Different roles of two groEL homologues in methylotrophic utiliser of dichloromethane Methylorubrum extorquens DM4
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The genome of methylotrophic bacteria Methylorubrum extorquens DM4 contains two homologous groESL operons encoding the 60-kDa and 10-kDa subunits of GroE heat shock chaperones with highly similar amino acid sequences. To test a possible functional redundancy of corresponding GroEL proteins we attempted to disrupt the groEL1 and groEL2 genes. Despite the large number of recombinants analysed and the gentle culture conditions the groEL1-lacking mutant was not constructed suggesting that the loss of GroEL1 was lethal for cells. At the same time the ∆groEL2 strain was viable and varied from the wild-type by increased sensitivity to acid, salt and desiccation stresses as well as by the impaired growth with a toxic halogenated compound—dichloromethane (DCM). The evaluation of activity of putative PgroE1 and PgroE2 promoters using the reporter gene of green fluorescent protein (GFP) showed that the expression of groESL1 operon greatly prevails (about two orders of magnitude) over those of groESL2 under all tested conditions. However the above promoters demonstrated differential regulation in response to stresses. The expression from PgroE1 was heat-inducible, while the activity of PgroE2 was upregulated upon acid shock and cultivation with DCM. Based on these results we conclude that the highly conservative groESL1 operon (old locus tags METDI5839-5840) encodes the housekeeping chaperone essential for fundamental cellular processes. On the contrary the second pair of paralogues (METDI4129-4130) is dispensable, but corresponding GroE2 chaperone promotes the tolerance to acid and salt stresses, in particular, during the growth with DCM.
KeywordsAerobic methylotrophic bacteria Chaperonins Dichloromethane 60-kDa heat shock protein Stress response
This work was supported by Russian Foundation for Basic Research (Grants 15-01-04458-a and 18-04-01148-a).
ML Torgonskaya designed the experiments, coordinated the study, analysed the DNA sequences of groESL operons and their upstream regions. YE Firsova carried out the generation of the mutant and reporter strains, characterised the phenotype of ∆groEL2 mutant and registered the GFP expression from promoters. Both authors contributed to data analysis and manuscript preparation. The final manuscript was reviewed and approved by both authors.
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Conflict of interest
The authors declare that they have no conflict of interest.
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