Dual control of Sinorhizobium meliloti RpoE2 sigma factor activity by two PhyR-type two-component response regulators

Abstract

RpoE2 is an extracytoplasmic function (ECF) sigma factor involved in the general stress response of Sinorhizobium meliloti, the nitrogen-fixing symbiont of the legume plant alfalfa. RpoE2 orthologues are widely found among alphaproteobacteria, where they play various roles in stress resistance and/or host colonization. In this paper, we report a genetic and biochemical investigation of the mechanisms of signal transduction leading to S. meliloti RpoE2 activation in response to stress. We showed that RpoE2 activity is negatively controlled by two paralogous anti-sigma factors, RsiA1 (SMc01505) and RsiA2 (SMc04884), and that RpoE2 activation by stress requires two redundant paralogous PhyR-type response regulators, RsiB1 (SMc01504) and RsiB2 (SMc00794). RsiB1 and RsiB2 do not act at the level of rpoE2 transcription but instead interact with the anti-sigma factors, and we therefore propose that they act as anti-anti-sigma factors to relieve RpoE2 inhibition in response to stress. This model closely resembles a recently proposed model of activation of RpoE2-like sigma factors in Methylobacterium extorquens and Bradyrhizobium japonicum, but the existence of two pairs of anti- and anti-anti-sigma factors in S. meliloti adds an unexpected level of complexity, which may allow the regulatory system to integrate multiple stimuli.

FIG. 1.

(A) Schematic representation of the S. meliloti chromosomal regions analyzed in this study. Arrows represent open reading frames, and the dotted arrow indicates the previously unannotated rsiA2 (SMc04884; chromosome coordinates 815835 to 816023). (B) DNA sequence of the 5′ region of rsiA1 (SMc01505), showing the previously annotated start codon (ATG in bold italic letters) and the first amino acids of the protein, as well as the 9-amino-acid N-terminal extension predicted from the present study (roman capital letters, with the new ATG start codon in bold). The position of the transcription start site mapped in the present study by 5′-RACE (see Materials and Methods) is indicated (+1), with the putative −35 and −10 promoter sequences recognized by RpoE2 underlined (33).

FIG. 2.

Induction of RpoE2-dependent transcriptional responses in various genetic backgrounds. The transcription level of the P_SMc00885-lacZ fusion carried on plasmid pMP220-885, used as a reporter of RpoE2 activity, was measured in the S. meliloti strain Rm1021 (WT), CBT208 (rpoE2), CBT557 (ΔrsiA2), CBT390 (ΔrsiB1), CBT392 (ΔrsiB2), or CBT430 (ΔrsiB1 ΔrsiB2), as indicated below the graph. β-Galactosidase activity was measured using aliquots of cultures grown to exponential phase at 28°C (white bars), after 1 h at 40°C (black bars), or to stationary phase at 28°C (dotted bars). Average values and standard deviations of results from at least three independent biological experiments are shown. Stars indicate significant induction relative to the log phase control at 28°C (Student's t test; P < 0.05).

FIG. 3.

RsiA1and RsiA2 are negative regulators of RpoE2. The transcription level of the P_SMc00885-lacZ fusion carried on plasmid pMP220-885 was measured in the wild-type S. meliloti strain Rm1021 carrying either the empty vector pMLBAD(−) or pMLBAD derivatives expressing RsiA1₅₅, RsiA1₆₄, or RsiA2, as indicated below the graph. Cultures were grown to exponential phase in the absence (white bars) or in the presence of either 0.2% (gray bars) or 2% (black bars) arabinose for 2 h and then incubated for a further 1 h at either 28°C or 40°C before β-galactosidase activity was measured. Results are expressed as the ratio of activities measured at 40°C versus 28°C. Average values and standard deviations of results from at least three independent biological experiments are shown. Stars indicate that the induction level is significantly lower relative to that of the control without inducer (Student's t test; P < 0.05). Note that the basal levels of expression of the fusion at 28°C were not significantly changed by the strain background or the presence of arabinose (Student's t test; P < 0.05).

FIG. 4.

Protein-protein interactions assessed by in vitro pull-down assays. RpoE2-HA (A), RsiB1-c-Myc (B), RsiB2 (C), RsiB1-D191A-c-Myc (D), or RsiB1-Nterm (E) was assayed for interaction with RsiA1 by loading corresponding E. coli cell lysates on a Strep-Tactin column bound or not with RsiA1-Strep (as indicated) and eluting with desthiobiotin as described in Materials and Methods. In panels B, C, and D, the tested lysates were preincubated or not with 100 mM acetyl-phosphate (acetyl∼P) as indicated. Elution fractions were separated by SDS-PAGE, and only the two fractions containing the largest amounts of proteins are shown (the same fractions are shown for all panels in a given experiment). Proteins were revealed either by Sypro Ruby staining (C and E) or by Western blotting using anti-HA or anti-c-Myc antibodies (A, B, and D). Every experiment was repeated at least twice independently.

FIG. 5.

RsiB1 and RsiB2 are positive regulators of RpoE2. The transcription level of the P_SMc00885-lacZ fusion carried on plasmid pMP220-885 was measured in the S. meliloti strains CBT390 (ΔrsiB1), CBT392 (ΔrsiB2), CBT430 (ΔrsiB1 ΔrsiB2), or CBT208 (rpoE2) carrying either the empty vector pMLBAD(−) or pMLBAD derivatives expressing RsiB1 (B1)_, RsiB2 (B2), the N-terminal domain of RsiB1 (B1-Nterm), or the D191A mutant derivative of RsiB1 (RsiB1-D191A), as indicated below the graphs. (A) β-Galactosidase activity was measured on aliquots of cultures grown to exponential phase in the presence of 2% arabinose for 2 h and then incubated for a further 1 h at either 28°C (white bars) or 40°C (black bars). In every strain at 40°C, the presence of either pMLBAD-RsiB1 or pMLBAD-RsiB2 led to an activity significantly higher than did the empty vector in the same condition (Student's t test; P < 0.05). (B) β-Galactosidase activity was measured on aliquots of the cultures before (0 h) (white bars) and after incubation for 2 h in the presence of 2% arabinose (2 h ara) (gray bars), as well as after a further 1 h of incubation at either 28°C (2 h ara + 1 h 28°C) (hatched bars) or 40°C (2 h ara + 1 h 40°C) (black bars), as described in Materials and Methods. In each panel, average values and standard deviations of results from at least three independent biological experiments are shown. Only the high β-galactosidase activity levels resulting from expression of RsiB1 or RsiB1-Nterm in the ΔrsiB1 ΔrsiB2 strain were significantly different from those measured in the same strain containing the empty vector (Student's t test; P < 0.05).

FIG. 6.

Model for the activation of S. meliloti RpoE2 in response to stress. In unstressed bacteria, RpoE2 is maintained in an inactive form by interaction with its anti-sigma factors RsiA1 and RsiA2. Under stress or starvation conditions, one or several yet-unknown histidine kinase(s) sense the stimuli and phosphorylate the C-terminal domain of RsiB1 and/or RsiB2. This results in the activation of the N-terminal “effector” domains of these proteins which become available to contact the anti-sigma factors RsiA1 and RsiA2 and relieve their inhibiting effect on RpoE2. Sharp and flat arrowheads represent positive and negative regulatory actions, respectively.