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Starvation-induced Mucts62-mediated coding sequence fusion: a role for ClpXP, Lon, RpoS and Crp
S Lamrani1, C Ranquet, M J Gama
1Laboratoire de Génétique des Procaryotes, Département de Biologie Moléculaire, Université Libre de Bruxelles, 67 rue des Chevaux, B1640 Rhode St Genèse, Belgium.
Abstract:
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 sigma 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.
Insights
Bacterial adaptation involves Mu bacteriophage rearrangements, triggered by starvation and phage induction. Host proteases and RpoS regulate this process, enabling controlled transposition for survival.
Area of Science:
- Bacteriology
- Molecular Biology
- Genetics
Background:
- Bacterial chromosomal rearrangements can be induced by bacteriophage Mu, specifically the Mucts62 mutant.
- Fusion formation is linked to host physiology, occurring after aerobic carbon starvation and requiring phage transposase pA.
- These conditions suggest a role for Mucts62 prophage induction in fusion formation.
Purpose of the Study:
- To investigate the role of thermal induction in accelerating Mucts62-induced araB-lacZ fusion formation.
- To elucidate the physiological regulation of fusion formation under starvation conditions.
- To identify host and phage factors involved in prophage derepression and maintenance of the derepressed state.
Main Methods:
- Utilized Mucts62, a thermoinducible mutant of bacteriophage Mu, for inducing chromosomal rearrangements.
- Employed a transcriptional fusion indicator system with Mu early lytic promoter fused to the lacZ gene.
- Assessed the requirement of host proteases (ClpXP, Lon), RpoS, Crp, and phage proteins (pA, pB, Ner) in fusion formation and prophage regulation.
Main Results:
- Thermal induction of Mucts62 prophage accelerated araB-lacZ fusion formation, indicating derepression is rate-limiting.
- Starvation remained essential for fusion completion, highlighting additional physiological regulation.
- Stationary phase (S) derepression of Mucts62 prophage at low temperature depended on ClpXP, Lon, and RpoS, but not Crp; thermal induction bypassed these requirements.
Conclusions:
- The Mucts62 mutation allows the prophage to respond to host growth phase signals, influencing transposition.
- Host proteases (ClpXP, Lon) and RpoS are crucial for S derepression and maintaining the derepressed state.
- Crp is required for fusion formation only when phage protein pB is present, demonstrating complex regulatory interplay for adaptation.