated with scepticism. However, even if this evidence is taken as accepted, is it necessary to invoke a special device to explain the observations? The central issue here is the state of the membrane in the coated regions. If the possibility that the membrane consists of closely packed proteins and iipids with some proteins
exhibiting a higher affinity for the membrane/adaptin/clathrin lattice is correct, it follows that others will be excluded for steric reasons, just as the majority of membrane proteins are excluded from the budding site of viruses. In such a system the exclusion observed does not involve any physicochemical principle other than the affinity of the membrane proteins for each other and/or the adaptin/clathrin lattice. In other words, there is no filter because the exclusion can be effected by the membrane molecules themselves. The consequence of these considerations is that the selectivity observed during protein trafficking along cellular pathways is the direct consequence of the selective affinity of proteins for each other at different points in the pathway. The exclusion of particular components is not the result of a filter, rather the simple consequence of the fact that there are spatial limitations at the assembly sites. The underlying principle is already well known in the secretory pathway, since secretory granules are believed to assemble in this way, i.e. condensation sorting 5. We have proposed that the selective assembly into supramolecular structures of varying stability also contributes to the sorting of resident luminal proteins such as reticuloplasmins in the ER6. Given these considerations, it seems to me that the term 'filtration' should be abandoned altogether in the context of selective membrane protein trafficking.
GORDON KOCH Laboratory of Molecular Biology, Hills Road, Cambridge, UK CB2 2QH.
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EVER SINCE DARWIN proposed his theory of natural selection, biological evolution has been viewed as a process with two distinct stages. First, variation accumulates in a population of related organisms. This is now thought to occur through random mutation and recombination of DNA sequences. Second, competition among members of the variant population leads the bestadapted individuals to thrive and displace less well-adapted individuals. This principle, that evolutionary processes have a basal rate of variation and selection, is not disputed and, in fact, underpins many genetic procedures in bacteria, yeast and higher eukaryotes. However, debate about rates of variation and mechanisms for generating mutants continuesL Darwin suggested that evolution works mainly through gradual diversification, which allows useful adaptations to be incorporated slowly but ceaselessly over long time-frames. Darwin recognized that evolution accelerates when organisms move into novel environments, but there was no visible mechanism. In 1972, Eldredge and Gould proposed an alternative to evolutionary gradualism called punctuated equilibrium 2, which accounted for two observations in paleontology. First, many species have been remarkably stable - species life spans of more than ten million years are not unusual in the geological record. However, there is also evidence of the appearance of many new species during a very short time span, with no sign of a gradual series of intermediaries. The proposal of Eldredge and Gould that the rate of evolution may be highly variable stimulated new ideas about the role of catastrophic extinctions in evolution. It also suggested that evidence for both punctuation and equilibrium in variation could be found in present-day biological systems. Most molecular theories about variation assume that evolution is driven
Death and transfiguration among bacteria
When bacteria are placed in sub-optimal environments, they can respond by increasing the frequency of mutants created by base substitution, frame-shift and transposition mutations. Also, during periods of restrictive growth, 'dead' bacterial cells may transfer genetic material to neighboring colony-forming cells. This can be beneficial, resulting in a heterogeneous population that may exhibit differentiation and even produce killer cells. These discoveries reveal several conundrums about the control of an organism over mutations and the supposed randomness of genetic variation.
by random changes occurring in reproductive populations. Few, if any, scientists challenge the fact that there is a basal rate of mutation. However, in addition to constant slow change, mechanisms for dramatic changes have been uncovered. For example, moveable segments of DNA known as transposons influence chromosome structure and function through transposition between chromosomes in all cellular organisms. They alter gene activity by modifying transcriptional regulation, by disrupting active genes and by creating new genes with novel activities. Gilbert suggests that re-assorting as few as 2000 small genes of 40-60 amino acids (exons) might be sufficient to generate the complete repertoire of modern proteins 3. Transposition provides a mechanism for this DNA shuffling.
Detectingtransposons
When do transposons act, and do they move at fixed rates or with punctuated rhythms? The first indication that transposition rates fluctuate was in the 1940s. McClintock discovered transposons while examining maize plants with special chromosomes that often break during the cell divisions that follow meiosis. In several cases, a transposon changed anthocyanin pigment N. P. Higgins is at the Department of production only in the aleurone layer of Biochemistry, University of Alabama at a corn kernel, which restricted the Birmingham, 464 Basic Health Sciences transposition to a period covering only Building, 1918 University Blvd, UAB Station, Birmingham, AL 35294-0005, USA. two cell mitoses after introduction of a © 1992,ElsevierSciencePublishers, (UK) 0376-5067/92/$05.00
chromatid break 4. Thus, one response of maize to a chromosome break was an increased transposition rate. This revolutionary discovery was not widely appreciated until transposons were found years later in bacteria. However, McClintock recognized two important properties of this unexpected genetic behavior: genes in chromosomes can be surprisingly mobile and they can be induced to move with a genomic shock or challenge. Obtaining temporal information about transposon movement is difficult in most traditional genetic systems. However, novel properties of phage Mu permit transposition events to be monitored in living bacterial populations. Mu is both a transposon and a temperate virus. It is the most efficient transposon known, capable of replicating itself to new chromosomal sites in a single cell 100 times an hour. Mu DNA is not cut out of the host chromosome during transposition, so it scrambles the host gene order by making deletions and inversions and by joining together distant segments of the bacterial chromosome 5 (see Fig. 1). However, Mu may also remain silent and at the same position for many generations. One way to examine the timing of Mu transposition is to track subsets of selectable Mu-driven rearrangements that benefit the host. Shapiro studied a notable case in E. coil6. A Mu prophage was located between two non-functional
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t10 6 Figure 1
Mu scrambles the host chromosome. Bacteriophage Mu is depicted as a bold arrow along a circular chromosome with gene order A-J. The consequence of replicative Mu transposition to a second site within the bacterial chromosome is either formation of a deletion, if Mu inserts in one orientation (left arrow), or an inversion, ifMu inserts in the opposite orientation (right arrow). Subsequent Mu transposition events may form more circular deletions or reform the large circle. An example of one gene order after nine cycles of Mu replication is shown in the bottom circle. In the lytic cycle Mu transposes to 100 positions within the host chromosome in an hour.
operons. On one side was an inactive lactose operon (lac) that contained functional genes for transporting and hydrolysing lactose but lacked a transcription promoter. On the other side of Mu was an arabinose operon (ara) bearing functional arabinose-sensitive
transcription control elements and an enzyme defective in its ability to hydrolyse arabinose. Thus, the parent strain would not grow on plates containing both arabinose and lactose as the carbon sources. However, Mu can generate ara-lac fusions that allow fermentation
R
lacZ L.
Y
I
lac g e n e s
A
Kan )
B k
A
i
c )
Mu g e n e s
Rgure 2 Genetic structure of Mudl1168I, a non-plaque-forming derivative of Mu carrying the Mu c repressor, the A transposase and B replication protein. A selectable neomycin phosphotransferase gene from Tn903 (Kan) is near the center of the element. Arrows below the genes indicate the direction of transcription. Transcription of lac genes must be initiated outside the transposon (hyphenated arrow) and production of functional ~-galactosidase requires fusion of lacZ in the proper reading frame to a protein outside the eJement.
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of lactose in the presence of inducing levels of arabinose. In these rearranged chromosomes, Mu transposition connected the lac genes to the ara regulatory sequences, so these strains grew only when both lactose and arabinose were present. Ara-lac fusions, did not appear on plates with an abundant carbon source. However, after starvation on minimal plates containing both arabinose and lactose, ara-lac-utilizing colonies were frequently detected. After several days, hundreds of independent colonies appeared on a single plate. Shapiro's results and careful analysis clearly showed that ara-lac fusion formation was caused by punctuated transposon-driven genetic rearrangements and that the carbon limitation history of the culture was also important. A more general way to detect transposition in colonies is with non-plaqueforming Mu derivatives like MudIl1681 (Ref. 7). MudII1681 contains the viral DNA sequences and proteins needed to regulate and mobilize a phage genome. but it lacks phage structural genes (A in Fig. 2). A lacZ gene that lacks both a transcription promoter and a ribosomebinding site is situated towards the left. Functional B-galactosidase (E.C. 3.2.1.23) can be made only after MudII1681 transposes into the correct reading frame of another gene and creates a protein chimera. The amino-terminal domain of the chimera comes from a host protein; the carboxy-terminal segment is an active domain of lacZ. Because Mu may land in either orientation in any of three reading frames, only one-sixth of all transposition events into transcribed genes produce a lacE, chimera. However, the random transposition products that generate active B-galactosidase can be visualized and quantitated due to the blue stain that results from cleavage of galactose residues (Fig. 3). Reproducible patterns, which are caused by temporal variation in MudII1681 transposition rates, have been observed in many bacterial strains. Some strains produce B-galactosidase fusions near the colony center, giving a blue bull's-eye pattern with white edges like those in Fig. 3b. However, most E. coil strains produce colonies with a white center and concentric blue rings and develop wedgeshaped blue sectors (Fig. 3c; Ref. 7). To date, the most significant determinants of the MudII1681 transposition rate have been found to be the nutritional conditions on the plate and regulatory
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&
Figure 3 Mudl11681 transposition in S. typhimurium and E. coil (a) This sectored colony pattern was caused by a duplication of the genes between minutes 45-80 of the standard S. typhimurium map11. Cells carrying the duplication have a stimulated Mu transposition (Smt) phenotype. (b) Cells streaked from the dark blue sector make two types of colonies. If the 45-80 minute duplication is present, the colonies spread rapidly, appear flattened, and have dark blue centers caused by frequent Mudl11681 transposition. The smaller, light-staining colonies have lost the duplicated region by homologous recombination. (Picture and information courtesy of R. Sonti and J. Roth, University of Utah.) (c) An E. coil colony producing sectors with new growth and enzyme phenotypes. This colony was grown from a spot of 105 cells containing a silent copy of Mudl11681. Dark rings and sectors have high levels of Mudl11681 replication. See Ref. 7 for details. (Picture courtesy of J. Shapiro, University of Chicago.)
genes in the host. Transposition rings and sectored patterns appear in colonies of most strains only after several days of growth on minimal plates with a single limited carbon source. Under these circumstances, Mu transposition can be exceedingly variable.
plication have a selective advantage on media containing low concentrations of a carbon source. Other examples of punctuated transposon activity are known. One case, reminiscent of McClintock's elements in maize, is activation of genes for degrading ~-glucosides in E. coll. No repressor Genetics of punctuated transposition or activator protein specific for the bgl A sector . signifies an inherited operon is known, but Reynolds et al. change, so the Mu phenotype provides found that transposon insertions near an opportunity to study genetic control the bgl promoter activate this cryptic of transposition. Mutants have been operon 12. The transposons, present at isolated from blue sectors that retain a other locations in the host chromohypertransposition phenotype and some, moved near bgl only after culform many new ~galactosidase fusions tures were placed under selective conduring growth. Genetic analyses are ditions and not during growth in rich just beginning, but two genetic changes media. Hall studied a case in which a increase the transposition frequency of disruptive copy of IS103 was present in Mu. First, mutants with an inactive hns one of the bgl operon genes. Efficient gene exhibit an increased Mudl11681 IS103 excision occurred after cells were transposition frequency during carbon incubated for several days under seleclimitation 8. The hns gene encodes an tion 13. These examples show that carintriguing chromosome-associated pro- bon starvation can modulate transpotein responsible for silencing numerous sition frequencies of elements unreunrelated operons in enteric bacteria 9,~°. lated to Mu. A second change that stimulates Mu transposition is duplication of the chro- Transposition in dead cells mosomal segment spanning minute 45 Many cells die when E. coil cultures to minute 80 of the S. typhimurium or enter stationary phase, and cells survivE. coil chromosome (Fig. 3a and b; R. V. ing in stationary phase were recently Sonti and J. R. Roth, pers. commun.). shown to kill log phase cells TM. A dead Duplication of this 35-minute chromo- cell is defined as one that has lost the some arc occurs repeatedly using a ability to form a new colony, although precise chromosomal joint point u. In biochemical activity may be retained. addition to having a high Mu trans- Surprisingly, studies of mini-MudlI1681 position rate, strains bearing this du- transposition revealed that the bulk of
gene-fusion products were in noncolony-forming cellsL Southern blots showed that hundreds of Mu-driven rearrangements were present in the blue sectors of a colony7. In contrast, MudI11681 was found only at the original position in white sectors. When cells from blue or white sectors were inoculated into rich medium, grown overnight and then examined by Southern blot analysisl only a single Mu located at the original position could be detected. Since there is no obvious way to delete Mu from a rearranged chromosome, the potential for hypertransposition must have been carried by colony-forming cells, and the bulk of chimeric proteins must have been made in dead cells that can synthesize RNA, protein and DNA (since Mu DNA is amplified). Can rearranged genes in dead cells affect the development of a colonyforming population? If true, this could modify the darwinian emphasis on survival of the fittest. There is currently no clear answer to this question with regard to cells that have undergone hypertransposition with Mu. However, there are examples of dead cells that benefit adapting bacterial cultures. In cyanobacteria, a terminally differentiated heterocyst is produced when cells become starved of nitrogen ~s. The heterocyst is a permissive anaerobic compartment for redox enzymes that are poisoned by oxygen. In Anabaena it is
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estimated that 1000 genes are differentially expressed in the non-dividing heterocyst. Another notable example is Bacillus subtilis, where starvation induces a differentiation leading to a heat-resistant spore. During sporulation, unequal cytokinesis leads to formation of two compartments - one containing the forespore and the other housing a dead mother cell. In both Anabaena heterocysts 15 and the noncolony-forming B. subtilis mother celP 6, chromosomal deletions occurring during the developmental program lead to formation of proteins needed for specific differentiated functions. Thus, genes rearranged within dead cells can have significant impact on their sibling neighbors.
P22, ~., P1 and Mu. An interesting example in photosynthetic bacteria is the genetic transfer factor (GTF) TM,which is a phage-like agent of Rhodobacter capsulatus that cuts the host chromosome to uniform size pieces and packages the DNA into a tiny phage-like particle. The head is too small to package DNA of the virus-like element, but cultures harboring GTF are continually infected by DNA from dead sister cells. Conjugation is the third mechanism for transferring DNA in bacteria harboring plasmids. Many plasmids, like F, make a special pilus for attaching to its mating partner. However, DNA transfer is not limited purely to exchanges involving bacteria; F+ strains of E. coil also transfer DNA to yeast cells ~.
Transfer of dead-cell genes
Do dead-cell mutations count?
If transposition rates vary in response to external or internal genomic shocks and cultures harboring Mu make cells with extensively rearranged chromosomes, can bacteria inherit rearranged dead-cell genes? If so, what are the consequences of hypermutation in dead cells? In bacteria, three mechanisms facilitate intercellular DNA transfer: transformation, transduction and conjugation. In transformation and transduction, DNA moves from dead cells to colonyforming cells. Some striking examples illustrate these phenomena. Neisseria gonorrhoeae changes its cell-surface antigens to escape an immune response of the host. One of the dominant surface antigens is pilin. Multiple pilin genes have different antigenic character, but only one type of pilin is made by a single cell because only one gene resides in the expression locus on the bacterial chromosome. Pilin types change over time because alternative genes present at silent loci will recombine non-reciprocally at the expression site. One path of recombination leading to pilin switching is via DNA transformation ~7. When gonococci die and lyse, colony-forming cells take DNA from the dead bacteria and insert alternative alleles into the expression locus, converting the cell from one antigen type to another. This transfer reactio.n is facilitated by an extremely efficient DNA transformation system. In this case, cell death precedes. (triggers?) one type of antigen switching. A second mechanism for transferring DNA from terminal cells is transduction. Phages capable of transducing E. coli or S. typhimurium genes include
What could be the consequence of transferring hypermutated DNA from dead cells to colony-forming bacteria? There has been recent interest in a process called 'directed' mutation. Directed mutations occur as punctuated events during complex physiological selections. Results from two types of experiments suggest that environmental conditions can direct the recovery of certain mutants. Cairns et al. 2°, Cairns and Foster21and Hall22, find that bacteria bearing nonsense or frameshift mutations in different lacZ genes show mutation rates to lactose-utilization that depend on growth conditions. One explanation is hypermutation, which is feasible since inducible mutator systems exist in E. coli. However, the mutation rate in non-selected genes was not highly elevated as a consequence of a lactose selection. Thus, recovery of mutants that restore the lac operon function seemed to be directed by the selection. Shapiro's ara-lac fusion experiment 6 described above is similar. Cairns et aL 2° repeated this selection and found that Mu-driven rearrangements allowing fermentation of lactose in the presence of arabinose were not recovered if either sugar was missing from the medium. Again, hypermutation by Mu induction does not explain the result because Mu transposition should not fuse the lac and ara operons only when both substrates are present. Several theories have been proposed to explain the apparent direction of mutant formation in these systems 23-2s. These explanations assume that mutations are generated in colony-forming units that eventually exhibit the trait. However, if mutations arise through
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hypermutation in dead cells and can be transferred to neighboring cells, there would be less mystery surrounding directed mutagenesis. According to this alternative scenario, starvation induces hypermutation of a fraction of the deadcell population. Mutagenesia occurs randomly but with frequencies at least 1000-fold higher than the spontaneous mutation rate in colony-forming cells. Once a beneficial mutation is fixed, the dead cell acquires a new energy source, which allows it to activate DNA transfer machinery and mobilize the active genes to a neighbor that can form colonies. Thus, after fixation, the population appears to have undergone a directed mutation. In this case, only DNA containing the adaptive mutation would have to be placed into the colony-forming recombinant. DNA transfer could occur by any mechanism. Strains used by Cairns et al. ~° have an F plasmid and Shapiro's strain 6 has Mu. What mechanisms for responding to environmental challenges are hidden within the complex evolutionary history of a modern organism? The ability to punctuate periods of genetic equilibrium with episodes of highly active variation may be a great advantage for organisms. Temporally modulated transposition and directed mutagenesis suggest that organisms have a more active role in creating diversity than previously thought. And if adaptive mutations pass from hypermutated dead cells to colony-forming cells, then many assumptions about genetic selection warrant re-evaluation. While the theory of natural selection is holding up amazingly well, it was proposed 100 years before the structure of DNA was understood. New discoveries mean that darwinian theory itself must evolve. This does not denigrate Darwin's original thinking, but it is a natural outgrowth of the explosion of new information about regulatory mechanisms, where no important biological pathway seems to be unregulated or completely random. In this regard, the words of another British author seem relevant: 'There are more things in heaven and earth, Horatio, than are dreamt of in your philosophy' 26.
Acknowledgements I thank J. Shapiro, R. Sonti and J. Roth for photographs and for sharing information about strains. Thanks also to J. Shapiro, T. Elliott and V. McGovern
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for constructive c o m m e n t s on t h e manuscript. Work in the Higgins laborat o r y is s u p p o r t e d by NSF grant DMB 9122048.
References 1 Drake, J. W. (1991) Annu. Rev. Genet. 25, 125-146 2 EIdredge, N. and Gould, S. J. (1972) in Models in Paleobiology(Schopf, T. J. M., ed.), pp. 88-115, Freeman, Cooper and Co. 3 Dorit, R. L., Schoenbach, L. and Gilbert, W. (1990) Science 250, 1377-1382 4 McClintock, B. (1984) Science 226, 792-801 5 Pato, M. L. (1989) in Mobile DNA (Berg, D. and Howe, M. M., eds), pp. 23-52, American Society for Microbiology
6 Shapiro, J. A. (1984) Mol. Gen. Genet. 194, 79-90 7 Shapiro, J. A. and Higgins, N. P. (1989) J. Bacteriol. 171, 5975-5986 8 Falconi, M. et al. (1991) New Biol. 3, 615-625 9 Hulton, C. S. J. et al. (1990) Cell 63, 631-642 10 May, G. et al. (1990) Mol. Gen. Genet. 224, 81-90 11 Sonti, R. V. and Roth, J. R. (1989) Genetics 123, 19-28 12 Reynolds, A. E., Mahadevan, S., LeGrice, S. F. and Wright, A. (1986) J. Mol. Biol. 191, 85-95 13 Hall, B. G. (1988) Genetics 120, 887-897 14 Kolter, R. (1992) Life and Death in Stationary Phase, Am. Soc. Microbiol. News 58, 75-79 15 Haselkorn, R. (1989) in Mobile DNA (Berg, D. and Howe, M. M., eds), pp. 735-742, American Society for Microbiology 16 Stragier, P., Kunkel, B., Kroos, L. and Losick, R.
(1989) Science 243, 507-512 17 Seifert, H. S. et al. (1988) Nature 336, 392-395 18 Scolnik, P. A. and Marrs, B. L. (1987) Annu. Rev. Microbiol. 41, 703-726 19 Heineman, J. A. and Sprague, G. F. (1989) Nature 340, 205-209 20 Cairns, J., Overbaugh, J. and Miller, S. (1988) Nature 335, 142-145 21 Cairns, J. and Foster, P. (1991) Genetics 128, 695-701 22 Hall, B. (1991) Proc. Natl Acad. Sci. USA 88,
5882-5886 23 Davis, B. D. (1989) Proc. Natl Acad. Sci. USA
86, 5005-5009 24 Stahl, F. W. (1988) Nature 335, 112-113 25 Shapiro, J. A. and Leach, D. (1990) Genetics
126, 293-299 26 Shakespeare, W. (1954) The Tragedy of Hamlet, Prince of Denmark I,v, p. 167, Folio Society
LETTERS Are molecular filters really necessary? The article by Hopkins 1 places great emphasis on the role of molecular filters in membrane protein trafficking. The molecular filters he refers to are supposed to be those originally proposed and documented by Brets'cher 2'3. However, the concept of the molecular filter as defined by Bretscher differs in at least one fundamental respect from that invoked by Hopkins. Bretscher proposed that the molecular filter was a device for preventing plasma membrane proteins from entering domains such as the coated pit~ so that these structures could preferentially internalize lipid for recycling and maintaining a directed lipid flow on the plasma membrane. This extreme concept was moderated to the idea that the coated pit operated to exclude most membrane proteins from the coated regions 3. Even in this attenuated form, however, the molecular filter was essentially a negative device for excluding membrane proteins from specific domains. In contrast, Hopkins employs the term almost entirely in the positive sense, that is, as a device for concentrating membrane proteins at specific sites, although this was not the sense in which it was 'originally proposed and documented by Bretscher'. Is our understanding of membrane protein sorting enhanced by the above concept, given the confusion surrounding the use of the term 'molecular filter'? In the positive filtering idea, it is unclear whether there is a special process involved or whether it is simply a different name for a well-known process,
in which case introducing a new term is confusing and unhelpful. As described by Hopkins, the coated-pit positive filter is thought to operate through a shell of adaptor complexes between the lipid bilayer of the membrane and the clathrin lattice, and that these complexes bind trafficking proteins. The underlying physicochemical principle leading to this chain of events is the well-known one of preferential affinity between macromolecules, which operates in a host of familiar processes such as the assembly of budding viruses, secretory granules and even organelles. It involves complex simultaneous interaction between arrays of macromolecules in a manner analogous to crystallization in solution. These latter processes exhibit the same selectivity in their assembly in that they focus specific molecular species (and exclude others) at a particular site, but it has never been necessary to refer to them as possessing a special filtering mechanism. Thus the use of the term filter in this context is unnecessary and confusing. This then leaves the question of the advantage of using the term in the context of the apparent exclusion of some components from the coated regions, i.e. the negative filter, as originally proposed. The evidence for the existence of selective exclusion is at best controversial 4 and could reasonably be treated with scepticism. However, even if this evidence is taken as accepted, is it necessary to invoke a special device to explain the observations? The central issue here is the state of the membrane in the coated regions. If the possibility that the membrane consists of closely packed proteins and iipids with some proteins
exhibiting a higher affinity for the membrane/adaptin/clathrin lattice is correct, it follows that others will be excluded for steric reasons, just as the majority of membrane proteins are excluded from the budding site of viruses. In such a system the exclusion observed does not involve any physicochemical principle other than the affinity of the membrane proteins for each other and/or the adaptin/clathrin lattice. In other words, there is no filter because the exclusion can be effected by the membrane molecules themselves. The consequence of these considerations is that the selectivity observed during protein trafficking along cellular pathways is the direct consequence of the selective affinity of proteins for each other at different points in the pathway. The exclusion of particular components is not the result of a filter, rather the simple consequence of the fact that there are spatial limitations at the assembly sites. The underlying principle is already well known in the secretory pathway, since secretory granules are believed to assemble in this way, i.e. condensation sorting 5. We have proposed that the selective assembly into supramolecular structures of varying stability also contributes to the sorting of resident luminal proteins such as reticuloplasmins in the ER6. Given these considerations, it seems to me that the term 'filtration' should be abandoned altogether in the context of selective membrane protein trafficking.
GORDON KOCH Laboratory of Molecular Biology, Hills Road, Cambridge, UK CB2 2QH.
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