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.

Mutational analysis of the bacteriophage P1 late promoter sequence Ps.

The bacteriophage P1 late promoter sequence Ps controls the expression of the genes in the tail-fibre operon. Transcription from Ps only occurs during the second half of the P1 vegetative growth cycle and is positively regulated by the product of the phage gene 10. In this study degenerate oligonucleotides were used as primers in site-directed mutagenesis reactions in order to construct a large set of point mutations within the late promoter sequence Ps. A total of 35 independent single point mutations was isolated and the mutants were tested for promoter activity. Mutations in the Escherichia coli-like -10 region and in a late operator sequence, containing a symmetric sequence centred around position -22, resulted in significant reductions in promoter strength. Most of these mutations alter base-pairs that are highly conserved among the known late promoter sequences of the P1 family. In addition, insertion mutants that change the spacing between the -10 and the late operator indicate that a special topological arrangement between the two boxes is crucial for late promoter function. These results suggest that the product of gene 10 binds specifically to a late operator in order to activate transcription from P1 late promoter sequences.

Physical analysis of the genomes of hybrid phages between phage P1 and plasmid p15B.

The genomes of three plaque-forming recombinant phages between phage P1 and plasmid p15B were characterized by restriction cleavage analysis and electron microscopic heteroduplex studies. The structure of all three P1-15 hybrid genomes differs from that of P1 DNA in the res mod region coding for restriction and modification systems EcoP15 and EcoP1, respectively. P1-15 hybrid 2 shows an additional major difference to P1 around the site of the residential IS1 element of P1 and it does not carry an IS1 in its genome.

Bent DNA is needed for recombinational enhancer activity in the site-specific recombination system Cin of bacteriophage P1. The role of FIS protein.

A series of recombinational enhancer mutants was constructed by manipulating the ClaI site between the two FIS binding sites of the Hin enhancer. These mutants include insertions from two to 12 base-pairs and two deletions of one or two base-pairs. Recombinational enhancer activity was found only with four mutants carrying either a four base-pair substitution, ten base-pair insertions or a one base-pair deletion, respectively; two other ten base-pair insertion mutants, however, were inactive, although FIS protein binding was unaffected. So, besides binding of FIS protein to its specific sites within the enhancer sequence and the correct helical positioning of these sites on the DNA, another criterion for enhancer activity must be fulfilled. DNA bending assays identify this requirement as a change of the enhancer DNA conformation, which FIS protein is able to induce and to stabilize. This conformational change of the DNA can be blocked by mutations in the central segment between the two FIS binding sites of the Hin enhancer. This sequence has special functions for the recombinational enhancer activity.

Bacteriophage P1 tail-fibre and dar operons are expressed from homologous phage-specific late promoter sequences.

Two plasmid systems, containing the easily assayable galK and lacZ functions, were employed to study the regulation of the bacteriophage P1 tail-fibre and dar operons. Various P1 DNA fragments carrying either the 5' end of lydA (the 1st gene in the dar operon) or the tail-fibre gene 19 precede the promoterless coding region of galK or were fused, in-frame, to the lacZ gene. In the presence of an induced P1 prophage, GalK and LacZ activities were both detected after a 20 to 30 minute lag period, indicating that the dar and tail-fibre operons are expressed from positively regulated, late promoters. The corresponding DNA operons are expressed from positively regulated, late promoters. The corresponding DNA region of the closely related p15B plasmid exhibits comparable promoter properties. Deletion analysis mapped the promoter of a gene 19-lacZ fusion to a DNA region upstream from gene R, an open reading frame that precedes the coding frame of gene 19. The tail-fibre gene thus forms the second gene in a three gene operon (genes R, 19 (S) and U). Sequence comparison between this promoter region, upstream sequences of the lydA gene and the corresponding portions of the p15B genome allowed the identification of a highly conserved 38 base-pair sequence, which most likely represents a P1-specific late promoter. This was confirmed by 5' mapping of P1 mRNA. Transcription of both the tail-fibre and dar operons is initiated at sites five and six base-pairs, respectively, downstream from the first conserved nucleotide of this sequence. The conserved motif consists of a standard Escherichia coli -10 region followed by a nine base-pair palindromic sequence located centrally about position -22.

DNA restriction--modification genes of phage P1 and plasmid p15B. Structure and in vitro transcription.

The EcoP1 and EcoP15 DNA restriction-modification systems are coded by the related P1 prophage and p15B plasmid. We have examined the organization of the genes for these systems using P1 itself, "P1-P15" hybrid phages expressing the EcoP15 restriction specificity of p15B and cloned restriction fragments derived from these phage DNAs. The results of transposon mutagenesis, restriction cleavage analysis. DNA heteroduplex analysis and in vitro transcription mapping allow the following conclusions to be drawn concerning the structural genes. (1) All of the genetic information necessary to specify either system is contained within a contiguous DNA segment of 5 x 10(3) bases which encodes two genes. One of them, necessary for both restriction and modification, we call mod and the other, required only for restriction (together with mod), we call res. (2) The res gene is about 2.8 x 10(3) bases long and at the heteroduplex level is largely identical for P1 and P15: it shows a small region of partial nonhomology and some restriction cleavage site differences. The mod gene is about 2.2 x 10(3) bases long and contains a 1.2 x 10(3) base long region of non-homology between P1 and P15 toward the N-terminus of the gene. The rest of the gene at this level of analysis is identical for the two systems. (3) Each of the genes is transcribed in vitro from its own promoter. It is possible that the res gene is also transcribed by readthrough from the mod promoter.

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.

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.

The characterization of terminators of RNA transcription on IS30 and an analysis of their role in IS element-mediated polarity.

Using expression vectors carrying the lacUV5 or Pgal promoters and the galK gene, we have studied terminators of transcription on the prokaryotic mobile genetic element IS30. The long open reading frame, ORF-A, of IS30 contains a relatively Rho-independent terminator, T30A, within its coding sequence. T30A terminates the majority of transcripts initiated at either an external promoter or the IS30-borne promoter P30A. No other terminator functions on this strand of IS30 (orientation left to right). In the orientation right to left, the previously identified terminator T30C, which follows ORF-C, is Rho-independent. T30C together with T30D, a newly identified, strong, partially Rho-dependent terminator near the left end of IS30, permits less than 2% read-through from external promoters. Neither ORF-A nor ORF-C appears to be protected from transcription by external promoters. As a consequence of the internal terminators, the insertion of IS30 into an operon can be expected to reduce the expression of genes downstream of the site of insertion weakly for one orientation of IS30 and strongly for the other orientation.

Characterization of in vitro constructed IS30-flanked transposons.

In order to facilitate functional studies on the mobile genetic element IS30, a resident of the Escherichia coli chromosome, transposon structures with two copies of IS30 flanking the chloramphenicol-resistance gene cat were constructed in vitro. Transposons containing IS30 as direct repeats (Tn2700 and Tn2702) transpose from multicopy plasmids into the genome of phage P1-15, thus giving rise to special transduction for cat with frequencies between 10(-5) and 10(-8)/plaque-forming unit. In contrast, transposon structures with IS30 in inverted repeat (Tn2701 and Tn2703) showed no detectable (less than 10(-9] transposition activity in vivo. By restriction analysis, two insertion sites of Tn2700 and Tn2702 on the phage P1-15 genome were indistinguishable from those observed earlier with a single copy of the IS30 element. These two insertion sites were used several times independently by Tn2700 and Tn2702. This confirms the non-random target selection by the element and it indicates that transposition of Tn2700 and Tn2702 follows the same rules as that of IS30.

Random mutagenesis using degenerate oligodeoxyribonucleotides.

An efficient method for random mutagenesis was applied to a 75-bp target sequence. The mutational changes in the target region are introduced by the technique of oligodeoxyribonucleotide(oligo)-directed, site-specific mutagenesis using mixtures of degenerate oligos. These are designed in such a way that they carry with a high probability randomly distributed substitutions, which are introduced into the oligos by utilizing appropriate concentrations of all four nucleotide precursors during each chain elongation step. These mixtures of degenerate oligos were hybridized to the appropriate M13-hybrid ss-template and then extended in vitro using PolIk. In order to avoid any bias artificially created by the Escherichia coli mismatch repair system, homoduplex molecules were synthesized in vitro according to the method of Taylor et al. [Nucleic Acids Res. 13 (1985) 8765-8785]. After transformation of the appropriate E. coli host, M13 plaques were randomly analysed by DNA sequencing. Using appropriate preparations of template DNA and oligos we attained mutagenesis efficiencies in the range of 20-50%. The analysis of 85 different mutants revealed that the distribution of the mutations is random and that all expected substitutions occur with about the same probability.

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.

Organization of the bacteriophage P1 tail-fibre operon.

The revised sequence of a bacteriophage P1 DNA fragment containing the 5' end of the tail-fibre gene, gene 19, revealed that this gene is closely preceded by another open reading frame (ORF) of 432 bp. We have designated this ORF as gene R. The tail-fibre gene and gene R are transcriptionally and translationally coupled. Thus, the tail-fibre operon of bacteriophage P1 consists of three genes: gene R, gene 19 (or gene S) and gene U.

Terminal inverted repeats of prokaryotic transposable element IS186 which can generate duplications of variable length at an identical target sequence.

The insertion element IS186, which resides in the chromosome of Escherichia coli K-12, is 1338 bp long. Its termini represent 23-bp perfectly inverted repeats, but a variant carries a mismatch at position 23. IS186 transposes preferentially into G + C-rich sequences and generates target duplications of variable length, even at the same integration site.

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.

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.

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.

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.