HspR mutations are naturally selected in Bifidobacterium longum when successive heat shock treatments are applied

Abstract

The development of molecular tools allowed light to be shed on several widespread genetic mechanisms aiming at limiting the effect of molecular damage on bacterial survival. For some bacterial taxa, there are limited tools in the genetic toolbox, which restricts the possibilities to investigate the molecular basis of their stress response. In that case, an alternative strategy is to study genetic variants of a strain under stress conditions. The comparative study of the genetic determinants responsible for their phenotypes, e.g., an improved tolerance to stress, offers precious clues on the molecular mechanisms effective in this bacterial taxon. We applied this approach and isolated two heat shock-tolerant strains derived from Bifidobacterium longum NCC2705. A global analysis of their transcriptomes revealed that the dnaK operon and the clpB gene were overexpressed in both heat shock-tolerant strains. We sequenced the hspR gene coding for the negative regulator of dnaK and clpB and found point mutations affecting protein domains likely responsible for the binding of the regulators to the promoter DNA. Complementation of the mutant strains by the wild-type regulator hspR restored its heat sensitivity and thus demonstrated that these mutations were responsible for the observed heat tolerance phenotype.

FIG. 1.

Cyclic selection of naturally occurring heat shock-tolerant mutants. The black bars represent the loss in viability at each heat shock treatment, expressed as a logarithm of the loss in viability (left-hand y axis). The limit of detection was reached at cycle 14. The black diamonds show the temperature of selection for each cycle (right-hand y axis).

FIG. 2.

Characterization of the newly isolated strains by measurement of their loss in viability after a 13-min heat shock at 59°C. The strains are shown in the figure without the NCC prefix. Strain NCC2705 is the wt strain. Strains NCC2912 and NCC2913 are independent heat shock-tolerant mutants. The bars show the mean values from 7 to 15 replicates (error bars show the 95% confidence intervals).

FIG. 3.

Global transcriptome analysis of the heat shock-tolerant strains compared to the wt strain NCC2705. The change in expression versus average intensity is plotted in gray for each gene, except for the dnaK operon (black) and the clpB gene (white). Measurements were performed in exponential phase before (A and D) or during (B and E) heat shock treatment and in stationary phase (stat.) (C and F) for both mutants versus the wt strain. The strains are shown in the figure without the NCC prefix. The BL numbers are gene locus tags.

FIG. 4.

Comparison of the changes in gene expression obtained with the two heat shock-tolerant mutants versus the wt strain. The change in expression for one strain versus the change in expression for the other strain is plotted in gray for each gene, except for genes showing more than threefold differences of transcript level in at least one of the strains (A) or in the two strains (B), which are plotted in black. Measurements were performed in exponential phase before (A) or during (B) heat shock treatment. The diagonal line depicts the position of genes showing the identical change in expression in both strains.

FIG. 5.

Positions of the mutations in the HspR regulator. (a) Schematic representation of the protein with the functional domains and the amino acid substitutions. N, N terminus; HTH, helix-turn-helix domain; W1 and W2, winged helix domains 1 and 2, respectively; C, C terminus. (b and c) Sequences of the mutations in strains NCC2912 (b) and NCC2913 (c). Underlined amino acids were conserved in Actinobacteridae. The boldface amino acids indicate that the residue interacts with the DNA molecule. Mutations in the DNA and protein sequences are indicated by white letters on black background.

FIG. 6.

Loss in viability of the complemented strains after a 13-min heat shock at 59°C. The strains are shown in the figure without the NCC prefix (NCC2705 is the wt strain, and NCC2912 is the complemented heat shock-tolerant mutant). The black triangles depict an increased expression of the wt HspR (hspR from strain NCC2705 [hspR-2705]) or mutated HspR (hspR from strain NCC2912 [hspR-2912]) obtained by three transcriptional levels of promoters. The bars show the means from four or five replicates (error bars show the 95% confidence intervals).

FIG. 7.

Analysis by qPCR of the dnaK-clpB regulon transcription level in the complemented strains in stationary phase without heat shock. The bars show the means from three biological replicates of the mutant strain NCC2912 without complementation (2912), with the highest expression of the wt HspR (hspR from strain NCC2705 [hspR-2705]) or the mutated HspR (hspR from strain NCC2912 [hspR-2912]) (error bars show the 95% confidence intervals). Results are expressed in comparison with three biological replicates of the wt strain NCC2705. Significant differences from the value for the wt strain are indicated when the P value is lower than 0.05 (*) or 0.01 (**).