Communicated by: Hiroji Aiba
Roles of cell division control factor SdiA: recognition of quorum sensing signals and modulation of transcription regulation targets
Version of Record online: 19 MAR 2014
© 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd
Genes to Cells
Volume 19, Issue 5, pages 405–418, May 2014
How to Cite
Shimada, T., Shimada, K., Matsui, M., Kitai, Y., Igarashi, J., Suga, H. and Ishihama, A. (2014), Roles of cell division control factor SdiA: recognition of quorum sensing signals and modulation of transcription regulation targets. Genes to Cells, 19: 405–418. doi: 10.1111/gtc.12139
- Issue online: 21 APR 2014
- Version of Record online: 19 MAR 2014
- Manuscript Accepted: 14 JAN 2014
- Manuscript Received: 31 OCT 2013
- Grant-in-Aid for Scientific Research. Grant Numbers: 21241047, 18310133, 25430173, 24710214
- MEXT (Ministry of Education, Culture, Sports, Science and Technology of Japan). Grant Number: S0801037
- Top of page
- Experimental procedures
- Supporting Information
In Gram-negative bacteria, N-acylhomoserine lactone (HSL) is used as a signal in cell–cell communication and quorum sensing (QS). The model prokaryote Escherichia coli lacks the system of HSL synthesis, but is capable of monitoring HSL signals in environment. Transcription factor SdiA for cell division control is believed to play a role as a HSL sensor. Using a collection of 477 species of chemically synthesized HSL analogues, we identified three synthetic signal molecules (SSMs) that bind in vitro to purified SdiA. After SELEX-chip screening of SdiA-binding DNA sequences, a striking difference was found between these SSMs in the pattern of regulation target genes on the E. coli genome. Based on Northern blot analysis in vivo, a set of target genes were found to be repressed by SdiA in the absence of effectors and derepressed by the addition of SSMs. Another set of genes were, however, expressed in the absence of effector ligands but repressed by the addition of SSMs. Taken together, we propose that the spectrum of taget gene selection by SdiA is modulated in multiple modes depending on the interacting HSL-like signal molecules.
- Top of page
- Experimental procedures
- Supporting Information
Quorum sensing (QS) is a bacterial cell–cell communication process by which bacteria communicate using extracellular signals called autoinducers (AIs). In the Gram-negative bacteria, three QS systems have been identified, which differ in the molecular species of QS signals. The best characterized QS signal is N-acylhomoserine lactone (HSL), referred to AI-1, which is involved as the language for intraspecies communication and in monitoring the own population density (Fuqua et al. 1996). The LuxI-family proteins synthesize these HSL signal molecules, whereas the LuxR-family proteins play a role in monitoring HSL signals (Waters & Bassler 2005). A variety of HSLs have been identified, each differing in length, oxidation, and saturation of the acyl chain. Each bacterium carries a pair of HSL synthetase and HSL sensor. For population density-dependent expression of luminescence, Vibrio fischeri LuxI produces AI-1 with the structure of N-3-oxohexanoy HSL (oxoC6-HSL) (Eberhard et al. 1981), whereas Vibrio harveyi produces AI-1 with the structure of N-butyryl HSL (C4 HSL) by LuxLM (Cao & Meighen 1989). Pseudomonas aeruginosa contains two pairs of HSL synthetase and HSL sensor, LasI/LasR, and RhlI/RhlR. LasI synthesizes N-(3-oxo-dodecanoyl)-L-HSL (oxoC12-HSL) (Pearson et al. 1994), whereas RhlI synthesizes N-butyryl-L-HSL (C4-HSL) (Pearson et al. 1995, 1997). The HSL sensors are considered to play roles in regulation of a wide range of environmental signal-response genes in a cell density-dependent manner.
Later another group of QS signals, referred to AI-2, was identified as the interspecies communication signal in a broad range of bacterial species (Bassler 2002). AI-2 is a furanosyl borate diester derived from cyclization of 4,5-dihydroxy-2,3-pentanedione (DPD), which is made from S-ribosylhomocysteine, an intermediate in the breakdown of S-adenosylhomocysteine (Schauder et al. 2001). Enterohemorrhagic E. coli (EHEC) is the agent responsible for outbreaks of bloody diarrhea. EHEC uses a QS regulatory system to sense the external condition within the host intestine and to activate the genes essential for intestinal colonization. EHEC produces another ill-characterized QS signal AI-3, which is used, together with the host-derived epinephrine, for cross-communication between E. coli and host animals. AI-3 is involved in the interkingdom communication with host animals (Sperandio et al. 2003). Indole was also predicted to be involved in cell–cell communication. Indole production is one of the unique characteristics of E. coli and is used as a diagnostic maker for distinguishing it from other enteric bacteria. Indole generated by E. coli inhibits QS system in P. aeruginosa, and E. coli mutants defective in indole production are not competitive in coculture with P. aeruginosa (Chu et al. 2012).
Bacterial species of the genera Escherichia and Salmonella are unique in that they have a LuxR homologue, SdiA, but they do not contain a LuxI homologue or any other enzyme family that can synthesize HSLs (Ahmer 2004). Because of the lack of LuxI-type enzyme for HSL synthesis, E. coli is considered to sense an as yet unidentified HSL-like signal produced by other bacteria in environment using SdiA sensor (Michael et al. 2001). SdiA was identified as the ‘suppressor of the cell division inhibitor’ that controls the transcription of the ftsQAZ operon involved in cell division (Wang et al. 1991; Garcia-Lara et al. 1996; Sitnikov et al. 1996; Weiss 2004). SdiA is composed of two domains, N-terminal effector-binding domain and C-terminal DNA-binding domain, and is considered to play as a transcription factor. Its crystal structure was solved using crystals that had a hexagonal space (Wu et al. 2008), and its direct interaction with HSL was proposed from modeling studies (Yao et al. 2006). Using the newly developed PS-TF (promoter-specific transcription factor) screening system, we have identified as many as 15 regulators, each monitoring a specific environmental factor or condition, are involved in regulation of the sdiA promoter (Shimada et al. 2013). This finding agrees with the observations that SdiA is involved in the variety of phenotypes including biofilm formation, motility, antibiotic sensitivity, and virulence expression (Eberl 1999; Wei et al. 2001b; Dyszel et al. 2010). Up to the present time, however, the only known regulation target of SdiA transcription factor is the ftsQAZ operon (Sitnikov et al. 1996; Yamamoto et al. 2001), which encodes the division assembly protein FtsQ, the protein FtsA for recruitment of FtsZ to Z-ring, and the tubulin-like cell division protein FtsZ, respectively (Hale & de Boer 1997).
In this study, we attempted to identify the full range of regulation targets of SdiA in E. coli. The most common signal-transduction mechanisms in E. coli are the one-component systems, in which a single polypeptide contains both an effector-binding sensory domain and a DNA-binding domain (Ishihama 2010, 2012). As the natural effector ligand for SdiA has not been identified, we first tried to identify an HSL-like effector ligand(s). For this purpose, we searched for effectors, which are recognized in vitro by purified SdiA, from a collection of 477 species of the chemically synthesized HSL analogues (Smith et al. 2003a; Igarashi & Suga 2011). This library was used for the selection of effectors that influence cell–cell communications in vivo of P. aeruginosa (Smith et al. 2003b; Chung et al. 2011). Here, we identify three ‘synthetic signal molecules’ (SSMs) that affect the binding in vitro of purified SdiA to the ftsQ promoter, the only known regulation target of SdiA. The influence of these three SSMs on the recognition pattern of SdiA on the E. coli genome was then analyzed using the improved genomic SELEX screening system (Shimada et al. 2005). As the recognition target genes of SdiA were found to be different depending on the effector species, transcription in vivo of the regulation target genes was examined in the presence and absence of the SSMs. Taken all the results together, we propose that SdiA is a unique transcription factor in E. coli, because SdiA interacts with multiple species of effector ligands and its promoter recognition specificity is modulated to various directions depending on the interacting effectors.
- Top of page
- Experimental procedures
- Supporting Information
Search for effector molecules affecting SdiA functions
Laboratory strains of E. coli do not synthesize HSL-type signals, the widely used QS signals in Gram-negative bacteria, but are capable of monitoring QS signals in environment supposedly produced by other bacteria. To identity HSL-type chemicals recognized by SdiA, we used a library of HSL analogues, which were chemically synthesized with the procedure of Igarashi and Suga (2011) (see Fig. S1 in supporting information for details of the library). In this study, we examined a total of 477 species from three libraries of HSL analogues: C4 library (96 different amines acylated with the C4 chain) (Fig. S1A in supporting information); C12 library (96 different amines acylated with the 3-oxoC12 HSL or C12 chain) (Fig. S1B in supporting information); and a total of 255 species of 5KH-13 library (52 lactone ring variants, each joined with the acyl chains of 4, 6, 8, 10, and 12 in length (Fig. S1C in supporting information).
The DNA-binding affinity and specificity of transcription factors often change after interaction with specific effector molecules, so-called inducers or corepressors. As an attempt for screening chemicals that affect SdiA functions, we tested the DNA-binding activity in vitro of purified SdiA to the only known target, ftsQAZ promoter. When 0.5 pmol of 300-bp-long fluorescent-labeled ftsQ promoter fragment was mixed with the increasing amounts (1–100 pmol) of SdiA for 15 min at 37 °C and subjected to PAGE, the fluorescent probe shifted to slowly migrating a smear band at the SdiA concentration above 15 pmol, indicating the formation of SdiA-ftsQ promoter complexes (Table 1A). We then examined possible influence of all the chemically synthesized HSL analogues on the gel-shift pattern of SdiA-promoter complexes. For this purpose, 5 pmol each of HSL analogues was added into the reaction mixture containing 0.5 pmol ftsQ promoter probe and a fixed amount (10 pmol) of SdiA protein. After 15-min incubation at 37 °C, the mixtures were directly subjected to gel-shift assay. Among a total of 477 species of HSL analogues tested, a significant difference in the gel-shift pattern was reproducibly observed for three HSL analogues, A4-30C12 (afterward referred to A), F12S-C12 (F), and K12-C12 (K) (Fig. 1). These HSL analogues with the binding activity to ftsQ promoter were hereafter referred to ‘synthetic signal molecules’ (SSMs). The smear gel pattern of SdiA-ftsQ promoter complexes implies either a weak affinity of SdiA to the ftsQ DNA probe or a conformational heterogeneity of SdiA-probe complexes. The SSM dose-depending increase in SdiA binding to the ftsQ DNA probe was observed for all three SSMs (Table 1B).
|SdiA (pmol)||ftsQ promoter (pmol)||SdiA-ftsQ promoter complex (%)|
|(A) In the absence of effectors|
|SdiA (pmol)||ftsQ promoter (pmol)||HSL analogues (pmol)||SdiA-ftsQ promoter complex|
|(B) In the presence of effectors|
|SdiA (pmol)||ftsQ promoter (pmol)||SSM-A SSM-F SSM-K (pmol)||SdiA-ftsQ promoter complex|
|(C) In the presence of two effectors|
In the presence of A, the SdiA-ftsQ promoter complex (SdiA-DNA complex-1) was observed apparently at the same position with that formed in the absence of SSMs (Fig. 2A), but the fluorescent intensity increased markedly (Table 1B), indicating that the affinity to the target promoter increased in the presence of A. Moreover, the set of DNA sequences recognized by SdiA-A complex was completely different with that by SdiA alone (see below). In the presence of authentic HSL (C4-HSL produced by Vibrio harveyi and Pseudomonas aeruginosa), the SdiA-HSL complex also moved to the same position with complex-1, and the affinity of SdiA to the ftsQ promoter probe increased significantly as in the case of effector A (Table 1B), suggesting that the influence of A on the structure of SdiA is similar to that of the authentic C4-HSL.
In contrast, the SdiA-ftsQ promoter complex formed in the presence of F migrated on PAGE to a different position (SdiA-DNA complex-2) (Fig. 2B). Likewise, the SdiA-ftsQ promoter complex formed in the presence of K moved to a different position (SdiA-DNA complex-3) (Fig. 2C). These findings altogether indicate the effector ligand-induced alteration in the form of SdiA-ftsQ promoter complex: the complex-1 (C1) in the presence of HSL and A; the complex-2 (C2) in the presence of F; and the complex-3 (C3) in the presence of K. The difference in mobility on PAGE might be due to the difference in the affinity of SdiA with different HSL chemicals or the difference in the structure of HSL-associated SdiA-DNA complexes.
After formation of C1 complex in the presence of A, it was converted into C2 by adding excess amounts of F (Fig. 2D). In contrast, the C2 complex was converted into C1 complex after subsequent addition of A. Likewise, the C1 complex was converted into C3 by adding excess amount of K, whereas the preformed C3 complex was converted into C1 by adding effector A (Fig. 2E). This finding suggests that all three SSMs (A, F, and K) bind to the same or over-lapping sites on the effector-binding N-terminal domain of SdiA protein. As an attempt to estimate the affinity difference between three SSMs, we next determined the SSM concentration-dependent conversion of SdiA-ftsQ promoter complexes. The conversion of C1 complex to C2 increased concomitant with the increase in SSM-F addition (Table 1C). Likewise, the conversion of C1 to C3 complex increased with the increase in SSM-K level (Table 1C). The concentration needed for conversion of the C1 complex was lower for SSM-F than SSM-K, indicating that the higher affinity to SdiA is higher for SSM-F than SSM-K.
The mobility shift of SdiA-ftsQ promoter complex was not observed in the presence of some well-known low-molecular-weight signal molecules such as cAMP, c-di-GMP, indole, and AI-2 (data not shown), furthermore supporting the conclusion that the binding of SSMs to SdiA is specific. Next possible influence of these SSMs on the DNA-binding specificity of SdiA was then examined by the genomic SELEX screening.
Genomic SELEX screening of SdiA-binding sequences
For the identification of genome DNA sequences that are recognized by E. coli SdiA, we employed the genomic SELEX screening system (Shimada et al. 2005), in which a library of E. coli genome DNA fragments of 200–300 bp in length was used for screening of DNA sequences with SdiA-binding activity instead of synthetic oligonucleotides with all possible sequences used in the original SELEX method (Ellington & Szostak 1990; Tuerk & Gold 1990; Singer et al. 1997). Into the mixture of E. coli genomic DNA fragments, fourfold molar excess of the purified His-tagged SdiA protein was added and the SdiA-DNA complexes were affinity purified. In the early stage of this genomic SELEX cycle, the SdiA-bound DNA fragments gave smear bands on PAGE as did the original genome fragment mixture. SdiA-associated DNA fragments were recovered and PCR amplified and subjected to next cycle of SELEX. After three SELEX cycles, DNA fragments with high affinity to SdiA were enriched, forming several discrete bands on PAGE.
For the identification of the whole set of SdiA-binding sequences, we then subjected the mixture of SELEX fragments to the SELEX-chip analysis. In this study, SELEX fragments were labeled with Cy5, whereas the original DNA library was labeled with Cy3. The mixture of fluorescent-labeled samples was hybridized to a DNA-tilling microarray (Oxford Gene Technology, Oxford, UK) (Shimada et al. 2008, 2011b; Teramoto et al. 2010). For elimination of the bias of library DNA, the ratio of fluorescence intensity bound to each probe between the test sample (Cy5) and the original library DNA (Cy3) was measured and plotted against the corresponding position on the E. coli genome (Fig. 3). On the DNA-tilling array used, the 60-bp-long probes are aligned along the E. coli genome at 105-bp intervals, and therefore approximately 300-bp-long SELEX fragments should bind to two or more consecutive probes. The height of SdiA-binding correlates with the affinity to the SdiA protein.
First, we carried out SELEX-chip analysis for SdiA alone in the absence of effector ligands. The location of SdiA-binding sites thus identified was classified into four groups: (i) type-A, intergenic spacers between bidirectional transcription units; (ii) type-B, intergenic spacers upstream of one transcription unit but downstream of another transcription unit; (iii) type-C, intergenic spacers located downstream of both transcription units; and (iv) type-D, inside open-reading frames. High-level peaks of SdiA binding are shown in Fig. 3A. The highest peak of SdiA binding was detected within type-A spacer between divergently transcribed yqjH and ygjI genes (69.27–69.29 min). YqjH is a FAD-containing NADPH-dependent ferric reductase that plays a role in the adaptation to iron starvation, whereas YqjI regulates expression of the yqjH gene together with the ferric uptake regulator Fur (Miethke et al. 2011; Wang et al. 2011). The binding of SdiA to this site was always detected in the absence and presence of SSMs albeit at different levels (see below). Besides the yqjH-yqjI operon, another type-A spacer between the yjiC and yjiD genes (98.14–98.18 min) was always detected in the absence and presence of SSMs. IraD, renamed from YjiD, interacts with RssB sRNA and inhibits proteolysis of RpoS sigma factor by ClpXP protease (Bougdour et al. 2008). In E. coli, transcription factors generally bind near the promoter region, and thus, we focused detailed analysis on the SdiA-binding sites located upstream of genes within 8 type-A and 10 type-B spacers.
By setting the cut-off level higher than 20% the level of the highest yqjH-yqjI peak, a total of 31 peaks were detected (Table 1A in supporting information), including the high-level peaks shown in Fig. 3A, of which 20 peaks were detected only in the presence of SdiA alone without HSL ligands. Among the total of 17 candidate genes (7 within type-A spacers and 10 within type-B spacers) under the control of SdiA alone, more than half encode the membrane-integrated transporters including yagG (sugar transporter), ydcS (polyamine transporter), gatA (galactitol PTS), mglC (methyl-galactoside transporter), ypjA (adhesion-like autotransporter), ascF (cellobiose/arbutin/salicin transporter), ygcS (transporter for unidentified substrate), yrbG (calcium/sodium:proton antiporter), and alsC (D-allose transporter). The genes coding for five transcription factors (YbeO, YgaV, AscG, YqjI, and GadW) are also included in this list, implying that a number of genes are indirectly controlled by SdiA. The ftsQAZ region formed a low-level peak (approximately 15% the highest peak at yqjH/yqjI) under the experimental conditions employed. Transcription of the ftsQAZ operon is also initiated together with the upstream mraZ operon consisting of 12 genes including the genes for cell division proteins, FtsL and FtsI. A low level of SdiA binding was also identified within this mraZ promoter region.
Influence of HSL analogues on the DNA recognition specificity of SdiA: in vitro SELEX-chip analyses
We next analyzed possible influence of the newly identified three SSMs: A, F, and K, as effectors on the DNA-binding activity and/or specificity of SdiA. For identification of the recognition targets of SdiA in the presence of each SSM, we again carried out the SELEX-chip analysis of SdiA-binding sequences in the presence of individual SSMs. In parallel, we also carried out SELEX-chip analysis of SdiA in the presence of authentic HSL. The set of high-level peaks of SdiA binding are shown in Fig. 3B–E (see also Table S1 in Supporting information). These SELEX-chip patterns are significantly different from that in the presence of SdiA alone (Fig. 3A), and moreover, a striking difference was observed between four SELEX-chip patterns in the presence of HSL and SSMs, indicating that each SSM modulates the DNA recognition properties of SdiA in a different mode.
In the presence of SSM-A, a total of 44 SdiA-binding sites were identified, of which 10 and 22 were located within type-A and type-B spacers, respectively (Table 1B in supporting information). In the presence of SSM-A, the pattern of SdiA-binding sites on the genome (Fig. 3B) was completely different from the SdiA pattern without the effector ligands (Fig. 3A). The list of 32 genes predicted to be under the control of SdiA-SSM-A complex includes a number of stress-response genes such as tig (peptidyl-prolyl cis/trans isomerase), rsfA (ribosome silencing factor), hchA (Hsp31 chaperone), yfcV (stress-response fimbriae), ypjA (stress-response adhesin), and sspA (stringent starvation protein). A number of the genes encoding cell surface components for cell–cell interaction are also included, such as hlyE (hemolysin E), yfcV (fimbrial-like adhesin), yqjH (siderophore interaction protein), ypfA (adhesion-like autotransporter), and fimA (major type-I fimbrin). Interestingly a high level of SdiA binding was detected in the promoter region of zipA encoding the Z-ring assembly factor, which initially interacts with FtsZ leading to assemble more then ten Z-ring components. The recognition pattern of SdiA-SSM-A complex (Fig. 3B) is similar to that in the presence of authentic HSL (Fig. 3E). Within the total of 44 peaks in the presence of SSM-A (Table S1B in supporting information) and of 39 peaks in the presence of authentic HSL (Table S1E in supporting information), overlapping was observed for 20 peaks. In good agreement with the similarity in DNA recognition patterns, the structure of SSM-A is similar to that of authentic HSL.
In the presence of SSM-F, a total of 26 SdiA-binding sites (5 sites within type-A spacers and 12 within type-B spacers [described in Table S1C in Supporting information]) with the peak level higher than 20% of the highest ygjH-ygjI peak were identified (Table S1C in supporting information), of which 7 were identified only in the presence of SSM-F. Noteworthy is that besides the set of stress-response genes, the genes related to translation were identified, including rsfA (ribosome silencing factor), glyW (tRNA-Gly), gltX (Glu-tRNA synthetase), and rrsG (16S rRNA). In the presence of SSM-K, a total of 37 SdiA-binding sites (14 sites within type-A spacers and 11 type-B spacers) were identified (Table S1D in Supporting information), of which 14 were detected only in the presence of SSM-K. As in the case of SSM-F, some genes related to translation appear to be under the control of SdiA, including thrS (Thr-tRNA synthetase), rrsG (16S rRNA), ileY (tRNA-Ile), rimP (ribosome maturation protein), rrsC (16S rRNA), and thrU (tRNA-Thr). These findings together suggest that upon expose to SSM-F and SSM-K, the DNA recognition properties of SdiA are modulated so as to control the genes for translation apparatus.
Taken together, we concluded that the target selection pattern of SdiA changes depending on the molecular species of HSL-like analogue. This finding indicates that SdiA is one of the unique transcription factors, because the activity and specificity of its regulatory function are under the control of multiple effector ligands. On the ftsQ promoter, we identified the SdiA-binding sequence of 29 bp in length, which contains a 18-bp-long core sequence, referred to SdiA box, with AAAA sequence at both ends, each being separated by a 10-bp-long spacer (Yamamoto et al. 2001). The SdiA-binding regions in the presence of HSL analogues contained SdiA box-like sequences with AT-rich segments, but the identification of SdiA-binding sites and of possible difference in the effector-dependent recognition sequences awaits foot-printing analyses.
Influence of HSL analogues on the DNA recognition specificity of SdiA: in vivo Northern blot analyses
To examine the influence of SSMs on the target selectivity of SdiA in vivo, we next carried out Northern blot analysis of mRNA levels for some representative target genes. Total RNA was extracted from E. coli grown in the absence and presence of each of three newly identified SSMs as well as the authentic HSL. On the basis of Northern blot pattern, the genes under the control of SdiA can be classified into two groups. The first group genes such as fimA (type-I bimbriae pilin), ypjA (adhesin-like autotransporer), zipA (cell division protein), and hchA (Hsp31 molecular chaperone) are transcribed in the absence of effector addition, but their mRNA levels decreased to various extent by the addition of SSMs (Fig. 4A). This group of genes must be expressed under the steady state of growing phase but repressed upon exposure to SSMs. In contrast, mRNA level of the second group genes including ybaO (uncharacterized DNA-binding transcription factor), yqiH (periplasmic pilin chaperone), acpP (acyl carrier protein for fatty acid biosynthesis), and rsfS (ribosomal silencing factor) was low in the absence of effectors but increased in the presence of HSL-like chemicals (Fig. 4B). This group of genes must be repressed under the normal growth conditions, but derepressed upon exposure to QS signals.
Two different modes of the regulation are generally in good agreement of the binding mode of SdiA as detected by SELEX-chip analysis. The level of SdiA binding in the absence of effector ligands is low for the first group targets that are expressed in the absence of SSMs, but high for the second group targets that are not expressed in the absence of SSMs (see Fig. 3). Taken together, we conclude that SdiA is a repressor for a set of target genes in the absence of effectors, but the expression of this group of genes is derepressed by the addition of SSMs; however, SdiA also plays as an activator for another set of genes but the expression of this group of genes becomes repressed after interaction with SSMs.
- Top of page
- Experimental procedures
- Supporting Information
Regulatory signals of SdiA function
Bacterial species of the genera Escherichia and Salmonella are unique in that they have a LuxR homologue, SdiA, but they do not contain a LuxI homologue or any other enzyme family that can synthesize HSLs (Ahmer 2004). SdiA was originally identified as a regulator of the ftsQAZ operon encoding the cell division apparatus, but several lines of evidence indicate the involvement of SdiA in control of various phenotypes such as biofilm formation and virulence expression (Eberl 1999; Wei et al. 2001b; Dyszel et al. 2010). The involvement of effector for control of the SdiA function was first indicated by the influence of a conditioned culture medium on the regulatory activity of SdiA (Garcia-Lara et al. 1996). Later, SdiA in Salmonella enterica serovar typhimurium was found to monitor AHLs produced by other bacterial genera (Michael et al. 2001; Smith & Ahmer 2003), and as a result, to activate the rck (resistance to complement killing) operon on a virulence plasmid and srg (SdiA-regulated gene) with unknown function (Ahmer et al. 1998; Smith & Ahmer 2003). These observations raised the hypothesis that E. coli also monitors an HSL-like signal(s) produced by other bacteria (Michael et al. 2001). Besides HSL, SdiA has been proposed to monitor other as yet unidentified external signals including indole (Lee et al. 2007). In this study, however, we failed to confirm the binding of a set of well-known signal molecules with SdiA protein.
One effective approach for the identification of signal molecules that are monitored by a LuxR-family protein is to examine the influence of synthetic HSL analogues on the expression of the genes under the control of test LuxR-family protein. Such approaches have been useful in other bacteria. For instance, Schaefer et al. (1996) examined a total of 25 species of chemically synthesized N-(3-oxohexanoyl)-HSL derivatives with alterations in the acyl side chain in luminescence induction in Vibrio fischeri and found that none showed a higher activity than the authentic AI-1, N-3-oxododecanoyl-HSL (3OC12-HSL). Passador et al. (1996) tested a total of 21 species of the acylated derivatives of L-homoserine lactone, L-homoserine thiolactone, and lactam analogues for their ability to act as autoinducers in Pseudomonas aeruginosa and concluded that the length of acyl chain is crucial in both in vivo activation of LasR-regulated target genes and in vitro binding to LasR.
Using a promoter trap library to screen for E. coli promoters whose expression is affected by synthetic N-hexanoyl-L-homoserine lactone (C6-HSL), six upregulated and nine downregulated promoters were identified, which also responded to synthetic 3-oxo-N-hexanoyl-L-homoserine lactone (3-oxo-C6-HSL) (Van Houdt et al. 2006). The HSL-dependent regulation of these promoters is eliminated by knockout of sdiA, supporting the functional role of different HSL signals on the regulation function of SdiA. By making a set of sdiA mutants in E. coli, Lee et al. (2009) identified that different sites on SdiA protein are involved in sensing HSL signals from other bacteria and its own signal indole. The three-dimensional structure of a complex between the N-terminal domain of E. coli SdiA and a candidate N-octanoyl-L-homoserine lactone (C8-HSL) has been calculated in solution from NMR data (Yao et al. 2006, 2007). The structural study indicated the association of SdiA with not only C8-HSL but also additional effectors such as xylose. Other HSL derivatives are also capable of acting as folding-switch effectors for SdiA. The observed structural heterogeneity of the bound ligand in the complex, together with the variety of HSL-type molecules that can apparently act as folding switches in the SdiA system, is consistent with the postulated biologic function of the SdiA protein as a detector of the presence of other species of bacteria.
One short-cut approach for the identification of the signal molecules that are recognized by E. coli SdiA is to test possible influence of HSL analogues on the DNA-binding activity in vitro of purified SdiA. In this study, we examined, for the first time, a systematic analysis of as many as 477 species of HSL analogues on the recognition and binding activity in vitro of DNA sequences by SdiA. Interestingly, two of three SSMs, F and K, were previously identified as QS inhibitors for P. aeruginosa (Smith et al. 2003a; J. Igarashi and H. Suga, unpublished data). In contrast, SSM-A was a novel compound, which was previously unidentified as neither QS agonist nor antagonist. In this study, these SSMs were examined for their influence on the gene selectivity by SdiA. The genomic SELEX screening herein described indicates that SdiA responds to various HSL analogues and alters its recognition targets depending on the signal species. This finding raises the possibility that in nature such as in host intestine, SdiA recognizes various species of the signal molecule released from co-existing bacteria, resulting in different mode of the genome expression for adaptation. Transcription in vivo of the candidate genes under the direct control of SdiA was then examined in the presence and absence of SdiA and in the presence and absence of effector ligands.
Functional response of SdiA to different signals
Microarray analysis in the presence of SdiA over-expression indicated more than threefold transcription elevation for as many as 75 genes including sdiA itself and the ftsQAZ genes and reduction for 62 genes including the motility and chemotaxis genes (Wei et al. 2001a). Over-expression of sdiA causes mytomycin resistance (Wei et al. 2001b). Likewise, SdiA over-expression triggers the resistance to some drugs such as fluoroquinolone and ceftazidime (Tavio et al. 2010). Over-expression of SdiA confers multidrug resistance and increased levels of AcrAB drug efflux pump (Rahmati et al. 2002). The change in genome expression pattern examined using plasmid-encoded over-expressed SdiA was, however, reported to be different from that observed with the genome-coded SdiA (Dyszel et al. 2010). The SdiA encoded by a single copy activates the genes (gadW, gadE, yhiD, and hdeA) for glutamate-dependent acid-resistance system and represses the genes encoding flagella and curli fimbriae. Accordingly, the bacterial motility and increased adherence to epithelial cells are enhanced in the sdiA mutant (Sharma et al. 2010). Several lines of evidence indicate that in response to HSLs, the genome expression pattern and phenotypical properties of E. coli are modulated by means of the HSL responsive transcriptional regulator SdiA. SdiA mutants with altered sensitivity to HSLs and indole reduced or increased biofilm formation (Lee et al. 2009). SdiA is necessary for enterohemorrhagic E. coli (EHEC) colonization of cattle (Hughes et al. 2010). Metagenome analysis indicated that HSLs within the mammalian gastrointestinal tract are produced by Bacteroidetes. One of the regulation targets of SdiA is the ydiV gene encoding an EAL domain-containing protein that is involved in degradation of c-di-GMP, a signal of cell–cell communication and growth switch between biofilm formation and planktonic growth (Jenal & Malone 2006). Expression of YdiV is induced by the addition of HSL (Zhou et al. 2008). These observations altogether indicate that SdiA regulates not only the ftsQAZ operon for cell division but also a number of genes for adaptation to the variety of stressful environmental conditions in nature and survival in host animals.
Transcription factors under the control of multiple effectors
Here, we provided both in vitro and in vivo evidence indicating the alteration of gene selectivity of SdiA by interaction with HSL analogues. Moreover, the spectrum of target gene selection by SdiA is modulated in multiple modes depending on the effector molecules. One unique finding of this study is that a single and the same transcription factor responds to multiple effector ligands.
Escherichia coli contains as many as 300 species of transcription factors, each monitoring a specific factor or condition in environments (Ishihama 2010, 2012). The majority of E. coli transcription factors belong to the one-component signal-transduction system, in which a single polypeptide contains both an effector-binding sensory domain and a DNA-binding domain. The activity of this group's transcription factors has been believed to be controlled by a single species of the effector ligand, that is, inducer or corepressor. Recently, however, the involvement of two effectors have been identified in a number of cases such as allantoin and glyoxalate for AllR (Hasegawa et al. 2008); arginine and lysine for ArgP (or IciA) (Marbaniang & Gowrishankar 2011); glyoxylate and pyruvate for IclR (Lorca et al. 2007); hypoxanthine and guanine for PurR (Houlberg & Jensen 1983); ATP and maltotriose for MalT (Schreiber & Richet 1999); and uracil and thymine for RutR (Shimada et al. 2007). One unique exception is TyrR that regulates a set of genes for synthesis and transport of aromatic amino acids. The activity and specificity of TyrR are regulated by tyrosine, phenylalanine, and tryptophan (Pittard 1996). Here, SdiA was identified as a novel type of transcription factors, of which the activity and specificity are controlled by multiple species of the effector ligand.
- Top of page
- Experimental procedures
- Supporting Information
Collection of HSL analogues
A library of AHL analogues was prepared by chemical synthesis according to the published procedure (Igarashi & Suga 2011). From this collection, we used in this study a total of 477 HSL analogues as shown in Supporting Information Fig. S1, which include AHL analogues with different chain length and different acyl conformation and AHL analogues with different lactone rings. The influence of these SSMs on the growth and pathogenicity of P. aeruginosa has been analyzed in detail (Smith et al. 2003b; Chung et al. 2011).
Bacterial strains and culture conditions
Escherichia coli BL21(DE3) [F- ompT hsd (rB- mB-) dcm gal λ(DE3)] (Studier & Moffatt 1986) was used for expression and purification of SdiA. E. coli K-12 BW25113 [lacIq rrnB lacZ hsdR araBAD rhabAD] and its otherwise isogenic mutant strain JW1901 lacking the sdiA gene were products of the Keio collection (Baba et al. 2006) and obtained from National Bio-Resource Center (National Institute of Genetics, Japan). Cells were cultured in LB medium or YESCA medium (Pratt & Silhavy 1998). When necessary, 100 μg/ml ampicillin and 50 μg/ml kanamycin were added in to the medium.
Expression and purification of the SdiA protein
The sdiA-coding sequence was amplified by PCR using E. coli K-12 W3350 genome DNA as a template, and after digestion with NdeI and NotI within the primer sequences, cloned into an expression vector pET21a(+) (Novagen) at the corresponding sites. His-tagged SdiA was expressed in pSdiA-transformed E. coli BL21(DE3) and affinity purified according to the standard purification procedure (Shimada et al. 2005; Yamamoto et al. 2005). The purity of SdiA was higher than 95% as judged by SDS-PAGE.
Gel-shift assay of SdiA-DNA complexes
For estimation of SdiA binding to the ftsQ promoter, the gel-shift assay was used. A fluorescent-labeled promoter fragment of the ftsQAZ operon, the only known target of SdiA, was generated by PCR amplification of using the ftsQ promoter assay vector as the template, a pair of primers (5′-FITC-labeled vector sequence and 3′-proximal vector sequence at the vector-fstQ junction), and Ex Taq DNA polymerase. The PCR product with FITC at 5′ termini was purified by PAGE, and then used for gel-shift assay under the standard conditions (Shimada et al. 2005). For estimation of the amount of SdiA protein for binding to the ftsQ promoter, a fixed amount of ftsQ promoter probe (0.5 pmol) and various amounts (1–100 pmol) of purified SdiA in 0.01 ml were incubated at 37OC for 15 min, and then directly subjected to PAGE. Next possible influence of HSL analogues on the binding of SdiA protein to the ftsQ promoter was examined in the presence of 0.5 pmol ftsQ promoter, 10 pmol SdiA protein, and 0.05 mm each of HSL analogues.
Genomic SELEX screening for SdiA-binding sequences
The improved genomic SELEX system was as described previously (Shimada et al. 2005). A mixture of DNA fragments of the E. coli K-12 W3110 genome was prepared after sonication of purified genome DNA and cloned into a multicopy plasmid pBR322. In each SELEX screening, the DNA mixture was regenerated by PCR. For SELEX screening, 5 pmol of the mixture of DNA fragments and 10 pmol His-tagged SdiA were mixed in a binding buffer (10 mm Tris-HCl, pH 7.8 at 4 °C, 3 mm magnesium acetate, 150 mm NaCl, and 1.25 mg/ml bovine serum albumin) and incubated for 30 min at 37 °C. The DNA-SdiA mixture was applied to a Ni-NTA column, and after washing out unbound DNA with the binding buffer containing 10 mm imidazole, DNA-SdiA complexes were eluted with an elution buffer containing 200 mm imidazole.
For SELEX-chip analysis, PCR-amplified SELEX-DNA fragments and the original DNA library were labeled with Cy3 and Cy5, respectively, and then combined. The fluorescent-labeled DNA mixtures were hybridized to a DNA-tilling array consisting of 43,450 species of 60-bp-long DNA probe, which are designed to cover the entire E. coli genome at 105-bp interval (Oxford Gene Technology, Oxford, UK) (Shimada et al. 2008, 2011a; Teramoto et al. 2010). The fluorescent intensity of test sample at each probe was normalized with that of the corresponding peak of original library. After normalization of each pattern, the Cy5/Cy3 ratio was measured and plotted along the E. coli genome.
Northern blot analysis
Total RNAs were extracted from E. coli cells by the hot phenol method (Aiba et al. 1981). RNA purity was checked by electrophoresis on 0.8% agarose gel in the presence of formaldehyde followed by staining with methylene blue. The amounts of RNAs used were measured by staining rRNAs with ethidium bromide. Northern blot analysis was carried out essentially as described previously (Shimada et al. 2011a,b). DIG-labeled probes were prepared by PCR amplification using E. coli AI-0053 (W3110) genomic DNA as template, DIG-11-dUTP (Roche), and dNTP as substrates, gene-specific forward and reverse primers, and Ex Taq DNA polymerase (Takara). Total RNAs were incubated in formaldehyde-MOPS (morpholinepropanesulfonic acid) gel-loading buffer for 10 min at 65° C for denaturation, subjected to electrophoresis on formaldehyde-containing 1.5% agarose gel, and then transferred to nylon membrane (Roche). Hybridization was carried out with DIG easy Hyb system (Roche) at 50 °C overnight with a DIG-labeled probe. For detecting the DIG-labeled probe, the membrane was treated with anti-DIG-AP Fab fragments and CDP-Star (Roche), and the image was scanned with LAS-4000 IR multicolor (Fuji Film).
- Top of page
- Experimental procedures
- Supporting Information
This work was supported by Grant-in-Aid for Scientific Research (A) (21241047), (B) (18310133) and (C) (25430173) to AI and Grant-in-Aid for Grants-in-Aid for Young Scientists (B) (24710214) to TS from MEXT (Ministry of Education, Culture, Sports, Science and Technology of Japan) and MEXT-Supported Program for the Strategic Research Foundation at Private Universities 2008-2012 (S0801037).
- Top of page
- Experimental procedures
- Supporting Information
- 2004) Cell-to-cell signaling in Escherichia coli and Salmonella enterica. Mol. Microbiol. 52, 933–945. (
- 1998) Salmonella typhimurium encodes an SdiA homolog, a putative quorum sensor of the LuxR family, that regulates genes on the virulence plasmid. J. Bacteriol. 180, 1185–1193. , , , & (
- 1981) Evidence for two functional gal promoters in intact Escherichia coli cells. J. Biol. Chem. 256, 11905–11910. , & (
- 2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 20006.0006. , , , , , , , , & (
- 2002) Small talk: Cell-to-cell communication in bacteria. Cell 109, 421–424. (
- 2008) Multiple pathways for regulation of sigmaS (RpoS) stability in Escherichia coli via the action of multiple anti-adaptors. Mol. Microbiol. 68, 298–313. , , , & (
- 1989) Purification and structural identification of an autoinducer for the luminescence system of Vibrio harveyi. J. Biol. Chem. 264, 21670–21676. & (
- 2012) Indole production promoters Escherichia coli mixed-culture growth with Pseudomonas aeruginosa by inhibiting quorum signaling. Appl. Environ. Microbiol. 78, 411–419. , , , , , , & (
- 2011) Small-molecule inhibitor binding to an N-acyl-homoserine lactone synthase. Proc. Natl Acad. Sci. USA 108, 12089–12094. , , , , , , , , , , & (
- 2010) E. coli K-12 and EHEC genes regulated by SdiA. PLoS ONE 5, e8946. , , , , & (
- 1981) Structural identification of autoinducer Photobacterium fischeri luciferase. Biochemistry 20, 2444–2449. , , , , & (
- 1999) N-Acyl homoserinelactone-mediated gene regulation in Gram-negative bacteria. Syst. Appl. Microbiol. 22, 493–506. (
- 1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822. & (
- 1996) Census and consensus in bacterial ecosystems; the LuxR-LuxI family of quorum sensing transcriptional regulators. Annu. Rev. Microbiol. 50, 727–751. & (
- 1996) An extracellular factor regulates expression of sdiA, a transcriptional activator of cell division genes in Escherichia coli. J. Bacteriol. 178, 2742–2748. , & (
- 1997) Direct binding of FtsZ to ZipA, an essential component of the septal ring structure that mediates cell division in E. coli. Cell 88, 175–185. & (
- 2008) The transcription regulator AllR senses both allantoin and glyoxylate and controls a set of genes for degradation and reutilization of purines. Microbiology 154, 3366–3378. , , , & (
- 1983) Role of hypoxanthine and guanine in regulation of Salmonella typhimurium pur gene expression. J. Bacteriol. 153, 837–845. & (
- 2010) Chemical sensing in mammalian host-bacterial commensal associations. Proc. Natl Acad. Sci. USA 107, 9831–9836. , , , , , , , , & (
- 2011) Custom synthesis of autoinducers and their analogs. Methods Mol. Biol. 692, 265–274. & (
- 2010) Prokaryotic genome regulation: multi-factor promoters, multi-target regulators and hierarchic networks. FEMS Microbiol. Rev. 34, 628–645. (
- 2012) Prokaryotic genome regulation: a revolutionary paradigm. Proc. Jpn Acad. Ser. B Phys. Biol. Sci. 88, 485–508. (
- 2006) Mechanisms of cyclic-di-GMP signaling in bacteria. Annu. Rev. Genet. 40, 385–407. & (
- 2007) Indole is an inter-species biofilm signal mediated by SdiA. BMC Microbiol. 7, 42. , & (
- 2009) Reconfiguring the quorum-sensing regulator SdiA of Escherichia coli to control biofilm formation via indole and N-acylhomoserine lactones. Appl. Environ. Microbiol. 75, 1703–1716. , , & (
- 2007) Glyoxylate and pyruvate are antagonistic effectors of the Escherichia coli IclR transcriptional regulator. J. Biol. Chem. 282, 16476–16491. , , , , , , , & (
- 2011) Role of ArgP (IciA) in lysine-mediated repression in Escherichia coli. J. Bacteriol. 193, 5985–5996. & (
- 2001) SdiA of Salmonella enterica is a LuxR homolog that detects mixed microbial communities. J. Bacteriol. 183, 5733–5742. , , , & (
- 2011) The siderophore-interacting protein YqjH acts as a ferric reductase in different iron assimilation pathways of Escherichia coli. Biochemistry 50, 10951–10964. , & (
- 1996) Functional analysis of the Pseudomonas aeruginosa atutoinducer PAI. J. Bacteriol. 178, 5995–6000. , , , , & (
- 1994) Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes. Proc. Natl Acad. Sci. USA 91, 197–201. , , , , , & (
- 1995) A second N-acylhomoserine lactone signal produced by Pseudomonas aeruginosa. Proc. Natl Acad. Sci. USA 92, 1490–1494. , , & (
- 1997) Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of elastase and rhamnolipid biosynthesis genes. J. Bacteriol. 179, 5756–5767. , & (
- 1996) The various strategies within the TyrR regulation of Escherichia coli to modulate gene expression. Genes Cells 1, 717–725. (
- 1998) Crl stimulates RpoS activity during stationary phase. Mol. Microbiol. 29, 1225–1236. & (
- 2002) Control of the AcrAB multidrug efflux pump by quorum-sensing regulator SdiA. Mol. Microbiol. 43, 677–685. , , & (
- 1996) Quorum sensing in Vibrio fischeri: probing autoinducer-LuxR interactions with autoinducer analogs. J. Bacteriol. 178, 2897–2901. , , & (
- 2001) The LuxS family of bacterial autoinducers: biosynthesis of a novel quorum-sensing signal molecule. Mol. Microbiol. 41, 463–476. , , & (
- 1999) Self-association of the Escherichia coli transcription activator MalT in the presence of maltotriose and ATP. J. Biol. Chem. 274, 33220–33226. & (
- 2010) Evaluation of the effects of sdiA, a luxR homologue, on adherence and motility of Escherichia coli O157:H7. Microbiology 156, 1303–1312. , & (
- 2013) Screening of promoter-specific transcription factors: multiple regulators for the sdiA gene involved in cell division control and quorum sensing. Microbiology 59, 2501–2512. , , , , , , , & (
- 2011a) Novel roles of LeuO in transcription regulation in E. coli gonome: antagonistic interplay with the universal silencer H-NS. Mol. Microbiol. 82, 378–397. , , & (
- 2005) Systematic search for the Cra-binding promoters using genomic SELEX system. Genes Cells 10, 907–918. , , & (
- 2011b) Novel roles of cAMP receptor protein (CRP) in regulation of transport and metabolism of carbon sources. PLoS ONE 6, e20081. , , & (
- 2007) RutR is the uracil/thymine-sensing master regulator of a set of genes for synthesis and degradation of pyrimdines. Mol. Microbiol. 66, 744–757. , , , & (
- 2008) The Escherichia coli RutR transcription factor binds at targets within genes as well as intergenic regions. Nucleic Acids Res. 36, 3950–3955. , , & (
- 1997) Libraries for genomic SELEX. Nucleic Acids Res. 25, 781–786. , , & (
- 1996) Control of cell division in Escherichia coli: regulation of transcription of ftsQA involves both rpoS and SdiA-mediated autoinduciton. Proc. Natl Acad. Sci. USA 93, 336–341. , & (
- 2003) Detection of other microbial species by Salmonella: expression of the SdiA regulon. J. Bacteriol. 185, 1357–1366. & (
- 2003a) Library screening for synthetic agonists of Pseudomonas aeruginosa autoinducer. Chem. Biol. 10, 563–571. , & (
- 2003b) Induction and inhibition of Pseudomonas aeruginosa quorum sensing by synthetic autoinducer analogs. Chem. Biol. 10, 81–89. , & (
- 2003) Bacteria-host communication: the language of hormones. Proc. Natl Acad. Sci. USA 100, 8951–8956. , , , & (
- 1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113–130. & (
- 2010) Quorum-sensing regulator sdiA and marA overexpression is involved in in vitro-selected multidrug resistance of Escherichia coli. J. Antimicrob. Chemother. 65, 1178–1186. , , , , & (
- 2010) A novel nucleoid protein of Escherichia coli induced under anaerobiotic growth conditions. Nucleic Acids Res. 38, 3605–3618. , , & (
- 1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505–510. & (
- 2006) N-acyl-L-homoserine lactone signal interception by Escherichia coli. FEMS Microbiol. Lett. 256, 83–89. , , , & (
- 2011) Fur and the novel regulator YqiI control transcription of the ferric reductase gene yqjH in Escherichia coli. J. Bacteriol. 193, 563–574. , & (
- 1991) A factor that positively regulates cell division by activating transcription of the major cluster of essential cell division genes of Escherichia coli. EMBO J. 10, 3363–3372. , & (
- 2005) Quorum sensing: cell-to-cell communication in bacteria. Annu. Rev. Cell Dev. Biol. 21, 319–346. & (
- 2001a) Global impact of sdiA amplification revealed by comprehensive gene expression profiling of Escherichia coli. J. Bacteriol. 183, 2265–2272. , , & (
- 2001b) In vivo titration of mytomycin C action by four Escherichia coli genomic regions on multicopy plasmids. J. Bacteriol. 183, 2259–2264. , & (
- 2004) Bacterial cell division and the septal ring. Mol. Microbiol. 54, 588–597. (
- 2008) Crystallization and preliminary X-ray studies of SdiA from Escherichia coli. Acta Crystallogr. 64, 19–21. , , , & (
- 2005) Functional characterization in vitro of all two-component signal transduction systems from Escherichia coli. J. Biol. Chem. 280, 1448–1456. , , , , & (
- 2001) Novel mode of transcription regulation by SdiA, an Escherichia coli homologue of the quorum-sensing regulator. Mol. Microbiol. 41, 1187–1198. , , & (
- 2007) NMR detection of adventitious xylose binding to the quorum-sensing protein SdiA of Escherichia coli. Bioorg. Med. Chem. Lett. 17, 6202–6205. , , & (
- 2006) Structure of the Escherichia coli quorum sensing protein SdiA: activation of the folding switch by acyl homoserine lactones. J. Mol. Biol. 355, 262–273. , , , , & (
- 2008) An EAL domain protein and cyclic AMP contribute to the interaction between the two quorum sensing systems in Escherichia coli. Cell Res. 18, 937–948. , & (
- Top of page
- Experimental procedures
- Supporting Information
Figure S1 List of HSL analogs.
Table S1 SdiA-binding sites on the E. coli genome
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.