Geneviève Maenhaut-Michel

Overproduction of the Lon protease triggers inhibition of translation in Escherichia coli: involvement of the yefM-yoeB toxin-antitoxin system

In Escherichia coli, the Lon ATP‐dependent protease is responsible for degradation of several regulatory proteins and for the elimination of abnormal proteins. Previous studies have shown that the overproduction of Lon is lethal. Here, we showed that Lon overproduction specifically inhibits translation through at least two different pathways. We have identified one of the pathways as being the chromosomal yefM‐yoeB toxin‐antitoxin system. The existence of a second pathway is demonstrated by the observation that the deletion of the yefM‐yoeB system did not completely suppress lethality and translation inhibition. We also showed that the YoeB toxin induces cleavage of translated mRNAs and that Lon overproduction specifically activates YoeB‐dependent mRNAs cleavage. Indeed, none of the other identified chromosomal toxin‐antitoxin systems (relBE, mazEF, chpB and dinJ‐yafQ) was involved in Lon‐dependent lethality, translation inhibition and mRNA cleavage even though the RelB and MazE antitoxins are known to be Lon substrates. Based on our results and other studies, translation inhibition appears to be the key element that triggers chromosomal toxin‐antitoxin systems. We propose that under Lon overproduction conditions, translation inhibition is mediated by Lon degradation of a component of the YoeB‐independent pathway, in turn activating the YoeB toxin by preventing synthesis of its unstable YefM antidote.

What Is the Benefit to Escherichia coli of Having Multiple Toxin-Antitoxin Systems in Its Genome?

The Escherichia coli K-12 chromosome encodes at least five proteic toxin-antitoxin (TA) systems. The mazEF and relBE systems have been extensively characterized and were proposed to be general stress response modules. On one hand, mazEF was proposed to act as a programmed cell death system that is triggered by a variety of stresses. On the other hand, relBE and mazEF were proposed to serve as growth modulators that induce a dormancy state during amino acid starvation. These conflicting hypotheses led us to test a possible synergetic effect of the five characterized E. coli TA systems on stress response. We compared the behavior of a wild-type strain and its derivative devoid of the five TA systems under various stress conditions. We were unable to detect TA-dependent programmed cell death under any of these conditions, even under conditions previously reported to induce it. Thus, our results rule out the programmed-cell-death hypothesis. Moreover, the presence of the five TA systems advantaged neither recovery from the different stresses nor cell growth under nutrient-limited conditions in competition experiments. This casts a doubt on whether TA systems significantly influence bacterial fitness and competitiveness during non-steady-state growth conditions.

GATC sequences, DNA nicks and the MutH function in Escherichia coli mismatch repair.

Circular heteroduplex DNAs of bacteriophage phi X174 have been constructed carrying either a G:T (Eam+/Eam3) or a G:A (Bam+/Bam16) mismatch and containing either two, one or no GATC sequences. Mismatches were efficiently repaired in wild‐type Escherichia coli transfected with phi X174 heteroduplexes only when two unmethylated GATC sequences were present in phi X174 DNA. The requirements for GATC sequences in substrate DNA and for the E. coli MutH function in E. coli mismatch repair can be alleviated by the presence of a persistent nick (transfection with nicked heteroduplex DNA in ligase temperature‐sensitive mutant at 40 degrees C). A persistent nick in the GATC sequence is as effective in stimulating mutL‐ and mutS‐dependent mismatch repair as a nick distant from the GATC sequence and from the mismatch. These observations suggest that the MutH protein participates in methyl‐directed mismatch repair by recognizing unmethylated DNA GATC sequences and/or stimulating the nicking of unmethylated strands.

Starvation-induced Mucts62-mediated coding sequence fusion: a role for ClpXP, Lon, RpoS and Crp

The formation of araB–lacZ coding sequence fusions in Escherichia coli is a particular type of chromosomal rearrangement induced by Mucts62, a thermoinducible mutant of mutator phage Mu. Fusion formation is controlled by the host physiology. It only occurs after aerobic carbon starvation and requires the phage‐encoded transposase pA, suggesting that these growth conditions trigger induction of the Mucts62 prophage. Here, we show that thermal induction of the prophage accelerated araB–lacZ fusion formation, confirming that derepression is a rate‐limiting step in the fusion process. Nonetheless, starvation conditions remained essential to complete fusions, suggesting additional levels of physiological regulation. Using a transcriptional fusion indicator system in which the Mu early lytic promoter is fused to the reporter E. coli lacZ gene, we confirmed that the Mucts62 prophage was derepressed in stationary phase (S derepression) at low temperature. S derepression did not apply to prophages that expressed the Mu wild‐type repressor. It depended upon the host ClpXP and Lon ATP‐dependent proteases and the RpoS stationary phase‐specific σ factor, but not upon Crp. None of these four functions was required for thermal induction. Crp was required for fusion formation, but only when the Mucts62 prophage encoded the transposition/replication activating protein pB. Finally, we found that thermally induced cultures did not return to the repressed state when shifted back to low temperature and, hence, remained activated for accelerated fusion formation upon starvation. The maintenance of the derepressed state required the ClpXP and Lon host proteases and the prophage Ner‐regulatory protein. These observations illustrate how the cts62 mutation in Mu repressor provides the prophage with a new way to respond to growth phase‐specific regulatory signals and endows the host cell with a new potential for adaptation through the controlled use of the phage transposition machinery.

SOS mutator effect in E. coli mutants deficient in mismatch correction

We have used bacteriophage lambda to characterize the mutator effect of the SOS response induced by u.v. irradiation of Escherichia coli. Mutagenesis of unirradiated phages grown in irradiated or unirradiated bacteria was detected by measuring forward mutagenesis in the immunity genes or reversion mutagenesis of an amber codon in the R gene. Relative to the wild‐type, the SOS mutator effect was higher in E. coli mismatch correction‐deficient mutants (mutH, mutL and mutS) and lower in an adenine methylation‐deficient mutant (dam3). We conclude that a large proportion of SOS‐induced ‘untargeted’ mutations are removed by the methyl‐directed mismatch correction system, which acts on newly synthesized DNA strands. The lower SOS mutator effect observed in E. coli dam mutants may be due to a selective killing of mismatch‐bearing chromosomes resulting from undirected mismatch repair. The SOS mutator effect on undamaged lambda DNA, induced by u.v. irradiation of the host, appears to result from decreased fidelity of DNA synthesis.

GATC sequence and mismatch repair in Escherichia coli.

The Escherichia coli mismatch repair system greatly improves DNA replication fidelity by repairing single mispaired and unpaired bases in newly synthesized DNA strands. Transient undermethylation of the GATC sequences makes the newly synthesized strands susceptible to mismatch repair enzymes. The role of unmethylated GATC sequences in mismatch repair was tested in transfection experiments with heteroduplex DNA of phage phi 174 without any GATC sequence or with two GATC sequences, containing in addition either a G:T mismatch (Eam+/Eam3) or a G:A mismatch (Bam+/Bam16). It appears that only DNA containing GATC sequences is subject to efficient mismatch repair dependent on E. coli mutH, mutL, mutS and mutU genes; however, also in the absence of GATC sequence some mut‐dependent mismatch repair can be observed. These observations suggest that the mismatch repair enzymes recognize both the mismatch and the unmethylated GATC sequence in DNA over long distances. The presence of GATC sequence(s) in the substrate appears to be required for full mismatch repair activity and not only for its strand specificity according to the GATC methylation state.

Nature of the SOS mutator activity: Genetic characterization of untargeted mutagenesis in Escherichia coli

SummaryIn Escherichia coli, induction of the SOS functions by UV irradiation or by mutation in the recA gene promotes an SOS mutator activity which generates mutations in undamaged DNA. Activation of RecA protein by the recA730 mutation increases the level of spontaneous mutation in the bacterial DNA. The number of recA730-induced mutations is greatly increased in mismatch repair deficient strains in which replication errors are not corrected. This suggests that the majority of recA730-induced mutations (90%) arise through correctable, i.e. non-targeted, replication errors. This recA730 mutator effect is suppressed by a mutation in the umuC gene. We also found that dam recA730 double mutants are unstable, segregating clones that have lost the dam or the recA mutations or that have acquired a new mutation, probably in one of the genes involved in mismatch repair. We suggest that the genetic instability of the dam recA730 mutants is provoked by the high level of replication errors induced by the recA730 mutation, generating killing by coincident mismatch repair on the two unmethylated DNA strands. The recA730 mutation increases spontaneous mutagenesis of phage λ poorly. UV irradiation of recA730 host bacteria increases phage untargeted mutagenesis to the level observed in UV-irradiated recA+ strains. This UV-induced mutator effect in recA730 mutants is not suppressed by a umuC mutation. Therefore UV and the recA730 mutation seem to induce different SOS mutator activities, both generating untargeted mutations.

Effect of umuC mutations on targeted and untargeted ultraviolet mutagenesis in bacteriophage λ

Mutagenesis of phage lambda towards clear-plaque phenotype (c+----c) results in two classes of mutants that can be distinguished genetically and morphologically. Indirect mutagenesis, i.e. mutagenesis of unirradiated phage lambda c+ stimulated by the ultraviolet irradiation of the Escherichia coli host, results in mixed bursts (c/c+) of turbid wild-type and clear-plaque mutant phages. Pure bursts of lambda c mutants are induced by irradiation of the phage genome. Irradiation of both phages and host bacteria stimulates the production of the two classes of mutant clones. We show that three different mutant alleles of the E. coli umuC gene only prevent the appearance of pure bursts of clear-plaque mutants, while mixed bursts are produced at least as frequently in umuC mutants as in the umuC+ parent.

Involvement of DNA polymerase III in UV-induced mutagenesis of bacteriophage lambda

SummaryIt has been proposed that the mutation fixation processes stimulated by SOS induction result from an induced infidelity of DNA replication (Radman 1974). The aim of this study was to determine if mutator mutations in the E. coli DNA polymerase III might affect UV-induced mutagenesis.Using a phage λ mutation assay which can discriminate between targeted and untargeted mutations, we show that the polC74 mutator mutation (Sevastopoulos and Glaser 1977) primarily affects untargeted mutagenesis, which occurs in a recA1 genetic background and is amplified in the recA+ genetic background. The polC74 mutation also increases the UV-induced mutagenesis of the bacterial chromosome. These results suggest that DNA polymerase III is involved in the process of UV-induced mutagenesis in E. coli.

Regulation of bacteriophage Mu transposition

Bacteriophage Mu is a transposon and a temperate phage which has become a paradigm for the study of the molecular mechanism of transposition. As a prophage, Mu has also been used to study some aspects of the influence of the host cell growth phase on the regulation of transposition. Through the years several host proteins have been identified which play a key role in the replication of the Mu genome by successive rounds of replicative transposition as well as in the maintenance of the repressed prophage state. In this review we have attempted to summarize all these findings with the purpose of emphasizing the benefit the virus and the host cell can gain from those phage-host interactions.