Temporal σB stress-response profiles impact Bacillus subtilis fitness

ABSTRACT The Gram-positive model organism Bacillus subtilis responds to environmental stressors by activating the alternative sigma factor σB. The sensing apparatus upstream of σB activation is thought to consist of cytoplasmic stressosomes—megadalton-sized protein complexes that include five paralogous proteins known as RsbRs. The RsbRs are presumed to be involved in stress sensing and the subsequent response. Perturbations to the RsbR complement in stressosomes by engineering cells that produce only one of the RsbR paralogs (“single-RsbR strains”) lead to altered σB response dynamics with respect to timing and magnitude. Here, we asked whether such changes to σB response dynamics impact the relative fitness of a strain. We competed strain pairs with different RsbR complements under ethanol and sodium chloride stress and found not only differences in relative fitness among wild-type and single-RsbR strains but also different relative fitness values in the two different stressors. We found that the presence of RsbRA, which dominates the wild-type σB response, enhances fitness in ethanol but is detrimental to fitness in NaCl. Meanwhile, RsbRD-only cells were among the most fit in NaCl. Strains producing hybrid RsbR fusion proteins displayed different fitness values that depended on the RsbR proteins from which they were derived. Our results here suggest that σB response dynamics can impact fitness, highlighting the physiological importance of the unusual stressosome-based general stress response system of B. subtilis. IMPORTANCE The model bacterium Bacillus subtilis uses cytoplasmic multiprotein complexes, termed stressosomes, to activate the alternative sigma factor σB when facing environmental stresses. We have previously shown that genetically manipulating the complement of putative sensor proteins in stressosomes can alter the dynamics of the σB response in terms of its magnitude and timing. However, it is unknown whether these response dynamics impact the fitness of cells challenged by environmental stressors. Here, we examine the fitness of strains with different σB responses by competing strain pairs in exponential-phase co-cultures under environmental stress. We find that strains with different response dynamics show different competitive indices that differ by stressor. These results suggest that the dynamics of the σB response can affect the fitness of cells facing environmental stress, highlighting the relevance of different σB dynamics.


Figure S2. Analysis of cell chaining in co-cultures treated with ethanol or
NaCl.An RsbRA-only strain carrying constitutive green fluorescent protein (GFP; CSS415) was co-cultured with an RsbRDonly strain carrying constitutive red fluorescent protein (mKate2; CSS412) in the presence of 4% ethanol or with 1 M NaCl for 9 h in exponential phase before fluorescence imaging on agarose pads. A.
Representative images of cells at t = 9 h of culture in ethanol or NaCl as noted.Both images are at the same scale.B. Histogram of cell chain length as manually determined in fields taken from the NaCltreated sample; a "particle" is a unit of one or more connected cells.

Detailed Modes of Strain Construction
The markerless gene replacement in the strains below were added in succession using the pminiMAD vector (gift of Daniel Kearns).A pminiMAD vector propagated in E. coli and containing the desired gene was directly transformed into PY79 via competence and selected on MLS (0.5 μg/ml erythromycin and 2.5 μg/ml lincomycin).A phage SPP1 lysate was prepared from that intermediate strain, and the recipient strain was phage-transduced with the PY79 strain containing the desired chromosomally integrated pMiniMAD vector and again selected on MLS.Five to 10 transductants were then inoculated into liquid LB and kept in exponential phase at approximately 25°C for several hours to permit plasmid excision before being repeatedly diluted and grown in liquid LB at 37°C (restrictive for plasmid replication) to promote loss of excised plasmid.The cells were then plated, and single colonies were screened for the successful replacement by colony PCR, restreaked for single colonies, patched on plain LB and LB/MLS plates to verify plasmid loss, restreaked, verified by PCR and stored at -80°C.Hybrid strain construction was performed as specified below using PY79 lysates from Hamm et al. (1).

CSS1264
MTC1693 was transduced with a lysate from a phage from PY79 pminiMAD-rsbRB/C as described above to produce 3610 ΔytvA ΔrsbRA ΔrsbRB ΔrsbRD rsbRC::RsbRB/C This strain was then transduced again with a constitutive amyE::Phyperspank-mKate2 reporter as described above.

CSS2066
MTC1697 was transduced with a lysate from a phage from PY79 pminiMAD-ΔsigB as described above to produce 3610 ΔsigB ΔytvA ΔrsbRB ΔrsbRC ΔrsbRD.This strain was then transduced again with a constitutive amyE::Phyperspank-mKate2 reporter as described above.

CSS2078
MTC1691 was transduced with a lysate from a phage from PY79 pminiMAD-ΔsigB as described above to produce 3610 ΔsigB ΔytvA ΔrsbRA ΔrsbRB ΔrsbRC.This strain was then transduced again with a constitutive amyE::Phyperspank-GFP reporter as described above.