Michel Blot

Mechanisms Causing Rapid and Parallel Losses of Ribose Catabolism in Evolving Populations of Escherichia coli B

Twelve populations of Escherichia coli B all lost D-ribose catabolic function during 2,000 generations of evolution in glucose minimal medium. We sought to identify the population genetic processes and molecular genetic events that caused these rapid and parallel losses. Seven independent Rbs(-) mutants were isolated, and their competitive fitnesses were measured relative to that of their Rbs(+) progenitor. These Rbs(-) mutants were all about 1 to 2% more fit than the progenitor. A fluctuation test revealed an unusually high rate, about 5 x 10(-5) per cell generation, of mutation from Rbs(+) to Rbs(-), which contributed to rapid fixation. At the molecular level, the loss of ribose catabolic function involved the deletion of part or all of the ribose operon (rbs genes). The physical extent of the deletion varied between mutants, but each deletion was associated with an IS150 element located immediately upstream of the rbs operon. The deletions apparently involved transposition into various locations within the rbs operon; recombination between the new IS150 copy and the one upstream of the rbs operon then led to the deletion of the intervening sequence. To confirm that the beneficial fitness effect was caused by deletion of the rbs operon (and not some undetected mutation elsewhere), we used P1 transduction to restore the functional rbs operon to two Rbs(-) mutants, and we constructed another Rbs(-) strain by gene replacement with a deletion not involving IS150. All three of these new constructs confirmed that Rbs(-) mutants have a competitive advantage relative to their Rbs(+) counterparts in glucose minimal medium. The rapid and parallel evolutionary losses of ribose catabolic function thus involved both (i) an unusually high mutation rate, such that Rbs(-) mutants appeared repeatedly in all populations, and (ii) a selective advantage in glucose minimal medium that drove these mutants to fixation.

Somatic intrachromosomal homologous recombination events in populations of plant siblings

Intrachromosomal homologous recombination in whole tobacco plants was analyzed using β-glucuronidase as non-selectable marker. We found that recombination frequencies were additive for transgenes in allelic positions and could be enhanced by treatment of plants with DNA-damaging agents. We compared the patterns of distribution of recombination events of different transgenic lines of tobacco and Arabidopsis with the respective Poisson distributions. Some lines showed Poisson-like distributions, indicating that recombination at the transgene locus was occurring in a random fashion in the plant population. In other cases, however, the distributions deviated significantly from Poisson distributions indicating that for specific transgene loci and/or configurations recombination events are not randomly distributed in the population. This was due to overrepresentation of plants with especially many as well as especially few recombination events. Analysis of one tobacco line indicated furthermore that the distribution of recombination events could be influenced by treating the seedlings with external factors. Our results suggest that different plant individuals, or parts of them, might exhibit different transient ‘states’ of recombination competence. A possible model relating ‘recombination silencing’ and transcription silencing to heterochromatization of the transgene locus is discussed.

Transposable elements and adaptation of host bacteria

A transposable element (TE) is a mobile sequence present in the genome of an organism. TEs can cause lethal mutations by inserting into essential, genes, promoting deletions or leaving short sequences upon excision. They therefore may be gradually eliminated from mixed populations of haploid micro-organisms such asEscherichia coli if they cannot balance this mutation load. Horizontal transmission between cells is known to occur and promote the transfer of TEs, but at rates often too low to compensate for the burden to their hosts. Therefore, alternative mechanisms should be found by these elements to earn their keep in the cells. Several theories have been suggested to explain their long-term maintenance in prokaryotic genomes, but little molecular evidence has been experimentally obtained. In this paper, the permanence of transposable elements in bacterial populations is discussed in terms of costs or benefits for the element and for the host. It is observed that, in all studies yet reported, the elements do not behave in their host as selfish DNA but as a co-operative component for the evolution of the couple.

Genomic comparisons among Escherichia coli strains B, K-12, and O157:H7 using IS elements as molecular markers

BackgroundInsertion Sequence (IS) elements are mobile genetic elements widely distributed among bacteria. Their activities cause mutations, promoting genetic diversity and sometimes adaptation. Previous studies have examined their copy number and distribution in Escherichia coli K-12 and natural isolates. Here, we map most of the IS elements in E. coli B and compare their locations with the published genomes of K-12 and O157:H7.ResultsThe genomic locations of IS elements reveal numerous differences between B, K-12, and O157:H7. IS elements occur in hok-sok loci (homologous to plasmid stabilization systems) in both B and K-12, whereas these same loci lack IS elements in O157:H7. IS elements in B and K-12 are often found in locations corresponding to O157:H7-specific sequences, which suggests IS involvement in chromosomal rearrangements including the incorporation of foreign DNA. Some sequences specific to B are identified, as reported previously for O157:H7. The extent of nucleotide sequence divergence between B and K-12 is <2% for most sequences adjacent to IS elements. By contrast, B and K-12 share only a few IS locations besides those in hok-sok loci. Several phenotypic features of B are explained by IS elements, including differential porin expression from K-12.ConclusionsThese data reveal a high level of IS activity since E. coli B, K-12, and O157:H7 diverged from a common ancestor, including IS association with deletions and incorporation of horizontally acquired genes as well as transpositions. These findings indicate the important role of IS elements in genome plasticity and divergence.

The Tn5 bleomycin resistance gene confers improved survival and growth advantage on Escherichia coli

The bleomycin resistance gene (ble) of transposon Tn5 is known to decrease the death rate of Escherichia coli during stationary phase. Bleomycin is a DNA-damaging agent and bleomycin resistance is produced by improved DNA repair which also requires the host genes aidC and polA coding, respectively, for an alkylation-inducible gene product and DNA polymerase I. In the absence of the drug, this DNA repair system is believed to cause the slower death rate of bleomycin-resistant bacteria. In this study, the effect of ble and aidC genes on the viability of bacteria and their growth rate in chemostat competitions was studied. The results indicate, that bleomycin-resistant bacteria display greater fitness under these conditions. Another beneficial effect of transposon Tn5 had been previously attributed to the insertion sequence IS50R. We were not able to reproduce this result with IS50R, however, the complete transposon was beneficial under similar conditions. Moreover, we showed the Tn5 fitness effect to be aidC-dependent. The ble gene was discovered after the fitness effect of IS50R had been established; it has not previously been considered to mediate the beneficial effect of Tn5. This possibility is discussed based on the molecular mechanism of bleomycin resistance.

Cytochrome c biogenesis is involved in the transposon Tn5‐mediated bleomycin resistance and the associated fitness effect in Escherichia coli

The transposon Tn5 ble gene and the Escherichia coli alkylation‐inducible aidC locus are co‐operatively involved in the resistance to the anti‐cancer drug and DNA‐cleaving agent bleomycin and enhance fitness of bacteria in the absence of the drug. In this report, we demonstrate that the aidC locus is identical to nrfGthe last gene of the nrf operon involved in the periplasmic formate‐dependent nitrite reduction. In the presence of Ble, NrfG expression is specifically induced and restores both bleomycin resistance and its associated beneficial growth effect in an aidC− strain. In vitro DNA protection assays reveal that purified Ble prevents bleomycin‐mediated DNA breakage, as do bleomycin‐binding proteins. Similarities between haems of the cytochrome c biogenesis nrf pathway and iron bleomycin suggest a DNA repair‐independent molecular mechanism for both bleomycin resistance and increased viability. The Ble protein binds bleomycin and prevents DNA breakage. It also induces the nrf locus that may assimilate bleomycin into haem for extracellular transport or inactivate bleomycin. Inactivation of potent DNA oxidants confers a better fitness to the bacterium carrying the transposon, suggesting a symbiotic relationship between host and transposon.

Tn5-mediated bleomycin resistance in Escherichia coli requires the expression of host genes

The transposon Tn5 expresses a gene, ble, whose product increases the viability of Escherichia coli and also confers resistance to the DNA‐cleaving antibiotic bleomycin and the DNA‐alkylating agent ethyl‐methanesulphonate. We find that the Ble protein induces expression of an alkylation inducible gene, aidC, and that both the AidC gene product and DNA polymerase I are required for Ble to confer bleomycin resistance. These findings support models in which Ble enhances DNA repair and suggest that Tn5 confers a fitness advantage to the host bacterium by increasing the repair of spontaneous DNA lesions. Such co‐operation between a transposon and its host suggests that Tn5 is a symbiotic rather than a selfish DNA element.

Insertion Sequences as Genomic Markers

Mobile genetic elements are widespread in almost all living organisms. This chapter will focus on insertion sequence (IS) elements, which are bacterial mobile genetic elements carrying genetic information devoted to their transposition and its regulation [1]. IS elements, and more generally mobile genetic elements, were first discovered by their ability to generate mutations [2, 3], and several studies suggested their significant contribution to spontaneous mutagenesis in bacteria [4, 5]. Transposition of an IS element can result in gene inactivation, polar effects [6, 7], activation of cryptic genes, and modification of gene expression (for review, see [1]). Besides these “simple” transposition events, IS elements can be involved in global restructuring of genomes, through homologous recombination events between homologous copies. Chromosomal rearrangements such as inversions, deletions, and duplications have been described [8, 9].