Evolution of prokaryotic genomes.
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Animals↗
Biomedical subjects
W Arber
Publications and source records attributed to W Arber.
Microbiology: impact on research in life sciences.
In this essay with a flavor of science history, the influence of imaging techniques, as compared to other research strategies, on microbiologic investigations is discussed. Using a few selected examples, to what degree microbiology became a leading science during the last 50 years in gaining knowledge in life sciences, particularly with regard to molecular genetics and more recently molecular evolution is also discussed.
Animals↗
Genetic variation: molecular mechanisms and impact on microbial evolution.
On the basis of established knowledge of microbial genetics one can distinguish three major natural strategies in the spontaneous generation of genetic variations in bacteria. These strategies are: (1) small local changes in the nucleotide sequence of the genome, (2) intragenomic reshuffling of segments of genomic sequences and (3) the acquisition of DNA sequences from another organism. The three general strategies differ in the quality of their contribution to microbial evolution. Besides a number of non-genetic factors, various specific gene products are involved in the generation of genetic variation and in the modulation of the frequency of genetic variation. The underlying genes are called evolution genes. They act for the benefit of the biological evolution of populations as opposed to the action of housekeeping genes and accessory genes which are for the benefit of individuals. Examples of evolution genes acting as variation generators are found in the transposition of mobile genetic elements and in so-called site-specific recombination systems. DNA repair systems and restriction-modification systems are examples of modulators of the frequency of genetic variation. The involvement of bacterial viruses and of plasmids in DNA reshuffling and in horizontal gene transfer is a hint for their evolutionary functions. Evolution genes are thought to undergo biological evolution themselves, but natural selection for their functions is indirect, at the level of populations, and is called second-order selection. In spite of an involvement of gene products in the generation of genetic variations, evolution genes do not programmatically direct evolution towards a specific goal. Rather, a steady interplay between natural selection and mixed populations of genetic variants gives microbial evolution its direction.
Bacteria↗
Lack of hotspot targets: a constraint for IS30 transposition in Salmonella.
IS30 is an insertion element common in E. coli strains but rare or absent in Salmonella. Transfer of the IS30-flanked transposon Tn2700 to Salmonella typhimurium was assayed using standard delivery procedures of bacterial genetics (conjugation and transduction). Tn2700 'hops' were rare and required transposase overproduction, suggesting the existence of host constraints for IS30 activity. Sequencing of three Tn2700 insertions in the genome of S. typhimurium revealed that the transposon had been inserted into sites with a low homology to the IS30 consensus target, suggesting that inefficient Tn2700 transposition to the Salmonella genome might be caused by a lack of hotspot targets. This view was confirmed by the introduction of an IS30 'hot target sequence', whose sole presence permitted Tn2700 transposition without transposase overproduction. Detection of IS30-induced DNA rearrangements in S. typhimurium provided further evidence that the element undergoes similar activities in E. coli and S. typhimurium. Thus, hotspot absence may be the main (if not the only) limitation for IS30 activity in the latter species. If these observations faithfully reproduce the scenario of natural populations, establishment of IS30 in the Salmonella genome may have been prevented by a lack of DNA sequences closely related to the unusually long (24 bp) IS30 consensus target.
Bacteriophage T4↗
Involvement of gene products in bacterial evolution.
Three strategies of different quality contribute in parallel to the natural formation of genetic variants in bacteria: (1) small local alterations of DNA sequences; (2) recombinational reshuffling of segments of the genome; and (3) acquisition of DNA sequences by horizontal gene transfer. Key enzymes involved in these processes often act as variation generators by making use of structural flexibilities of biological macromolecules and of the effect of random encounter. In the theory of molecular evolution, genetic determinants of variation generators as well as of modulators of the frequency of genetic variation are defined as evolutionary genes. This postulate is consistent with the notion that spontaneous mutagenesis is in general not adaptive and that the direction of evolution depends on natural selection exerted on populations of genetic variants.
Bacteria↗
Genomic evolution during a 10,000-generation experiment with bacteria.
Molecular methods are used widely to measure genetic diversity within populations and determine relationships among species. However, it is difficult to observe genomic evolution in action because these dynamics are too slow in most organisms. To overcome this limitation, we sampled genomes from populations of Escherichia coli evolving in the laboratory for 10,000 generations. We analyzed the genomes for restriction fragment length polymorphisms (RFLP) using seven insertion sequences (IS) as probes; most polymorphisms detected by this approach reflect rearrangements (including transpositions) rather than point mutations. The evolving genomes became increasingly different from their ancestor over time. Moreover, tremendous diversity accumulated within each population, such that almost every individual had a different genetic fingerprint after 10,000 generations. As has been often suggested, but not previously shown by experiment, the rates of phenotypic and genomic change were discordant, both across replicate populations and over time within a population. Certain pivotal mutations were shared by all descendants in a population, and these are candidates for beneficial mutations, which are rare and difficult to find. More generally, these data show that the genome is highly dynamic even over a time scale that is, from an evolutionary perspective, very brief.
Biological Evolution↗
Accessory genes in the darA operon of bacteriophage P1 affect antirestriction function, generalized transduction, head morphogenesis, and host cell lysis.
Bacteriophage P1 mutants with the 8.86-kb region between the invertible C-segment and the residential IS1 element deleted from their genome are still able to grow vegetatively and to lysogenize stably, but they show several phenotypic changes. These include the formation of minute plaques due to delayed cell lysis, the abundant production of small-headed particles, a lack of specific internal head proteins, sensitivity to type I host restriction systems, and altered properties to mediate generalized transduction. In the wild-type P1 genome, the accessory genes encoding the functions responsible for these characters are localized in the darA operon that is transcribed late during phage production. We determined the relevant DNA sequence that is located between the C-segment and the IS1 element and contains the cin gene for C-inversion and the accessory genes in the darA operon. The darA operon carries eight open reading frames that could encode polypeptides containing >100 amino acids. Genetic studies indicate that some of these open reading frames, in particular those residing in the 5' part of the darA operon, are responsible for the phenotypic traits identified. The study may contribute to a better comprehension of phage morphogenesis, of the mobilization of host DNA into phage particles mediating generalized transduction, of the defense against type I restriction systems, and of the control of host lysis.
Bacteriolysis↗
Target specificity of insertion element IS30.
The Escherichia coli resident mobile element IS30 has pronounced target specificity. Upon transposition, the element frequently inserts exactly into the same position of a preferred target sequence. Insertion sites in phages, plasmids and in the genome of E. coli are characterized by an exceptionally long palindromic consensus sequence that provides strong specificity for IS30 insertions, despite a relatively high level of degeneracy. This 24-bp-long region alone determines the attractiveness of the target DNA and the exact position of IS30 insertion. The divergence of a target site from the consensus and the occurrence of 'non-permitted' bases in certain positions influence the target activity. Differences in attractiveness are emphasized if two targets are present in the same replicon, as was demonstrated by quantitative analysis. In a system of competitive targets, the oligonucleotide sequence representing the consensus of genomic IS30 insertion sites proved to be the most efficient target. Having compared the known insertion sites, we suppose that IS30-like target specificity, which may represent an alternative strategy in target selection among mobile elements, is characteristic of the insertion sequences IS3, IS6 and IS21, too.
Binding Sites↗
Mutations in the carboxy-terminal part of IS30 transposase affect the formation and dissolution of (IS30)2 dimer.
The transposase of IS30 catalyses different transpositional rearrangements via the dimer (IS30)2 intermediate structure. Mutation analysis provides evidence that the C-terminal part of IS30 transposase is required for the formation and dissolution of (IS30)2 dimer. C-terminal mutants are also defective in transpositional fusion; however, this deficiency can be 'suppressed' by addition of the final product of site-specific dimerisation, the core (IS30)2 intermediate structure. The transposase part studied shows significant homologies in three highly conserved regions to proteins of IS30-related mobile elements.
Amino Acid Sequence↗
Terminal inverted repeats of insertion sequence IS30 serve as targets for transposition.
In the present study, we demonstrate that the terminal inverted repeats of the Escherichia coli insertion sequence IS30 are functional target sites for the transposition of the (IS30)2 dimer, which represents an intermediate structure in the transposition of IS30. Comparative analysis of various target regions revealed that the left and right ends differ in their "attractivity." In our experiments, the joined left and right ends, i.e., the (IS30)2 intermediate structure, was found to be the most preferred target. It was also shown that flanking sequences can influence the target activity of the terminal repeats. The functional part of the target region was localized in the inverted repeats by means of mutational analysis, and it corresponds to the binding site of IS30 transposase. Insertion of 1 bp into the right inverted repeat resulted in unusual target duplication accompanied by gene conversion. The choice of the terminal inverted repeats as targets in transposition leads to the reconstruction of the (IS30)2 structure, which may induce a cascade of further rearrangements. Therefore, this process can play a role in the evolution of the genome.
Binding Sites↗
An antisense RNA in IS30 regulates the translational expression of the transposase.
Two functional promoters had previously been identified in the mobile genetic element IS30 of Escherichia coli. One, P30A, controls the transcription of ORF-A, whose product is the transposase; the other, located within the ORF-A sequence but on the opposite strand, is called P30C, but the nature and function of its product had remained unknown. We identified this product as an RNA about 150 nucleotides long (called RNA-C) that functions as an untranslated antisense transcript. Indeed, biochemical evidence indicates that ORF-C, which is completely contained on RNA-C, is not translated at detectable levels. Mutational analysis of P30C revealed that overproduction of RNA-C resulted in a decrease of IS30 transposition, while a reduction in the promoter strength resulted in an increase of transposition, as measured by the rate of cointegrate formation. We showed that the translation of ORF-A, but not transcription, is negatively affected by the presence of antisense RNA-C. In contrast to other antisense RNAs acting inhibitorily on translation, RNA-C does not seem to affect translation initiation. Most likely its hybridization to the transposase mRNA in the complementary region located in the central part of ORF-A inhibits the ribosomes in their progression, thus reducing the number of completely translated transposase molecules.
DNA Transposable Elements↗
Vector for IS element entrapment and functional characterization based on turning on expression of distal promoterless genes.
We constructed and characterized a novel trap vector for rapid isolation of insertion sequences. The strategy used for the isolation of IS elements is based on the ability of many IS elements to turn on the expression of otherwise silent genes distal to some sites of insertion. The simple transposition of an IS element can sometimes cause the constitutive expression of promoterless antibiotic resistance genes resulting in selectable phenotypes. The trap vector pAW1326 is based on a pBR322 replicon, it carries ampicillin and streptomycin resistance genes, and also silenced genes that confer chloramphenicol and kanamycin resistance once activated. The trap vector pAW1326 proved to be efficient and 85 percent of all isolated mutations were insertions. The majority of IS elements resident in the studied Escherichia coli strains tested became trapped, namely IS2, IS3, IS5, IS150, IS186 and Tn1000. We also encountered an insertion sequence, called IS10L/R-2, which is a hybrid of the two IS variants IS10L and IS10R. IS10L/R-2 is absent from most E. coli strains, but it is detectable in some strains such as JM109 which had been submitted to Tn10 mutagenesis. The distribution of the insertion sequences within the trap region was not random. Rather, the integration of chromosomal mobile genetic elements into the offered target sequence occurred in element-specific clusters. This is explained both by the target specificity and by the specific requirements for the activation of gene transcription by the DNA rearrangement. The employed trap vector pAW1326 proved to be useful for the isolation of mobile genetic elements, for a demonstration of their transposition activity as well as for the further characterization of some of the functional parameters of transposition.
DNA Transposable Elements↗
Three functions of bacteriophage P1 involved in cell lysis.
Amber and deletion mutants were used to assign functions in cell lysis to three late genes of bacteriophage P1. Two of these genes, lydA and lydB of the dar operon, are 330 and 444 bp in length, respectively, with the stop codon of lydA overlapping the start codon of lydB. The third, gene 17, is 558 bp in length and is located in an otherwise uncharacterized operon. A search with the predicted amino acid sequence of LydA for secondary motifs revealed a holin protein-like structure. Comparison of the deduced amino acid sequence of gene 17 with sequences of proteins in the SwissProt database revealed homologies with the proteins of the T4 lysozyme family. The sequence of lydB is novel and exhibited no known extended homology. To study the effect of gp17, LydA, and LydB in vivo, their genes were cloned in a single operon under the control of the inducible T7 promoter, resulting in plasmid pAW1440. A second plasmid, pAW1442, is identical to pAW1440 but has lydB deleted. Induction of the T7 promoter resulted in a rapid lysis of cells harboring pAW1442. In contrast, cells harboring pAW1440 revealed only a small decrease in optical density at 600 nm compared with cells harboring vector alone. The rapid lysis phenotype in the absence of active LydB suggests that this novel protein might be an antagonist of the holin LydA.
Amino Acid Sequence↗
Dynamics of IS-related genetic rearrangements in resting Escherichia coli K-12.
An analysis of restriction fragment length polymorphism (RFLP) using eight residential insertion sequence (IS) elements as hybridization probes reveals that the genome of resting bacteria is more dynamic than it was long believed. Escherichia coli strains stored in agar stabs for up to 30 yr accumulate a genetic variation which is correlated to time of storage. This spontaneous mutagenesis is often IS-specific, with particularly high activity for IS5, and thus suggests that transpositional DNA rearrangements are a major cause for the observed genetic polymorphism. The RFLP patterns indicate a burst of IS30 transposition to occur occasionally. Mutation rate is estimated by two different methods to roughly 10(-5) IS-related DNA rearrangements per bacterial chromosome per hour of storage for the eight IS elements studied. A pedigree derived from the RFLP data reveals that populations had evolved independently in each stab and showed no signs of convergence. Relics of an assumed ancestral population were still present in the stab cultures, but the elder stabs provided mostly mutants. These results indicate that cells placed under nutritional deprivation might have a highly plastic genome and suggest that such plasticity might play an adaptive role.
DNA Probes↗
Cin-mediated recombination at secondary crossover sites on the Escherichia coli chromosome.
The Cin recombinase is known to mediate DNA inversion between two wild-type cix sites flanking genetic determinants for the host range of bacteriophage P1. Cin can also act with low frequency at secondary (or quasi) sites (designated cixQ) that have lower homology to either wild-type site. An inversion tester sequence able to reveal novel operon fusions was integrated into the Escherichia coli chromosome, and the Cin recombinase was provided in trans. Among a total of 13 Cin-mediated inversions studied, three different cixQ sites had been used. In two rearranged chromosomes, the breakpoints of the inversions were mapped to cixQ sites in supB and ompA, representing inversions of 109 and 210 kb, respectively. In the third case, a 2.1-kb inversion was identified at a cixQ site within the integrated sequences. This derivative itself was a substrate for a second inversion of 1.5 kb between the remaining wild-type cix and still another cixQ site, thus resembling a reversion. In analogy to that which is known from DNA inversion on plasmids, homology of secondary cix sites to wild-type recombination sites is not a strict requirement for inversion to occur on the chromosome. The chromosomal rearrangements which resulted from these Cin-mediated inversions were quite stable and suffered no growth disadvantage compared with the noninverted parental strain. The mechanistic implications and evolutionary relevance of these findings are discussed.
Bacterial Outer Membrane Proteins↗
Insertion sequence-related genetic variation in resting Escherichia coli K-12.
Bacterial subclones recovered from an old stab culture of Escherichia coli K-12 revealed a high degree of genetic diversity, which occurred in spite of a very reduced rate of propagation during storage. This conclusion is based on a pronounced restriction fragment length polymorphism (RFLP) detected upon hybridization with internal fragments of eight resident insertion sequences (IS). Genetic diversity was dependent on the IS considered and, in many cases, a clear consequence of IS transposition. IS5 was particularly active in the generation of variation. All subclones in which IS30 had been active testify to a burst of IS30 transposition. This was correlated with a loss of prototrophy and a reduced growth on rich media. A pedigree of the entire clone could be drawn from the RFLP patterns of the subclones. Out of 118 subclones analyzed, 68 different patterns were found but the putative ancestral population had disappeared. A few patterns were each represented by several subclones displaying improved fitness. These results offer insights into the role of IS elements in the plasticity of the E. coli genome, and they further document that enzyme-mediated DNA rearrangements do occur in resting bacterial cultures.
Biological Evolution↗
Evolution of prokaryotic genomes.
Molecular genetics, which has its roots mainly in the development of microbial genetics in the middle of this century, not only greatly facilitates investigations of essential cellular functions, but also offers a means to better understand evolutionary progress. Spontaneous mutagenesis, the driving force of biological evolution, depends on a multitude of mechanistically distinct processes, many of which are already quite well understood. Often, enzymes act as variation generators, and natural gene vectors help to spread functional domains, entire genes and groups of genes across natural isolation barriers. In this overview, particular attention is given to comparing three selected natural strategies for the generation of genetic diversity: nucleotide substitution, DNA rearrangements, and gene acquisition. All of these mechanisms, as well as many others, appear to fulfill their specific roles in microbial evolution. Rather than being the result of an accumulation of errors, biological evolution may depend on a multitude of specific biological functions, as well as on a certain degree of intrinsic structural flexibility of biological molecules.
Biological Evolution↗
Formation of the tandem repeat (IS30)2 and its role in IS30-mediated transpositional DNA rearrangements.
Plasmids carrying two IS30 elements in the same orientation, as in the composite transposon Tn2706, are structurally unstable in Escherichia coli. A primary segregation product is formed by site-specific deletion of the sequences carried between the two IS30 elements. The resulting covalently closed replicon carries the two IS30 elements as tandem repeats separated by only 2 bp. This (IS30)2 structure is extremely unstable, but it can nevertheless be isolated on its vector plasmid and, after purification, can be reintroduced into host cells by transformation. Among the descendants of transformants of recA- bacteria, replicated copies of the introduced (IS30)2 structure are still present, together with various kinds of segregation products which provide evidence for the efficient generation of DNA rearrangements. Most abundant is the product of another site-specific recombination between two identical ends of the IS30 elements involved, which results in the presence of just one intact IS30 on the plasmid. Apart from this, and depending on the presence of appropriate targets for IS30 transposition, various transposition products of (IS30)2 are also seen. Intramolecular reactions lead to DNA inversions and deletions with breakpoints other than IS30 ends. In intermolecular reactions inverse transposition occurs at high frequency and one also obtains simple transposition and cointegration. A mutational study revealed the requirement in cis of one intact IS30 transposase gene and of both proximal ends of the two IS30 elements concerned not only for the formation of (IS30)2, but also for its further rearrangement reactions, including the efficient formation of site-specific deletions. A model is proposed, which postulates that (IS30)2 intermediates play a key role in IS30 transposition pathways in which the formation of (IS30)2 may be rate-limiting. Once this structure is formed, it gives rise to a burst of transpositional rearrangements in the subclone carrying (IS30)2. Evolutionary implications of these findings are discussed.
Base Sequence↗