Biochemical characterization of RssA-RssB, a two-component signal transduction system regulating swarming behavior in Serratia marcescens

Abstract

Our previous study had identified a pair of potential two-component signal transduction proteins, RssA-RssB, involved in the regulation of Serratia marcescens swarming. When mutated, both rssA and rssB mutants showed precocious swarming phenotypes on LB swarming agar, whereby swarming not only occurred at 37 degrees C but also initiated on a surface of higher agar concentration and more rapidly than did the parent strain at 30 degrees C. In this study, we further show that the predicted sensor kinase RssA and the response regulator RssB bear characteristics of components of the phosphorelay signaling system. In vitro phosphorylation and site-directed mutagenesis assays showed that phosphorylated RssA transfers the phosphate group to RssB and that histidine 248 and aspartate 51 are essential amino acid residues involved in the phosphotransfer reactions in RssA and RssB, respectively. Accordingly, while wild-type rssA could, the mutated rssA(H248A) in trans could not complement the precocious swarming phenotype of the rssA mutant. Although RssA-RssB regulates expressions of shlA and ygfF of S. marcescens (ygfF(Sm)), in vitro DNA-binding assays showed that the phosphorylated RssB did not bind directly to the promoter regions of these two genes but bound to its own rssB promoter. Subsequent assays located the RssB binding site within a 63-bp rssB promoter DNA region and confirmed a direct negative autoregulation of the RssA-RssB signaling pathway. These results suggest that when activated, RssA-RssB acts as a negative regulator for controlling the initiation of S. marcescens swarming.

FIG. 1.

Comparison of RssA blocks and RssB domains with two-component family proteins and purification of proteins used in this study. (A) Comparison of S. marcescens RssA with the conserved H, N, D/F, and G blocks of sensor proteins of two-component systems, including those of Bordetella pertussis BvgS, Neisseria gonorrhoeae BasS, and E. coli EvgS. (B) Comparison of S. marcescens RssB with the conserved phosphorylation and DNA-binding motifs of E. coli response regulators CpxR, OmpR, and PhoB. Asterisks indicate identical amino acids, and encircled asterisks indicate essential amino acid residues H248 in RssA and D51 in RssB for phosphorylation and phosphorelay reactions. (C) cRssA, RssB, cRssA(H248A), and RssB(D51E) were overexpressed and purified. Lanes 1, 8, and 10, protein molecular mass standards expressed in kDa; lanes 2 and 3, whole-cell lysates of E. coli BL21(DE3)/pET28cRssA before and after IPTG induction, respectively; lane 4, purified cRssA; lanes 5 and 6, whole-cell lysates of E. coli BL21(DE3)/pET28RssB before and after IPTG induction, respectively; lane 7, purified RssB; lanes 9 and 11, purified cRssA(H248A) and RssB(D51E), respectively. All proteins were purified from crude cell extracts after elution from Ni²⁺-nitrilotriacetic acid column affinity chromatography.

FIG.2.

Autophosphorylation of cRssA and cRssA(H248A) and transphosphorylation of RssB and RssB(D51E) by cRssA. (A) Purified cRssA was incubated with [γ-³²P]ATP, and samples were harvested at the time points indicated. At 45 min, an equal amount of RssB was added. Samples harvested were separated by 15% SDS-polyacrylamide gel electrophoresis before autoradiography. (B) The result of autoradiography was quantified by PhosphorImager analysis. (C) In vitro phosphorylation of the indicated proteins by [γ-³²P]ATP was carried out as described in Materials and Methods. Molecular mass markers (kDa) are shown, and proteins contained (+) in the reaction mixtures are shown in the list at the top. cRssA and cRssA(H248A) at concentrations of 10 μM were incubated with [γ-³²P]ATP for 1 hour, and an equal amount RssB or RssB(D51E) was added into the reaction mixtures for another hour. Reactions were stopped by the addition of 4× SDS sample buffer. Each reaction mixture (5 μl) was subjected to separation by 15% SDS-polyacrylamide gel electrophoresis, followed by visualization using Coomassie blue staining (upper panel) or autoradiography (lower panel). (D) Swarming phenotypes of S. marcescens strains CH-1 (I), CH-1ΔA (II), CH-1ΔA complemented with wild-type RssA (pBAD18RssA) (III), and CH-1ΔA complemented with mutant RssA [pBAD18RssA(H248A)] (IV) after 10 h of incubation on LB swarming plates at 37°C.

FIG. 3.

In vitro RssB-DNA gel mobility shift assay. (A) Purified RssB was used to test whether it interacted with DIG-labeled predicted promoter DNA regions, including shlB (I), ygfF_Sm (II), PrssBF-1/PrssBR-1 (III), PrssBF-2/PrssBR-2 (IV), and PrssBF-2/PrssBR-1 (V). Lane 1, DNA fragments only; lane 2, DNA fragments with purified RssB; lane 3, DNA fragments with purified RssB phosphorylated by Ac-P. (VI) Cold competition assay by unlabeled DNA fragment PrssBF-4/PrssBR-4 (lane 3) or ygfF_Sm (lane 4). Lane 1, labeled PrssBF-3/PrssBR-3 DNA fragment only; lane 2, PrssBF-3/PrssBR-3 with RssB-P only. (VII) Cold competition assay by unlabeled DNA fragment PrssBF-3/PrssBR-3. Lane 1, labeled PrssBF-3/PrssBR-3 DNA fragment only; for lanes 2 to 5, the competitive cold fragment concentrations were decreased from 50×, 10×, and 1×, respectively, to 0×. (B) rssB promoter region, where the locations of primers designed are shown with solid arrows. Arrows with filled circles indicate the predicted transcription and translation start sites. RssB-P binding DNA regions (63 bp) are shadowed.

FIG. 4.

Effect of in vitro phosphorylation to RssB on DNA-binding capability. We used the DIG-labeled 104-bp rssB promoter region amplified by PrssBF-3/PrssBR-3 to test the effect of RssB phosphorylation on binding efficiency and DNA mobility shift by (A) RssB and (B) RssB-P. Protein concentrations used are indicated at the bottom. (C) DNA mobility shift by RssB-P (0.02 μM) (lane 2), RssB (1.5 μM) (lane 3), and RssB (D51E; 10 μM) (lane 4) with the same DNA fragment. Lane 1, target DNA only, serving as the mobility shift control. Positions of DNA-protein complexes CI and CII are indicated with arrows.

FIG. 5.

rssB transcription level is elevated in S. marcescens CH-1ΔA. Primer extension (A) and RT-PCR (B) assays were performed for the quantification of rssB transcripts. Total cellular RNA was extracted from S. marcescens CH-1 and CH-1ΔA grown to log phase (OD₆₀₀ = 0.5) (lanes 1 and 3) and early stationary phase (OD₆₀₀ = 1.2) (lanes 2 and 4). Lanes 1 and 2, S. marcescens CH-1; lanes 3 and 4, S. marcescens CH-1ΔA.