A Two-Component Regulatory System Controls Autoregulated Serpin Expression in Bifidobacterium breve UCC2003

ABSTRACTThis work reports on the identification and molecular characterization of a two-component regulatory system (2CRS), encoded byserRK, which is believed to control the expression of theser2003locus inBifidobacterium breveUCC2003. Theser2003locus consists of two genes, Bbr_1319 (sagA) and Bbr_1320 (serU), which are predicted to encode a hypothetical membrane-associated protein and a serpin-like protein, respectively. The response regulator SerR was shown to bind to the promoter region ofser2003, and the probable recognition sequence of SerR was determined by a combinatorial approach ofin vitrosite-directed mutagenesis coupled to transcriptional fusion and electrophoretic mobility shift assays (EMSAs). The importance of theserRK2CRS in the response ofB. breveto protease-mediated induction was confirmed by generating aB. breve serRinsertion mutant, which was shown to exhibit alteredser2003transcriptional induction patterns compared to the parent strain, UCC2003. Interestingly, the analysis of aB. breve serUmutant revealed that the SerRK signaling pathway appears to include a SerU-dependent autoregulatory loop.

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A Conserved Two-Component Signal Transduction System Controls the Response to Phosphate Starvation in Bifidobacterium breve UCC2003

Applied and Environmental Microbiology ◽

10.1128/aem.00804-12 ◽

2012 ◽

Vol 78 (15) ◽

pp. 5258-5269 ◽

Cited By ~ 14

Author(s):

Pablo Alvarez-Martin ◽

Matilde Fernández ◽

Mary O'Connell-Motherway ◽

Kerry Joan O'Connell ◽

Nicolas Sauvageot ◽

...

Keyword(s):

Response Regulator ◽

Regulatory Protein ◽

Gc Content ◽

Regulatory System ◽

Bioinformatic Analysis ◽

Insertion Mutant ◽

Recognition Sequence ◽

Content Type ◽

Bifidobacterium Breve ◽

Two Component

ABSTRACTThis work reports on the identification and molecular characterization of the two-component regulatory system (2CRS) PhoRP, which controls the response to inorganic phosphate (Pi) starvation inBifidobacterium breveUCC2003. The response regulator PhoP was shown to bind to the promoter region ofpstSCAB, specifying a predicted Pitransporter system, as well as that ofphoU, which encodes a putative Pi-responsive regulatory protein. This interaction is assumed to cause transcriptional modulation under conditions of Pilimitation. Our data suggest that thephoRPgenes are subject to positive autoregulation and, together withpstSCABand presumablyphoU, represent the complete regulon controlled by thephoRP-encoded 2CRS inB. breveUCC2003. Determination of the minimal PhoP binding region combined with bioinformatic analysis revealed the probable recognition sequence of PhoP, designated here as the PHO box, which together withphoRPis conserved among many high-GC-content Gram-positive bacteria. The importance of thephoRP2CRS in the response ofB. breveto Pistarvation conditions was confirmed by analysis of aB. brevephoPinsertion mutant which exhibited decreased growth under phosphate-limiting conditions compared to its parent strain UCC2003.

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Identification of Genes Controlled by the Essential YycG/YycF Two-Component System of Staphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.186.4.1175-1181.2004 ◽

2004 ◽

Vol 186 (4) ◽

pp. 1175-1181 ◽

Cited By ~ 152

Author(s):

Sarah Dubrac ◽

Tarek Msadek

Keyword(s):

Staphylococcus Aureus ◽

Regulatory System ◽

Inducible Promoter ◽

Motif Analysis ◽

Two Component System ◽

Recognition Sequence ◽

Mobility Shift ◽

Component System ◽

Two Component

ABSTRACT The YycG/YycF essential two-component system (TCS), originally identified in Bacillus subtilis, is very highly conserved and appears to be specific to low-G+C gram-positive bacteria, including several pathogens such as Staphylococcus aureus. By studying growth of S. aureus cells where the yyc operon is controlled by an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoter, we have shown that this system is essential in S. aureus during growth at 37°C and that starvation for the YycG/YycF regulatory system leads to cell death. During a previous study of the YycG/YycF TCS of B. subtilis, we defined a potential YycF consensus recognition sequence, consisting of two hexanucleotide direct repeats, separated by five nucleotides [5′-TGT(A/T)A(A/T/C)-N5-TGT(A/T)A(A/T/C)-3′]. A detailed DNA motif analysis of the S. aureus genome indicates that there are potentially 12 genes preceded by this sequence, 5 of which are involved in virulence. An in vitro approach was undertaken to determine which of these genes are controlled by YycF. The YycG and YycF proteins of S. aureus were overproduced in Escherichia coli and purified. Autophosphorylation of the YycG kinase and phosphotransfer to YycF were shown in vitro. Gel mobility shift and DNase I footprinting assays were used to show direct binding in vitro of purified YycF to the promoter region of the ssaA gene, encoding a major antigen and previously suggested to be controlled by YycF. YycF was also shown to bind specifically to the promoter regions of two genes, encoding the IsaA antigen and the LytM peptidoglycan hydrolase, in agreement with the proposed role of this system in controlling virulence and cell wall metabolism.

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Identification of a Second Two-Component Signal Transduction System That Controls Fosfomycin Tolerance and Glycerol-3-Phosphate Uptake

Journal of Bacteriology ◽

10.1128/jb.02491-14 ◽

2014 ◽

Vol 197 (5) ◽

pp. 861-871 ◽

Cited By ~ 4

Author(s):

Kumiko Kurabayashi ◽

Yuko Hirakawa ◽

Koichi Tanimoto ◽

Haruyoshi Tomita ◽

Hidetada Hirakawa

Keyword(s):

Phosphate Uptake ◽

Response Regulator ◽

Anaerobic Respiration ◽

Regulatory System ◽

Carbon Substrate ◽

Two Component System ◽

Component System ◽

Content Type ◽

Two Component ◽

Biological Cost

Particular interest in fosfomycin has resurfaced because it is a highly beneficial antibiotic for the treatment of refractory infectious diseases caused by pathogens that are resistant to other commonly used antibiotics. The biological cost to cells of resistance to fosfomycin because of chromosomal mutation is high. We previously found that a bacterial two-component system, CpxAR, induces fosfomycin tolerance in enterohemorrhagicEscherichia coli(EHEC) O157:H7. This mechanism does not rely on irreversible genetic modification and allows EHEC to relieve the fitness burden that results from fosfomycin resistance in the absence of fosfomycin. Here we show that another two-component system, TorSRT, which was originally characterized as a regulatory system for anaerobic respiration utilizing trimethylamine-N-oxide (TMAO), also induces fosfomycin tolerance. Activation of the Tor regulatory pathway by overexpression oftorR, which encodes the response regulator, or addition of TMAO increased fosfomycin tolerance in EHEC. We also show that phosphorylated TorR directly represses the expression ofglpT, a gene that encodes a symporter of fosfomycin and glycerol-3-phosphate, and activation of the TorR protein results in the reduced uptake of fosfomycin by cells. However, cells in which the Tor pathway was activated had an impaired growth phenotype when cultured with glycerol-3-phosphate as a carbon substrate. These observations suggest that the TorSRT pathway is the second two-component system to reversibly control fosfomycin tolerance and glycerol-3-phosphate uptake in EHEC, and this may be beneficial for bacteria by alleviating the biological cost. We expect that this mechanism could be a potential target to enhance the utility of fosfomycin as chemotherapy against multidrug-resistant pathogens.

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Polymyxin B Resistance and Biofilm Formation in Vibrio cholerae Are Controlled by the Response Regulator CarR

Infection and Immunity ◽

10.1128/iai.02700-14 ◽

2015 ◽

Vol 83 (3) ◽

pp. 1199-1209 ◽

Cited By ~ 38

Author(s):

Kivanc Bilecen ◽

Jiunn C. N. Fong ◽

Andrew Cheng ◽

Christopher J. Jones ◽

David Zamorano-Sánchez ◽

...

Keyword(s):

Vibrio Cholerae ◽

Biofilm Formation ◽

Lipid A ◽

Response Regulator ◽

Regulatory Region ◽

Regulatory System ◽

Polymyxin B ◽

Content Type ◽

Two Component ◽

Antimicrobial Peptide Resistance

Two-component systems play important roles in the physiology of many bacterial pathogens.Vibrio cholerae's CarRS two-component regulatory system negatively regulates expression ofvps(Vibriopolysaccharide) genes and biofilm formation. In this study, we report that CarR confers polymyxin B resistance by positively regulating expression of thealmEFGgenes, whose products are required for glycine and diglycine modification of lipid A. We determined that CarR directly binds to the regulatory region of thealmEFGoperon. Similarly to acarRmutant, strains lackingalmE,almF, andalmGexhibited enhanced polymyxin B sensitivity. We also observed that strains lackingalmEor thealmEFGoperon have enhanced biofilm formation. Our results reveal that CarR regulates biofilm formation and antimicrobial peptide resistance inV. cholerae.

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Regulation of osmC Gene Expression by the Two-Component System rcsB-rcsC inEscherichia coli

Journal of Bacteriology ◽

10.1128/jb.183.20.5870-5876.2001 ◽

2001 ◽

Vol 183 (20) ◽

pp. 5870-5876 ◽

Cited By ~ 73

Author(s):

Marcela Davalos-Garcia ◽

Annie Conter ◽

Isabelle Toesca ◽

Claude Gutierrez ◽

Kaymeuang Cam

Keyword(s):

Response Regulator ◽

Regulatory Element ◽

In Vitro Transcription ◽

Proximal Promoter ◽

Two Component System ◽

Mobility Shift ◽

Component System ◽

Two Component ◽

Gel Mobility Shift

ABSTRACT The Escherichia coli osmC gene encodes an envelope protein of unknown function whose expression depends on osmotic pressure and growth phase. The gene is transcribed from two overlapping promoters, osmCp 1 andosmCp 2. Several factors regulating these promoters have been reported. The leucine-responsive protein Lrp represses osmCp 1 and activatesosmCp 2, the nucleoid-associated protein H-NS represses both promoters, and the stationary-phase sigma factor ςs specifically recognizesosmCp 2. This work reports the identification of an additional regulatory element, the two-component systemrcsB-rcsC, affecting positively the distal promoter osmCp 1. The response regulator of the system, RcsB, does not affect expression of the proximal promoter osmCp 2. Deletion analysis located the site necessary for RcsB activation just upstream ofosmCp 1. In vitro transcription experiments and gel mobility shift assays demonstrated that RcsB stimulates RNA polymerase binding at osmCp 1.

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DevR–DevS is a bona fide two-component system of Mycobacterium tuberculosis that is hypoxia-responsive in the absence of the DNA-binding domain of DevR

Microbiology ◽

10.1099/mic.0.26218-0 ◽

2004 ◽

Vol 150 (4) ◽

pp. 865-875 ◽

Cited By ~ 112

Author(s):

Deepak Kumar Saini ◽

Vandana Malhotra ◽

Deepanwita Dey ◽

Neha Pant ◽

Taposh K. Das ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Pathogenic Bacteria ◽

Response Regulator ◽

Regulatory System ◽

Site Directed Mutagenesis ◽

Phosphoryl Transfer ◽

Dependent Manner ◽

Two Component System ◽

Component System ◽

Two Component

Two-component systems play a central role in the adaptation of pathogenic bacteria to the environment prevailing within host tissues. The genes encoding the response regulator DevR (Rv3133c/DosR) and the cytoplasmic portion (DevS201) of the histidine kinase DevS (Rv3132c/DosS), a putative two-component system of Mycobacterium tuberculosis, were cloned and the protein products were overexpressed, purified and refolded as N-terminally His6-tagged proteins from Escherichia coli. DevS201 underwent autophosphorylation and participated in rapid phosphotransfer to DevR in a Mg2+-dependent manner. Chemical stability analysis and site-directed mutagenesis implicated the highly conserved residues His395 and Asp54 as the sites of phosphorylation in DevS and DevR, respectively. Mutations in Asp8 and Asp9 residues, postulated to form the acidic Mg2+-binding pocket, and the invariant Lys104 of DevR, abrogated phosphoryl transfer from DevS201 to DevR. DevR–DevS was thus established as a typical two-component regulatory system based on His-to-Asp phosphoryl transfer. Expression of the Rv3134c–devR–devS operon was induced at the RNA level in hypoxic cultures of M. tuberculosis H37Rv and was associated with an increase in the level of DevR protein. However, in a devR mutant strain expressing the N-terminal domain of DevR, induction was observed at the level of RNA expression but not at that of protein. DevS was translated independently of DevR and induction of devS transcripts was not associated with an increase in protein level in either wild-type or mutant strains, reflecting differential regulation of this locus during hypoxia.

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Identification of cpxR as a Positive Regulator Essential for Expression of the Shigella sonnei virF Gene

Journal of Bacteriology ◽

10.1128/jb.180.14.3522-3528.1998 ◽

1998 ◽

Vol 180 (14) ◽

pp. 3522-3528 ◽

Cited By ~ 70

Author(s):

Shu-ichi Nakayama ◽

Haruo Watanabe

Keyword(s):

Response Regulator ◽

Binding Capacity ◽

Regulatory System ◽

Upstream Region ◽

Dependent Manner ◽

Acetyl Phosphate ◽

Two Component ◽

Transcription In Vitro ◽

Cognate Response Regulator

ABSTRACT virF is the master regulator which activates the virulence determinant genes of Shigella spp. such asipaBCD and virG. We previously reported that expression of virF itself is regulated in a pH-dependent manner and that cpxA, a sensor of a two-component regulatory system, is involved in this regulation (S. Nakayama and H. Watanabe, J. Bacteriol. 177:5062–5069, 1995). Disruption of cpxR, which has been thought to be the cognate response regulator of cpxA (J. Dong, S. Iuchi, H.-S. Kwan, Z. Lue, and E. C. C. Lin, Gene 136:227–230, 1993), abolishedvirF expression almost completely. Purified CpxR bound directly to the upstream region of virF. Binding capacity was enhanced when CpxR was phosphorylated by coincubation with acetyl phosphate in vitro. Furthermore, we observed that phosphorylated CpxR could activate virF transcription in vitro. These results clearly indicated that CpxR was an essential activator for virF expression and strongly suggested that the binding of phosphorylated CpxR to the target site upstream of the virF gene induced a direct activation of virF transcription.

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The Two-Component System PhoPR of Clostridium acetobutylicum Is Involved in Phosphate-Dependent Gene Regulation

Journal of Bacteriology ◽

10.1128/jb.00574-08 ◽

2008 ◽

Vol 190 (20) ◽

pp. 6559-6567 ◽

Cited By ~ 11

Author(s):

Tomas Fiedler ◽

Maren Mix ◽

Uta Meyer ◽

Stefan Mikkat ◽

Michael O. Glocker ◽

...

Keyword(s):

Clostridium Acetobutylicum ◽

Response Regulator ◽

Specific Binding ◽

Expression Patterns ◽

Regulatory System ◽

Fold Increase ◽

Growth Conditions ◽

Two Dimensional Gel Electrophoresis ◽

Two Component

ABSTRACT The phoPR gene locus of Clostridium acetobutylicum ATCC 824 comprises two genes, phoP and phoR. Deduced proteins are predicted to represent a response regulator and sensor kinase of a phosphate-dependent two-component regulatory system. We analyzed the expression patterns of phoPR in Pi-limited chemostat cultures and in response to Pi pulses. A basic transcription level under high-phosphate conditions was shown, and a significant increase in mRNA transcript levels was found when external Pi concentrations dropped below 0.3 mM. In two-dimensional gel electrophoresis experiments, a 2.5-fold increase in PhoP was observed under Pi-limiting growth conditions compared to growth with an excess of Pi. At least three different transcription start points for phoP were determined by primer extension analyses. Proteins PhoP and an N-terminally truncated *PhoR were individually expressed heterologously in Escherichia coli and purified. Autophosphorylation of *PhoR and phosphorylation of PhoP were shown in vitro. Electromobility shift assays proved that there was a specific binding of PhoP to the promoter region of the phosphate-regulated pst operon of C. acetobutylicum.

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Characterization of the CpxRA Regulon in Haemophilus ducreyi

Infection and Immunity ◽

10.1128/iai.00678-10 ◽

2010 ◽

Vol 78 (11) ◽

pp. 4779-4791 ◽

Cited By ~ 32

Author(s):

Maria Labandeira-Rey ◽

Chad A. Brautigam ◽

Eric J. Hansen

Keyword(s):

Stress Response ◽

Response Regulator ◽

Cell Envelope ◽

Bacterial Species ◽

Regulatory System ◽

Promoter Regions ◽

Mobility Shift ◽

Envelope Stress ◽

Two Component ◽

Genes Encoding

ABSTRACT The H aemophilus ducreyi 35000HP genome encodes a homolog of the CpxRA two-component cell envelope stress response system originally characterized in E scherichia coli. CpxR, the cytoplasmic response regulator, was shown previously to be involved in repression of the expression of the lspB-lspA2 operon (M. Labandeira-Rey, J. R. Mock, and E. J. Hansen, Infect. Immun. 77:3402-3411, 2009). In the present study, the H. ducreyi CpxR and CpxA proteins were shown to closely resemble those of other well-studied bacterial species. A cpxA deletion mutant and a CpxR-overexpressing strain were used to explore the extent of the CpxRA regulon. DNA microarray and real-time reverse transcriptase (RT) PCR analyses indicated several potential regulatory targets for the H. ducreyi CpxRA two-component regulatory system. Electrophoretic mobility shift assays (EMSAs) were used to prove that H. ducreyi CpxR interacted with the promoter regions of genes encoding both known and putative virulence factors of H. ducreyi, including the lspB-lspA2 operon, the flp operon, and dsrA. Interestingly, the use of EMSAs also indicated that H. ducreyi CpxR did not bind to the promoter regions of several genes predicted to encode factors involved in the cell envelope stress response. Taken together, these data suggest that the CpxRA system in H. ducreyi, in contrast to that in E. coli, may be involved primarily in controlling expression of genes not involved in the cell envelope stress response.

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The BaeSR Two-Component Regulatory System Activates Transcription of the yegMNOB (mdtABCD) Transporter Gene Cluster in Escherichia coli and Increases Its Resistance to Novobiocin and Deoxycholate

Journal of Bacteriology ◽

10.1128/jb.184.15.4168-4176.2002 ◽

2002 ◽

Vol 184 (15) ◽

pp. 4168-4176 ◽

Cited By ~ 174

Author(s):

Natalya Baranova ◽

Hiroshi Nikaido

Keyword(s):

Escherichia Coli ◽

Response Regulator ◽

Regulatory System ◽

Major Facilitator Superfamily ◽

Multicopy Plasmid ◽

Active Efflux ◽

Membrane Fusion Protein ◽

Two Component ◽

Major Facilitator

ABSTRACT Screening of random fragments of Escherichia coli genomic DNA for their ability to increase the novobiocin resistance of a hypersusceptible ΔacrAB mutant resulted in the isolation of a plasmid containing baeR, which codes for the response regulator of the two-component regulatory system BaeSR. When induced for expression, baeR cloned in multicopy plasmid pTrc99A significantly increased the resistance of the ΔacrAB host strain to novobiocin (16-fold) and to deoxycholate (8-fold). Incubation of cells with novobiocin followed by a chromatographic assay for intracellular drug showed that overproduced BaeR decreased drastically the drug accumulation, presumably via increased active efflux. The genes baeSR are part of a putative operon, yegMNOB baeSR. Direct binding of BaeR to the yegM promoter was demonstrated in vitro by gel retardation assay. The gene yegB, which codes for a major facilitator superfamily transporter, was not necessary for increased resistance, but deletion of yegO or an in-frame deletion of yegN, both of which code for resistance-nodulation-cell division-type multidrug transporters, abolished the BaeR-induced increase in resistance. It is likely that both YegN and YegO produce a complex(es) with the membrane fusion protein family member YegM and pump out novobiocin and deoxycholate. We accordingly propose to rename yegMNOB as mdtABCD (mdt for multidrug transporter). Finally, the expression of two other genes, yicO and ygcL, was shown to be regulated by BaeR, but it is not known if they play any roles in resistance.

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