An iron detection system determines bacterial swarming initiation and biofilm formation

Iron availability affects swarming and biofilm formation in various bacterial species. However, how bacteria sense iron and coordinate swarming and biofilm formation remains unclear. Using Serratia marcescens as a model organism, we identify here a stage-specific iron-regulatory machinery comprising a two-component system (TCS) and the TCS-regulated iron chelator 2-isocyano-6,7-dihydroxycoumarin (ICDH-Coumarin) that directly senses and modulates environmental ferric iron (Fe3+) availability to determine swarming initiation and biofilm formation. We demonstrate that the two-component system RssA-RssB (RssAB) directly senses environmental ferric iron (Fe3+) and transcriptionally modulates biosynthesis of flagella and the iron chelator ICDH-Coumarin whose production requires the pvc cluster. Addition of Fe3+, or loss of ICDH-Coumarin due to pvc deletion results in prolonged RssAB signaling activation, leading to delayed swarming initiation and increased biofilm formation. We further show that ICDH-Coumarin is able to chelate Fe3+ to switch off RssAB signaling, triggering swarming initiation and biofilm reduction. Our findings reveal a novel cellular system that senses iron levels to regulate bacterial surface lifestyle.

Swarming initiation is associated with changes in the expression of genes involved in the metabolism, acquisition and transport of iron in various bacterial species 14,22,23 . Iron limitation induces cell differentiation in swarming 24 , and disruption of the iron acquisition system affects swarming 25 . Additionally, iron chelation reduces biofilm formation, whereas iron overloading promotes biofilm formation [26][27][28] . Further identification of the sensor in response to environmental iron and downstream signaling may help us to understand the transition between swarming and biofilm formation.
Two-component systems (TCSs), typically composed of histidine sensor kinases and cognate response regulators, are among the most sophisticated signaling systems used by bacteria to sense and react to environmental stimuli. Control of phosphotransfer from membrane-bound histidine sensor kinases to response regulator in TCSs offers bacteria the ability to adapt to a wide range of environmental conditions [29][30][31] . We previously identified a TCS, called RssA-RssB (RssAB), whose activation is involved in coordinating the development of surface multicellularity as well as virulence in Serratia marcescens [32][33][34][35][36] . Here we show that RssA directly senses ferric iron (Fe 3+ ) via its periplasmic region, and that this interaction leads to RssB phosphorylation. The Fe 3+ chelator 2-isocyano-6,7dihydroxycoumarin (ICDH-Coumarin), whose biosynthesis is under transcriptional control of RssAB signaling, is shown to fine-tune RssAB-modulated swarming initiation and biofilm formation by controlling extracellular Fe 3+ availability. Our results show that extracellular iron sensing by a TCS regulates multicellular behaviors in bacteria.

Results
Fe 3+ regulates swarming initiation and biofilm formation. To examine whether iron regulates multicellular behavior, we used the wild-type (WT) S. marcescens CH-1 strain, which exhibits canonical swarming consisting of a non-motile lag phase and a motile migration phase 35 on Luria-Bertani (LB) swarming plates. We observed that ferric iron (Fe 3+ ) availability determines the timing of swarming initiation in the WT strain ( Fig. 1a,b), without affecting swarming expansion rate ( Supplementary Fig. 1). Fe 3+ depletion by the Fe 3+ chelator deferoxamine mesylate (DFO) reduced lag phase duration and induced early swarming initiation (Fig. 1a,b). On the other hand, Fe 3+ supplementation (100 μ M) prolonged the lag phase and delayed swarming initiation, which was restored by co-administration of Fe 3+ and DFO (Fig. 1a,b). Of note, when growing on iron-limiting, defined minimal medium (DMM) swarming plates, no swarming lag phase was observed, while addition of Fe 3+ dose-dependently extended the lag phase to 2 hrs in WT bacteria (Fig. 1c). These results suggest that Fe 3+ may control swarming initiation.
The inverse relationship between swarming motility and biofilm formation 8 led us to examine the effects of iron on biofilm formation. As expected, Fe 3+ supplementation increased biofilm formation in a dose-dependent manner, and the effect of Fe 3+ supplementation was inhibited by the addition of the Fe 3+ chelator DFO (Fig. 1d). We concluded that extracellular Fe 3+ concentration controls swarming initiation and biofilm formation in S. marcescens.
The TCS RssAB is required for Fe 3+ -mediated regulation of swarming initiation and biofilm formation in S. marcescens. We previously reported that the TCS RssAB is temporally activated during the swarming lag phase and delays swarming initiation in S. marcescens 35 . We thus investigated whether RssAB mediates the effect of Fe 3+ on swarming initiation. Deletion of the rssBA locus in WT S. marcescens abolished the effects of both iron and iron chelators on swarming initiation, and the rssBA deletion mutant (Δ rssBA) constitutively displayed early swarming initiation on LB swarming plates, 1 hr earlier than the WT strain (Fig. 1a,b and Supplementary Fig. 2; Δ rssBA). In addition, the ability of Fe 3+ to prolong the swarming lag phase on iron-limiting DMM swarming plates and to induce biofilm formation were not detected in the Δ rssAB strain (Fig. 1c,d;  ΔrssBA). Episomal expression of a wild-type RssB-RssA construct in Δ rssBA bacteria complemented the iron responsiveness for both swarming ( Fig. 1e and Supplementary Fig. 3) and biofilm formation (Fig. 1f). However, expression of RssB-RssA constructs harboring mutations at conserved phosphorylation sites, either at aspartate 51 (D51) for RssB or histidine 248 (H248) for RssA 36 , failed to rescue iron irresponsiveness in Δ rssBA bacteria, when either swarming ( Fig. 1e and Supplementary Fig. 3) or biofilm formation was analyzed (Fig. 1f). These results demonstrate that RssAB signaling is responsible for the effects of Fe 3+ on swarming initiation and biofilm formation in S. marcescens.
Fe 3+ activates RssAB signaling during swarming and biofilm formation. To investigate whether environmental Fe 3+ modulates RssAB, we monitored RssAB signaling in response to Fe 3+ availability by examining the cytolocalization of enhanced green fluorescent protein (EGFP)-tagged RssB during swarming and biofilm formation (Fig. 2a) 35 . On LB swarming plates, dispersal of EGFP-RssB in the cytosol, which indicates activation of RssAB signaling (ON), was observed 2 hr in the swarming lag phase ( Fig. 2b; LB-2 hr). During the surface migration phase of swarming, EGFP-RssB was detected at the cell membrane, which represents the resting state (OFF) of RssAB ( Fig. 2b; LB-4, 6, 8 hr). While Fe 3+ supplementation extended RssAB signaling activation to 4 hr RssA whose periplasmic domain was replaced with the periplasmic domain of QseC. The results represent means ± SEM from three independent experiments (n = 3). Statistical analysis was performed using one-way ANOVA. For Fig. 1d, *, ** and *** represent P < 0.05, 0.01, 0.001, and 0.0001, respectively, compared to the untreated sample. # and ## represent P < 0.05 and 0.01, respectively, compared to the group treated with DFO but without Fe 3+ . For Fig. 1f, *P < 0.05; **P < 0.01 compared to LB group.
( Fig. 2b; Fe 3+ ), DFO-mediated Fe 3+ depletion resulted in constitutive OFF signaling during the entire swarming period ( Fig. 2b; DFO). Addition of both Fe 3+ and DFO in LB swarming plates did not affect RssAB signaling state, indicating that the lag period was specifically extended by Fe 3+ on LB swarming plates ( Fig. 2b; Fe 3+ /DFO). In contrast to LB swarming plates, RssAB signaling was constitutively OFF on iron-limiting DMM swarming plates (Fig. 2c), where no lag phase was observed (Fig. 1c). Iron supplementation induced RssAB signaling and dose-dependently extended the duration of RssAB activation up to 2 hrs (Fig. 2c), which also prolonged the duration of the lag phase in swarming development (Fig. 1c).
We previously showed 35 that RssAB signaling is specifically activated during the early stage of biofilm formation ( Fig. 2d; LB-12 hr) and is deactivated in mature biofilms ( Fig. 2d; LB-24, 36 and 48 hr). Here, we found that, while addition of Fe 3+ did not change the timing of RssAB activation ( Fig. 2d; Fe 3+ ), Fe 3+ depletion by DFO abrogated activation of RssAB signaling ( Fig. 2d; DFO), and this effect could be restored by Fe 3+ supplementation ( Fig. 2d; Fe 3+ /DFO). Together with the observation that RssAB signaling is required for Fe 3+ -mediated modulatory effects on swarming and biofilm formation (Fig. 1e,f and Supplementary Fig. 3), we conclude that Fe 3+ controls RssAB signaling to regulate swarming and biofilm formation.
RssAB directly senses Fe 3+ at the nanomolar level. To examine how Fe 3+ affects RssAB signaling, we first studied RssAB signaling in response to Fe 3+ addition in iron-limiting DMM broth, which allowed us to assess the status of RssAB signaling in real time. While RssAB signaling was constitutively OFF in DMM broth ( Supplementary Fig. 4a), addition of Fe 3+ immediately activated RssAB signaling for at least 60 min, and this effect was reverted by DFO ( Supplementary Fig. 4b). Of note, replacement of Fe 3+ -treated bacterial culture broth with mock-treated culture broth deactivated RssAB signaling ( Supplementary Fig. 4c), indicating that extracellular Fe 3+ alters the state of RssAB signaling. We further demonstrated that Fe 3+ at a concentration of 50 nM was sufficient to activate RssAB signaling ( Supplementary Fig. 4d), consistent with the observation that 50 nM Fe 3+ could prolong the duration of the lag phase on DMM swarming plates ( Fig. 1c; WT).
To address whether Fe 3+ triggers RssAB transphosphorylation, we performed liposome-based radiography phosphorylation assays by reconstituting purified His-tagged RssA into liposomes under various iron conditions. We found that as soon as 1 min after exposure to [γ 32 P]ATP, autophosphorylation of RssA occurred in the presence of Fe 3+ , followed by phosphotransfer to RssB (Fig. 3a). Fe 3+ -induced RssAB transphorylation was inhibited by co-treatment with the Fe 3+ chelator DFO (Fig. 3b). While the presence of Fe 2+ triggered RssA autophosphorylation, co-treatment with the reducing reagent ASC or the Fe 3+ chelator DFO (but not the Fe 2+ chelator 2,2′-DP) prevented RssA autophosphorylation (Fig. 3b). Importantly, Fe 3+ -induced phosphorelay was largely dependent on the conserved phosphorylation sites of RssA and RssB (Fig. 3b), consistent with our observation that only the expression of functional RssAB could restore the effects of Fe 3+ on swarming migration ( Fig. 1e and Supplementary Fig. 3), biofilm formation (Fig. 1f), and signaling activation ( Supplementary Fig. 5) in Δ rssBA bacteria.
As the periplasmic region of sensor kinases is generally responsible for sensing environmental cues 31 , we prepared a plasmid construct harboring RssA without the periplasmic domain (RssA ΔPPD ) to test its function. Expression of RssA without the periplasmic domain (RssA ΔPPD ) failed to rescue the phenotypes of Δ rssBA to Fe 3+ in swarming initiation ( Fig. 1e and Supplementary Fig. 3), biofilm formation (Fig. 1f), and RssAB signaling ( Supplementary Fig. 5). Using the 55 FeCl 3 binding assay, we observed that full-length RssA could bind to Fe 3+ , whereas RssA ΔPPD could not (Fig. 3c,d). Additionally, free Fe 3+ , in the absence of DFO or ASC, could directly bind to the purified periplasmic domain of RssA, indicating that the periplamic domain of RssA is responsible for Fe 3+ binding (Fig. 3d). To test the specificity of the RssA periplasmic domain to Fe 3+ , we constructed a chimeric RssA (RssA chimeric ) in which the periplasmic region of RssA was replaced by the corresponding region of QseC, a sensor kinase not involved in iron sensing 38 . RssA chimeric did not interact with Fe 3+ (Fig. 3c) and failed to restore the swarming lag period (Fig. 1e, Supplementary Fig. 3), biofilm formation (Fig. 1f), or Fe 3+ responsiveness ( Supplementary Fig. 5) in Δ rssBA bacteria. Collectively, these results indicate that Fe 3+ directly and specifically binds to the periplasmic region of RssA, thereafter triggering RssAB signaling and regulating swarming initiation and biofilm formation.
Identification of the RssAB-regulated pvc cluster and its involvement in swarming and biofilm formation. To investigate whether RssAB signaling regulates extracellular Fe 3+ availability, we performed an in vitro protein-DNA pull-down screening assay to identify genes involved in iron metabolism. We identified the promoter of the gene sma0021, annotated as pvcA, which is the first gene of the putative pvc cluster (Fig. 4a). The pvc cluster in S. marcescens is a homologue of the pvc operon in Pseudomonas aeruginosa, which was previously found to be involved in biosynthesis of pseudoverdine, a metabolite that possesses Fe 3+ chelation activity 39 . Clarke-Pearson et al. observed that the pvc operon is responsible for the production of 2-isocyano-6, 7-dihydroxycoumarin (ICDH-Coumarin), named by the authors as paerucumarin 40,41 , which regulates biofilm formation in P. aeruginosa 42 . Using an electrophoretic mobility shift assay (EMSA), we confirmed direct binding of the pvcA promoter to phosphorylated GST-RssB-P, instead of unphosphorylated GST-RssB D51E or GST protein (Fig. 4b). We further showed that expression of pvcA in WT S. marcescens is down-regulated during the lag phase (2 hr), whereas it increases during the migration phase (4 hr) ( Fig. 4c and Supplementary Fig. 7), in agreement with our previous observation that RssAB signaling is specifically activated in the lag phase to act as a transcriptional repressor (Fig. 2b). During swarming development, iron supplementation prolonged downregulation of the RssAB downstream genes flhDC 34 and pvcA in an RssAB-dependent manner ( Fig. 4c and Supplementary Fig. 7).
To understand the function of the pvc cluster in multicellular behavior, we constructed a whole pvc cluster deletion mutant (Fig. 4a). Compared to the WT strain, the pvc cluster deletion mutant (Δ pvc) showed delayed swarming initiation (Fig. 4d) and increased biofilm formation (Fig. 4e), with both processes being reversed by episomal expression of the pvc cluster (Fig. 4d,e; Δ pvc/pPvc). In contrast, the rssBA and pvc cluster double-deletion mutant exhibited early swarming initiation and reduced biofilm formation as observed in Δ rssBA (Fig. 4d,e; Δ rssBA-pvc). These data suggest that a metabolite produced by the pvc cluster may inhibit RssAB activation.
The pvc cluster is responsible for ICDH-Coumarin production in S. marcescens. To determine whether the pvc cluster in S. marcescens is responsible for the production of a molecule similar to pseudoverdine or paerucumarin identified in P. aeruginosa, we used liquid chromatography-mass spectrometry (LC-MS) (Fig. 5a, Supplementary Fig. 8a) and nuclear magnetic resonance (NMR) (Supplementary Fig. 8b) to identify the compounds that were enriched in S. marcescens over-expressing the pvc cluster (pPvc). A compound corresponding to 2-isocyano-6,7-dihydroxycoumarin (ICDH-Coumarin) (with the same structure as paerucumarin) was identified ( Fig. 5a; highlighted as *; Supplementary Fig. 8) 40 ; the ICDH-Coumarin compound was not observed in Δ pvc bacteria expressing the vector only. The purified ICDH-Coumarin harbored Fe 3+ chelation activity similar to that of DFO but to a lesser extent (Fig. 5b). ICDH-Coumarin prevented direct binding of Fe 3+ to the periplasmic domain of RssA (Fig. 5c). These findings demonstrate that the pvc cluster is implicated in ICDH-Coumarin production and that ICDH-Coumarin can chelate Fe 3+ to abolish RssA-Fe 3+ binding.

ICDH-Coumarin controls RssAB signaling and multicellular behaviors by modulating extracel-
lular Fe 3+ availability. We aimed to determine whether ICDH-Coumarin might alter extracellular iron availability and subsequently regulate RssAB signaling as well as multicellular behaviors. While addition of 30 μ M ICDH-Coumarin restored the delayed swarming migration phenotype of Δ pvc and induced early swarming migration in WT S. marcescens similar to DFO ( Fig. 6a; 30 μ M), supplementation of 300 μ M ICDH-Coumarin induced early swarming migration even in Δ pvc bacteria ( Fig. 6a; 300 μ M ICDH-Coumarin for Δ pvc). Moreover, early swarming initiation induced by ICDH-Coumarin was accompanied by deactivation of RssAB signaling in both WT and Δ pvc bacteria (Fig. 6b). The effects of ICDH-Coumarin supplementation on swarming initiation and RssAB signaling could also be observed by overexpression of the pvc cluster ( Supplementary Fig. 9a,b). Conversely, regulation of swarming initiation by ICDH-Coumarin or pvc overexpression was completely abolished in the absence of rssBA (Fig. 6a and Supplementary Fig. 9a,b). On the other hand, addition of ICDH-Coumarin induced early swarming initiation (Fig. 6c) and impaired biofilm formation similar to DFO treatment (Fig. 6d), and these effects could be restored by addtion of Fe 3+ . Taken together, our results demonstrate that ICDH-Coumarin is produced by the RssAB-regulated pvc cluster and that it regulates swarming and biofilm formation by altering extracellular Fe 3+ availability and RssAB signaling.

Discussion
Swarming and biofilm formation are two opposite but inter-related bacterial behaviors that are also among the most ancient features of living cells 10 . Here, we demonstrate that environmental Fe 3+ availability controls the transition between swarming initiation and biofilm formation through an RssAB signaling system in S. marcescens. We further determine that the RssAB-modulated pvc cluster produces the Fe 3+ chelator ICDH-Coumarin to regulate extracellular iron availability and RssAB signaling (Fig. 7). Our results show that RssAB signaling is off at low Fe 3+ concentrations, during which the pvc cluster is expressed to produce ICDH-Coumarin and chelate extracellular Fe 3+ (Fig. 7a). In an environment in which Fe 3+ is abundant, Fe 3+ directly binds to RssA, leading to RssA autophosphorylation and RssAB transphosphorylation, resulting in downregulated expression of the pvc cluster, reduced ICDH-Coumarin production, and decreased extracellular Fe 3+ chelation (Fig. 7b).

Figure 4. pvc cluster regulated by RssB is involved in regulating swarming and biofilm formation. (a)
Schematic map of the pvc cluster, RssB-P binding site, and pvc cluster deletion mutant. Red dash lines represent RssB-P binding sites (− 349 to + 38) of the pvcA promoter region. For construction of pvc cluster deletion mutants (Δ pvc), genomic region between two asterisks (*) was replaced with Sm r cassette. (b) EMSA was employed to confirm the interaction between phosphorylated RssB (RssB-P) and promoter region of pvcA (P pvcA ). Digoxigenin (DIG)-labeled DNA fragments were incubated with purified GST, GST-RssB D51E or GST-RssB-P, followed by analysis by non-denaturing PAGE. Negative control (NC) was performed by incubating GST-RssB-P with the DNA sequence between M13F/M13R in the plasmid pBIISK. Bacteria utilize a broad array of strategies to control the timing and duration of TCS signaling events in order to precisely control cellular processes based on extracellular signals 43 . In the context of the RssAB-ICDH-Coumarin-iron regulation circuit (Fig. 7), S. marcescens actively regulates extracellular iron availability through RssAB-modulated production of ICDH-Coumarin. Upon sensing high extracellular Fe 3+ concentration, the decrease in ICDH-Coumarin production by transcriptional repression of phosphorylated RssB in turn maintains active RssAB signaling, which restricts bacterial migration and promotes biofilm formation. Of note, we previously reported that RssAB activation suppresses bacterial swarming motility by repressing flhDC expression 34 , whereas overexpression of flhDC reduces biofilm formation 33 . Together with the results presented in this study that functional RssAB signaling is required for iron to downregulate flhDC expression, restrict swarming migration and promote biofilm formation, we highlight the pivotal role of iron-RssAB-FlhDC signaling in regulation of swarming and biofilm formation (Fig. 7). These results also indicate that tight regulation of flagellum production by RssAB-ICDH-Coumarin-iron is crucial for the development of multicellular behavior in S. marcescens.
Based on LC-MS and NMR analyses, ICDH-Coumarin secreted by S. marcescens (Fig. 5 and Supplementary  Fig. 8) and identified in this study has the same molecular structure (2-isocyano-6,7-dihydroxycoumarin) as paerucumarin in P. aeruginosa 40,41 . While ICDH-Coumarin (paerucumarin) enhances biofilm formation in P. aeruginosa by upregulating the fimbrial synthesis pathway 42 , we demonstrated here that ICDH-Coumarin reduces biofilm formation in S. marcescens. The different roles of this iron-chelating molecule in controlling multicellular behavior in P. aeruginosa and S. marcescens indicate that different cellular machineries may have evolved in response to a specific extracellular signal. Conservation of the pvc cluster across various bacterial species 40 and the function of ICDH-Coumarin in regulating bacterial behavior suggest that ICDH-Coumarin may be involved in interspecies communication.
Competition for iron between microbes in the environment usually involves the coordination of various bacterial activities, including oxidative stress response, antibiotics resistance, virulence and multicellular behavior [44][45][46][47] . Previously, the TCS PmrA-PmrB was found to be vital for survival of Salmonella enterica under high iron stress through direct sensing of extracellular iron 29 . It was further shown that high iron resistance is mediated by lipopolysaccharide modifications 48 . Here we show that RssAB participates in a sophisticated control system to regulate multicellular behavior without conferring iron resistance since rssBA deletion does not affect growth in either iron-abundant or iron-limiting conditions. The presence of multiple sensing systems in deciphering iron availability may provide flexibility for bacteria to thrive under changing environments. In summary, this study identifies a cellular mechanism underlying the transition between bacterial motility and static colonization, which are associated with acute and chronic infection, respectively, in response to extracellular iron availability. Our findings should prove helpful to understand the factors that determine bacterial acute or chronic infection as well as for the development of novel treatments against pathogenic bacteria.

Methods
Bacterial strains and culture conditions. S. marcescens strains were derived from S. marcescens CH-1 (WT). Bacteria were routinely cultured with agitation in LB broth (BD Difco TM , U.S.A.) at 30 °C or 37 °C. M9 salt (BD Difco TM , U.S.A.) solution 49 was used to make defined minimal medium (DMM) containing 1× M9 salts, 2 mM magnesium sulfate, 100 μ M calcium chloride, 0.8% glycerol, and 0.2% casamino acid. DMM was used as iron-limiting medium. When strains harboring pBAD series of plasmids were used, arabinose was added into the medium at the indicated concentrations. Bacterial strains and plasmids are summarized in Supplementary Table  1   (d) Biofilm of WT and Δ pvc in LB condition supplemented with or without Fe 3+ (100 μ M) and ICDH-Coumarin (300 μ M) was determined by monitoring absorbance at 630 nm. The results shown represent means ± SEM from three independent experiments (n = 3). One-way ANOVA with * and ** represent P values < 0.05 and < 0.01 compared to LB group; # represents P-value < 0.05 compared to ICDH-Coumarin group.
In vitro protein-DNA pull-down assay. The assay used was modified from Dietz et al. 50 . Briefly, WT S. marcescens chromosomal DNA was digested by Sau3AI and resuspended in 1 ml of interaction buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 100 mM KCl) containing 25 mM acetyl-phosphate, 5 mM EDTA and 10 μ g/ml BSA. GST-RssB was phosphorylated by 50 mM acetyl-phosphate at 37 °C for 1 hr, prior to addition into the mixture containing WT S. marcescens chromosomal DNA fragments. After incubation at room temperature for 20 min, 30 μ L glutathione sepharose-4B beads equilibrated with PBS were added into the mixture. The whole mixture was placed at 4 °C with constant shaking for 30 min. The beads bound to GST-RssB with binding DNA fragments were recovered by low-speed centrifugation. After three washing steps with 500 μ l of interaction buffer, the DNA was purified by phenol extraction and precipitated with isopropanol. Following precipitation, the bound DNA was analyzed on 2% agarose gel, and cloned into the BamHI site of pBluscriptIISK. The GenBank accession number of pvcABC is KC291199.
Electrophoretic mobility shift assay (EMSA). Promoter region of pvcA (P pvcA ) and rssB (P rssB ) was cloned into the BamHI site on pBluescript II SK+ to generate pBSK-P pvcA and pBSK-P rssB, respectively. DNA fragments for electrophoretic mobility shift assay were amplified by PCR using the M13F-DIG/M13R primer pairs using pBSK-P pvcA , pBSK-P rssB or pBluescript II SK+ (negative control, NC) as a template. GST, GST-RssB and GST-RssB D51E protein purification, and GST-RssB phosphorylation using acetyl-phosphate (Sigma-Aldrich, U.S.A.) were performed as described in our previous report 35 . Phosphorylated GST-RssB or GST-RssB D51E was diluted in binding reaction buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 100 mM KCl) before binding assay. The binding reaction was performed in binding reaction buffer, comprising the protein as indicated and 0.5 ng DIG labeled DNA fragments supplemented with 30 μ g/ml poly (dI-dC) and 1 μ g/μ l bovine serum albumin. The reaction mixtures were incubated for 30 min at room temperature before being loaded onto 6% nondenaturing polyacrylamide gels containing 0.5× TBE buffer. Electrophoresis was performed at 100 V for 1-4 hr. The DNA-protein complexes were then electroblotted onto a positively charged Hybond-N nylon membrane (Amersham, U.S.A.) and detected using alkaline phosphatase conjugated anti-DIG antibodies (Roche Life Science, U.S.A.). CSPD (Roche Life Science, U.S.A.) was used for substrate as described by the manufacturer. Membranes were exposed to X-ray film at room temperature for 2 to 30 min.
Evaluation of gene expression. Total bacterial RNA was extracted using a Trizol kit (Invitrogen).
After verifying the quality (A260/A280 = 1.8-2.0) and concentration, 200 ng of RNA was subjected to reverse-transcription into cDNA with a SuperScript III First-Strand Synthesis System kit (Invitrogen, U.S.A.) according to the manufacturer's instructions. 5 ng of cDNA was then applied to KAPA SYBR FAST Master Mix (2X) qPCR kit (Kapa Biosystems, South Africa). Expression level of target genes tested was verified by real-time quantitative PCR detection system (Roche LightCycler 480, U.S.A.). Melting curves and Ct values were analyzed using the LightCycler ® 480 SW version 1.5 (Roche, U.S.A.). The data were analyzed using the 2 −ΔΔC T method 51 .
Relative expression of target genes was normalized to 16S rRNA (Fig. 4c) or rpoD (Supplementary Fig. 7). The procedures used for qRT-PCR followed the MIQE guidelines 52 . The primers used in this study are summarized in Supplementary Table 2.