Embed Size (px)
Citation preview
HcpR of Porphyromonas gingivalis Is Required for Growth underNitrosative Stress and Survival within Host Cells
Janina P. Lewis,a,b Sai S. Yanamandra,a,b and Cecilia Anaya-Bergmana
Philips Institute of Oral and Craniofacial Molecular Biologya and Department of Microbiology and Immunology,b Virginia Commonwealth University, Richmond, Virginia,USA
Although the Gram-negative, anaerobic periodontopathogen Porphyromonas gingivalis must withstand nitrosative stress, whichis particularly high in the oral cavity, the mechanisms allowing for protection against such stress are not known in this organism.In this study, microarray analysis of P. gingivalis transcriptional response to nitrite and nitric oxide showed drastic upregula-tion of the PG0893 gene coding for hybrid cluster protein (Hcp), which is a putative hydroxylamine reductase. Although regula-tion of hcp has been shown to be OxyR dependent in Escherichia coli, here we show that in P. gingivalis its expression is depen-dent on the Fnr-like regulator designated HcpR. Growth of the isogenic mutant V2807, containing an ermF-ermAM insertionwithin the hcpR (PG1053) gene, was significantly reduced in the presence of nitrite (P < 0.002) and nitric oxide-generating ni-trosoglutathione (GSNO) (P < 0.001), compared to that of the wild-type W83 strain. Furthermore, the upregulation of PG0893(hcp) was abrogated in V2807 exposed to nitrosative stress. In addition, recombinant HcpR bound DNA containing the hcp pro-moter sequence, and the binding was hemin dependent. Finally, V2807 was not able to survive with host cells, demonstratingthat HcpR plays an important role in P. gingivalis virulence. This work gives insight into the molecular mechanisms of protec-tion against nitrosative stress in P. gingivalis and shows that the regulatory mechanisms differ from those in E. coli.
The oral cavity has nitrite concentrations ranging from 10 �Mto greater than 1 mM (34). Such high nitrite concentrations
are due to high dietary nitrate intake, which is then reduced by oralnitrate-reducing bacteria (25, 59, 60). Furthermore, following in-take of foods rich in sucrose, the pH in the oral cavity drops dras-tically due to metabolic conversion of the sugar to acid by oralmicrobiota, such as streptococci and lactobacilli, thus creatingconditions that are favorable for the chemical generation of nitricoxide from nitrite (44). Nitric oxide may also be generated fromnitrite by oral microbiota during metabolism through the respi-ratory nitrite reductase system, NrfHA (51). Another source ofnitrite and nitric oxide in the oral cavity, as well as in other sites ofhuman hosts, is eukaryotic cells, which respond to microbial in-fection by producing nitric oxide and O2-related species (16). Thisresponse is part of the innate immune response and is mediated byinducible nitric oxide synthases (iNOS) by both immune andnonimmune cells (35, 40).
Despite the high concentrations of nitrite/nitric oxide in theoral cavity, the mechanisms used by oral bacteria to tolerate nitro-sative stress are poorly understood. One of the major pathogens inthe oral cavity is Porphyromonas gingivalis (24). This is a Gram-negative anaerobic bacterium that plays a role in the developmentand progression of chronic adult periodontitis (15, 31). It is alsofound in other parts of the body, such as the cardiovascular systemand umbilical cord (5, 13, 14). Despite studies regarding the viru-lence aspects present in this organism, mechanisms of nitrite/ni-tric oxide detoxification in P. gingivalis are poorly understood.
Examination of the genomic sequence of this bacterium hasallowed us to predict possible mechanisms involved in nitrite/nitric oxide detoxification processes (42). P. gingivalis is an anaer-obic organism, and like other anaerobic bacteria, it codes for pu-tative genes playing a role in respiration using alternative electronacceptors such as in nitrite ammonification. Thus, the cyto-chrome c nitrite reductase system, NrfAH, which converts nitriteinto ammonia, has been identified on the genome of this bacte-
rium (PG1820 and PG1821 [PG1820-1]) (Oralgen) (37). Also, asmall protein with putative nitrite reductase activity is encoded byPG2213 (Oralgen). These mechanisms may have a role not only inmetabolism but also in detoxification of nitrosative stress (37), butso far those for P. gingivalis remain to be established.
A redox enzyme known as hybrid cluster protein, Hcp, ispresent in anaerobic bacteria (Desulfovibrio spp.) and faculta-tive anaerobic bacteria (Escherichia coli, Salmonella enterica se-rovar Typhimurium, Acidothiobacillus ferrooxidans, Rhodobac-ter capsulatus, and Shewanella oneidensis) (reviewed in reference46). Hcp is induced by nitrite, indicating that it has a role in ni-trogen metabolism. The proposed role of Hcp is to reduce hydrox-ylamine generated from nitrite or nitric oxide into ammonia andwater (8, 46, 58). As such, it plays a significant role in toxic nitro-gen compound detoxification. Indeed, recent studies have shownthat Hcp plays a role in the protection of a variety of bacteria fromnitrosative stress as well as from nitrosative stress-based killing bymacrophages (48). Hcp is also encoded on the genome of P. gingivalisby PG0893 and protects the bacterium from exposure to nitric oxide(7). However, the biological function and the regulatory mechanismsgoverning expression of this protein remain unknown.
Hcp expression in E. coli under anaerobic conditions in thepresence of nitrate is regulated by OxyR (48). However, the re-sponse to nitrate/nitrite stress is also regulated by fumarate-nitrate
Received 4 June 2012 Returned for modification 28 June 2012Accepted 2 July 2012
Published ahead of print 9 July 2012
Editor: J. B. Bliska
Address correspondence to Janina P. Lewis, [email protected].
Supplemental material for this article may be found at http://iai.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/IAI.00561-12
September 2012 Volume 80 Number 9 Infection and Immunity p. 3319–3331 iai.asm.org 3319
on May 21, 2021 by guest
http://iai.asm.org/
Dow
nloaded from
regulator (Fnr)-like proteins both in E. coli and in other bacteria(17, 46, 51). This family of broad-spectrum regulators mediatesadaptation to a variety of stimuli, including lack of oxygen (Fnr),toxin products, and exposure to nitric oxide (Dnr) (30, 36, 45). Inaddition, a novel Fnr-like regulator, designated HcpR, has beenidentified using in silico analysis and is predicted to regulate theexpression of hcp and other genes involved in response to nitrite(9, 46). This putative regulator was proposed to be present ingammaproteobacteria (Desulfovibrio spp., Geobacter spp.), clos-tridia, the Cytophaga-Flavobacteria-Bacteroides (CFB) group ofbacteria (Bacteroidetes), Fusobacterium nucleatum, Treponemadenticola, and Thermotogales (e.g., Thermotoga maritima). Thepresence of the novel regulator HcpR in these anaerobic bacteriasuggests that distinct mechanisms may regulate expression of thecritical nitrosative stress defense player hcp in those organisms,and the role of OxyR in this process marks just the beginning ofour understanding of hcp regulation, which is possibly limited tofacultatively anaerobic bacteria. Such an assumption prompts ex-perimental verification of the role of HcpR in the regulation of hcpexpression and in the response to nitrite.
In this study, microarray and bioinformatics analyses of P. gin-givalis transcriptional response to nitrite and nitric oxide wereperformed. In addition, an Fnr-like protein involved in the adapta-tion of P. gingivalis to nitrosative stress was identified. Our data showthat this novel protein is indispensable for bacterial growth with ni-trite and nitric oxide, and this adaptation is mediated by its ability toactivate hcp expression. Thus, we designated the regulator HcpR. Thiswork gives insight into the molecular mechanisms of protectionagainst nitrosative stress in P. gingivalis and marks the beginning ofexperimental characterization of the HcpR family of regulators.
MATERIALS AND METHODSBacterial strains and growth conditions. The bacterial strains used in thisstudy are listed in Table S1 in the supplemental material. Primarily, P.gingivalis strain W83 and its derivatives were used in this study. P. gingi-valis strain W83 is a virulent, encapsulated strain (49). The bacteria weregrown anaerobically (80% N2, 10% H2, and 10% CO2) at 37°C in ananaerobic chamber (Coy Manufacturing, Ann Arbor, MI). Blood agarplates (TSA II plus 5% sheep blood; BBL, Cockeysville, MD) or brain heartinfusion (BHI) broth containing hemin (5 �g/ml; Sigma, St. Louis, MO)were used to maintain the anaerobic bacteria. One Shot Top10 chemicallycompetent Escherichia coli cells (Invitrogen, Life Technologies, Grand Is-land, NY) and E. coli BL21(DE3 pLys) (Novagen, EMD4Biosciences,Merck KGaA, Darmstadt, Germany) were used for cloning and proteinexpression, respectively. Kanamycin, erythromycin, and clindamycinwere used to select E. coli transformants (50 �g/ml of kanamycin sulfate inLuria-Bertani medium [LB; Gibco, BRL Inc., Gaithersburg, MD] with orwithout 1.5% agar or 300 �g/ml erythromycin in LB broth or agar) and P.gingivalis mutants (0.5 �g/ml in BHI agar medium).
Growth studies. A single colony from a blood agar plate (BBL, BD,Franklin Lakes, NJ) served to inoculate 3 ml of mycoplasma medium (BD,Franklin Lakes, NJ) broth. The cultures were grown to confluence (ap-proximately 48 h) under anaerobic conditions. The cultures were thendiluted 1:10 with fresh mycoplasma broth and incubated for 18 h underanaerobic conditions. These cultures were used for our growth studies.
(i) Effect of oxygen. Cultures were prepared by inoculating myco-plasma broth (kept under anaerobic conditions or equilibrated with 6%oxygen overnight) to an optical density at 660 nm (OD660) of 0.05.Growth studies were conducted under anaerobic and aerobic conditionsin the presence of 6% oxygen (microaerophilic atmosphere consisting of6% O2, 80% N2, 7% CO2, and 7% H2 generated using Anoxomat Mark II[Mart Microbiology B.V., Netherlands]). Aliquots (500 �l) were removedat various time points, and growth was monitored by measuring the
OD660. Three independent cultures grown under both conditions wereprepared on different days to ensure biological significance of the results.
(ii) Growth with nitrogen species (nitrate [NaNO3], nitrite[NaNO2], S-nitrosoglutathione [GSNO]). Cultures were prepared usingmycoplasma broth and grown in the presence of various concentrations ofnitrosative stress-generating species. The growth studies were also done inmycoplasma broth supplemented with 5 �g/ml hemin.
Generation of HcpR-deficient strain. A fragment of P. gingivalis W83genomic DNA coding for the entire HcpR protein was PCR amplifiedusing forward (PG1053F) and reverse (PG1053R) primers (see Table S2 inthe supplemental material) and cloned into the pCR2.1 vector accordingto the manufacturer’s instructions (Invitrogen, Life Technologies, GrandIsland, NY). The ermF-ermAM cassette was inserted into the SspI site ofthe cloned hcpR gene. The DNA fragment containing the mutagenizedhcpR was released by digestion with EcoRI and used to electroporate P.gingivalis W83 according to a previously published protocol (19). Trans-formants were selected on BHI agar plates supplemented with 0.5 �g/mlof clindamycin. The expected insertion of the ermF-ermAM cassette on thegenome of the mutant strains was examined by PCR with PG1053F andPG1053R primers and confirmed by PCR fragment sequencing. The mutantwas designated V2807. The OxyR-deficient strain was generated similarly: theermF-ermAM cassette was inserted into the HincII site present at 556 bp of theoxyR gene. The OxyR-deficient mutant was designated V2798.
Complementation of HcpR-deficient strain. The mutation in V2807was reverted as described previously (27). Briefly, the DNA fragment con-taining an intact copy of the hcpR gene was electroporated into the HcpRmutant V2807 strain. The mixture was anaerobically grown in the pres-ence of 1.5 ml of BHI supplemented with 5 �g/ml hemin for 5 h. Rever-tants were then selected by plating on mycoplasma agar containing 500nM nitrite. As a negative control, V2807 was electroporated without DNAaddition. Reversion of the hcpR mutation was verified by the absence ofgrowth on BHI plates supplemented with clindamycin (0.5 �g/ml) andPCR amplification of hcpR followed by sequencing. One of the nitrite-resistant revertants, designated V2835, was chosen for further studies.
RNA preparation. RNA was isolated from bacteria grown to an earlylogarithmic phase (OD660, 0.3 to 0.6) using an RNeasy minikit (Qiagen,Valencia, CA) according to the manufacturer’s instructions. ResidualDNA was removed using the DNA-free kit (Ambion, Austin, TX) follow-ing the manufacturer’s instructions.
Microarray analysis. P. gingivalis cultures were grown to the midloga-rithmic phase of growth in mycoplasma broth and then supplementedwith 0.2 mM NaNO2 or 20 nM GSNO, and the cultures were grown for anadditional 60 min. Cells were harvested, and total RNA was isolated asdescribed above. Total RNA (10 �g) was reverse transcribed with Array-Script reverse transcriptase (Applied Biosystems/Ambion, Austin, TX).The cDNA was labeled with dyes (Cy3 and Cy5; GE Healthcare, Piscat-away, NJ) using the Array 900 MPX kit from Genisphere (Genisphere,Hatfield, PA). Differentially labeled cDNAs were hybridized to glassgenomic microarrays (obtained from the J. Craig Venter Institute [JCVI];formerly The Institute for Genomic Research [TIGR]) containing 70-meroligonucleotide probes for all predicted open reading frames (ORFs) pres-ent on the P. gingivalis W83 genome. Microarray hybridization and wash-ing were done as described by Genisphere (Genisphere, Hatfield, PA).Dithiothreitol (DTT) (0.1 mM) was added to washes following the finalhybridization, and the slides were protected from the impact of ozone bythe application of DyeSaver (Genisphere, Hatfield, PA). The microarrayswere scanned with an Axon 4200A microarray scanner (Molecular De-vices, Downingtown, PA) to detect the hybridized cDNA. The obtainedimages were inspected for quality and quantified using GenePix v6.0 soft-ware (Molecular Devices, Downingtown, PA), and the .gpr files were an-alyzed for significant differences using the Significance Analysis for OralPathogen Microarrays (SAOPMD) tools available at the BioinformaticsResource for Oral Pathogens (BROP) website provided by The ForsythInstitute (http://www.brop.org). Thus, results for four technical replicatesfor nitrosative stress exposure tested were averaged. Ratios of the average
Lewis et al.
3320 iai.asm.org Infection and Immunity
on May 21, 2021 by guest
http://iai.asm.org/
Dow
nloaded from
values of samples exposed to nitrosative stress versus controls were gen-erated. Genes with fold change of �1.5 at P � 0.05 were considered to besignificantly regulated in our study.
qRT-PCR analysis of gene expression. Real-time quantitative reversetranscriptase PCR (qRT-PCR) was done with the SYBR green-based de-tection system on an Applied Biosystems 7500 fast real-time PCR system(Applied Biosystems, Austin, TX). Primers were designed using Primer3software (http://frodo.wi.mit.edu). The primers used in this study arelisted in Table S2 in the supplemental material. The cDNA was generatedwith ArrayScript (Ambion, Austin, TX) using 10 ng of total RNA andrandom hexamers (for 16S rRNA, 0.02 ng of total RNA was used). qPCRwas done using the SYBR green qPCR mix (Applied Biosystems, Austin,TX). Experimental samples were tested in triplicate using gene-specificprimers, and samples in which reverse transcriptase was omitted served asnegative controls. Quantitative PCR was done using the standard curvemode protocol (a calibration curve was constructed using serial 5-folddilutions of cDNA obtained using 50 ng of total RNA) and a thermalprofile consisting of one cycle of 10 min at 95°C and 40 cycles of 15 s at95°C and 1 min at 60°C. The amounts of RNA used for the analysis werenormalized using a probe specific for 16S rRNA.
Survival in the presence of host cells. Human umbilical vein endo-thelial cells (HUVECs) (Lifeline Cell Technology, Frederick, MD) andhuman oral keratinocytes (HOKs) (ScienCell, Carlsbad, CA) were grownto confluence in 6-well tissue culture plates in the presence of gammainterferon (2 ng/ml) (Peprotech). The cells were infected with the parentalW83 and hcpR mutant V2807 P. gingivalis strains at a multiplicity of in-fection (MOI) of 100. Infection was conducted for 30 min under anaero-bic conditions at 37°C. For association of bacteria with HUVECs, theinfection medium was removed and the cells were washed twice withphosphate-buffered saline (PBS) and lysed to release intracellular bacte-ria. For microbial invasion studies, washed cells were incubated for 60 minwith a cell-specific medium (e.g., endothelial growth medium [EGM] forHUVECs) supplemented with 300 �g/ml gentamicin and 400 �g/ml met-ronidazole to kill extracellular bacteria. Next, the cells were washed withPBS and lysed by the addition of 1 ml of BHI supplemented with 1%saponin. Serial dilutions of lysed eukaryotic cells prepared in anaerobicBHI were plated to count colony-forming units (CFU), which are repre-sentative of a single surviving cell. Two biological and two technical re-peats were used to examine survival of bacterial cells in each cell type.
Expression of HcpR in E. coli. The coding sequence of the hcpR genewas obtained from http://www.oralgen.lanl.gov. The gene was PCR am-plified from the chromosomal DNA of P. gingivalis W83 with primers FnF(with an NcoI site) and FnR (with an XhoI site) (see Table S2 in thesupplemental material). The amplified DNA was cloned into the pET30avector (Novagen, EMD4Biosciences, Merck KGaA, Darmstadt, Ger-many), which attached a tail of six histidines to the 3= end of the gene. Theresulting construct, pET30-hcpR, was introduced into E. coli BL21(DE3)cells (Novagen, EMD4Biosciences, Merck KGaA, Darmstadt, Germany).To purify the recombinant protein, an overnight culture of E. coli-pET30-hcpR was diluted 1:100 in LB broth supplemented with 50 �g/ml of kana-mycin, and the culture was grown until midlog phase (OD660 � 0.6). Forgeneration of cell lysate, the cells were suspended in phosphate-bufferedsaline (PBS), disrupted by sonication, and centrifuged to remove the cel-lular debris. The supernatant was aspirated, and protein concentrationwas determined with the Bradford assay (Bio-Rad). For protein purifica-tion the cells were suspended in a buffer consisting of 50 mM NaH2PO4,pH 8.0, 300 mM NaCl, and 20 mM imidazole. Recombinant HcpR(rHcpR) was purified under native conditions using Ni-agarose(Qiagen) as described in the manufacturer’s protocol. The purity ofrHcpR was assessed using SDS-PAGE, and the protein concentration wasdetermined with a Bradford assay (Bio-Rad).
EMSA. A 200-bp DNA fragment containing the promoter region ofhcp was amplified by PCR using primers IGSF and IGSR (see Table S2 inthe supplemental material). The fragment was labeled with biotin usingthe biotin 3= end DNA labeling kit (Thermo Scientific, Rockford, IL), and
then the labeled DNA was used in an electrophoretic mobility gel shiftassay (EMSA) using the LightShift chemiluminescent EMSA kit (ThermoScientific, Rockford, IL). Briefly, a 20-�l reaction mixture consisting ofcell lysate proteins or rHcpR, 100 mM NaCl, 10% glycerol, and 2 �g ofdI-dC was incubated for 10 min at ambient temperature. The sampleswere subjected to electrophoresis using a 2% agarose gel in Tris-HCl buf-fer containing 2 mM EDTA and 5% glycerol. Following electrophoresis,DNA was transferred to nylon membranes (GE Healthcare, Piscataway,NJ), and the membrane was developed with the LightShift chemilumines-cent EMSA kit (Thermo Scientific, Rockford, IL).
Determination of PG0893 transcriptional start site. The P. gingivalisW83 transcriptome was determined as described previously (61). Readswere aligned to the reference genome using the CLC Genomics Work-bench (CLC Bio). The transcriptional start site was further verified usingqRT-PCR analysis with the primers shown (see Fig. 6B and Table S2 in thesupplemental material).
Bioinformatics analysis. The Basic Local Alignment Search Tool(BLAST) was used to find protein sequences similar to HcpR, and domainorganization was examined using the National Center for BiotechnologyInformation (NCBI) search tools (2) (http://www.ncbi.nlm.nih.gov). AnHcpR three-dimensional (3-D) structural model was generated using theSWISS-MODEL homology modeling server (4, 6, 47). The probablestructure was visualized and aligned with other similar structures usingthe PyMOL molecular graphics system, version 1.r1 (http://www.pymol.org). Search for HcpR binding sites was done using PRODORIC (38).
Microarray data accession number. Microarray data were depositedin the GEO database; the accession number is GSE38220.
RESULTSSensitivity of P. gingivalis to nitrosative stress. The oral cavitycontains high nitrite concentrations. To determine the suscepti-bility of P. gingivalis to nitrosative stress, we compared its ability togrow in various concentrations of nitrate (NaNO3), nitrite(NaNO2), and S-nitrosogluthathione (GSNO). As shown inFig. 1A, 40 mM nitrate reduced bacterial growth. However, P.gingivalis was still able to grow with such a high nitrate concentra-tion. No growth was observed in the presence of 8 mM or 4 mMnitrite, although P. gingivalis did grow with 1 mM nitrite (Fig. 1A).In addition, no growth inhibition was observed in the presence of0.2 mM nitrite. These results show that P. gingivalis is highly tolerantto nitrate and nitrite stress. Next, we examined the ability of P. gingi-valis to grow with nitric oxide stress. A nitric oxide donor, GSNO,inhibited P. gingivalis growth when 0.7 � 10�3 mM was used, andreduced bacterial growth was observed even in the presence of lowconcentrations (0.07 � 10�3 mM) of GSNO (Fig. 1B), thus indicat-ing that the pathogen is highly sensitive to nitric oxide stress.
Transcriptional response to nitrosative stress. To gain in-sight into the adaptive response of P. gingivalis to nitrosativestress, we examined its transcriptional response to nitrite orGSNO exposure. For exposure to nitrite, we used 0.2 mM NaNO2,which was shown in our studies to have no effect on bacterialgrowth (Fig. 1A). Using a 1.5-fold change in expression level wefound that 36 genes were upregulated and 105 genes were down-regulated (Table 1; see Table S3 in the supplemental material).The most drastically upregulated gene was hcp (PG0893), whichcodes for hydroxylamine reductase (Table 1). Other regulatedgenes included PG0616, coding for thioredoxin, PG1227, encod-ing LysR-type regulator, PG0776 and PG0900, coding for oxi-doreductases, PG1318, encoding ECF sigma protein, PG1129, en-coding ribonucleotide reductase, and PG1321, coding for aformate-tetrahydrofolate ligase. Several genes coding for hypo-thetical putative proteins were also upregulated. Downregulated
Identification and Role of HcpR in P. gingivalis
September 2012 Volume 80 Number 9 iai.asm.org 3321
on May 21, 2021 by guest
http://iai.asm.org/
Dow
nloaded from
genes included ones coding for putative nitrite reductase(PG2213), fumarate reductase (PG1614), and alkyl hydroperox-ide reductase (PG0618-9) (see Table S3 in the supplemental ma-terial).
Exposure to GSNO resulted in a much more altered transcrip-tional response; 538 genes were upregulated 1.5-fold, and 628
genes were downregulated 1.5-fold (Table 1; see Table S4 in thesupplemental material). This response could be predicted basedon the reduced growth of P. gingivalis in the presence of GSNO. Asobserved upon exposure to nitrite, the most drastically upregu-lated gene was hcp (PG0893) encoding putative hydroxylaminereductase (Tables 1 and 2). However, there was no significant
FIG 1 Sensitivity of P. gingivalis to nitrosative stress. P. gingivalis W83 was grown to midlogarithmic phase under anaerobic conditions in mycoplasma brothwithout hemin and was diluted to an OD660 of 0.1. Various concentrations of nitrite or nitrate (A) (amount given in mM) or GSNO (B) (amount given inmM � 10�3) were added to the cultures. Unsupplemented cultures served as controls. The cultures were then grown for an additional 48 h at 37°C underanaerobic conditions. Means from the experiment performed in triplicate are shown.
TABLE 1 P. gingivalis genes upregulated by nitrite
Locusa Common namee Mf Fldb Pc Repeatd
PG0893 Prismane protein 1.614092 3.061189 0.000026 4PG0616 Thioredoxin, putative 1.248251 2.375533 0.000293 4PG1237 Transcriptional regulator, LuxR family 0.941990 1.921176 0.007825 4PG1185 Hypothetical protein 0.904399 1.871765 0.021101 4PG0776 Electron transfer flavoprotein, alpha subunit 0.869507 1.827039 0.003561 4PG1335 Membrane protein, putative 0.837913 1.787463 0.001958 4PG1860 Conserved hypothetical protein 0.837301 1.786705 0.000603 4PG2027 Hypothetical protein 0.833295 1.781750 0.009845 4PG1318 RNA polymerase sigma-70 factor, ECF subfamily 0.809544 1.752657 0.004291 4PG0605 Hypothetical protein 0.792700 1.732314 0.000734 4PG0900 Cytochrome d ubiquinol oxidase, subunit I 0.780032 1.717169 0.000727 4PG0752 Uracil phosphoribosyltransferase, putative 0.764700 1.699016 0.006656 4PG1129 Ribonucleotide reductase 0.763810 1.697968 0.005135 4PG1321 Formate-tetrahydrofolate ligase 0.759365 1.692746 0.001151 4PG0456 PHP N-terminal domain protein 0.734354 1.663653 0.001138 4PG0931 DNA-binding protein, histone-like family 0.725102 1.653018 0.018597 4PG2167 Immunoreactive 53-kDa antigen PG123 0.677790 1.599688 0.032629 4PG2132 Fimbrilin 0.670559 1.591690 0.040085 4PG0313 Hypothetical protein 0.667261 1.588055 0.025777 4PG1171 Oxidoreductase, putative 0.665062 1.585636 0.047665 4PG2164 Peptidyl-prolyl cis-trans isomerase 0.663684 1.584123 0.013981 4PG1759 Adhesion protein, putative 0.625403 1.542642 0.008795 4PG0055 Conserved domain protein 0.622741 1.539798 0.008063 4PG1012 tRNA-i(6)A37 modification enzyme MiaB 0.615015 1.531573 0.026784 4PG0705 Glutamate racemase 0.612681 1.529098 0.006633 4PG0758 Peptidyl-dipeptidase Dcp 0.610164 1.526432 0.005511 4PG1340 L-Lactate permease 0.605587 1.521598 0.047160 4PG0722 Hypothetical protein 0.599049 1.514718 0.045470 4a Locus, Gene ID according to JCVI/NCBI.b Fld, ratio of transcript level in the presence of 0.2 mM NaNO2 to transcript level in the absence of NaNO2.c A P value of at least �0.05 was used to select the regulated genes.d Number of replicates used in the analysis.e See footnote a to Table 2 for abbreviations.f M � transcript level in the presence of nitrosative stress (NaNO2)/transcript level in the absence of nitrosative stress.
Lewis et al.
3322 iai.asm.org Infection and Immunity
on May 21, 2021 by guest
http://iai.asm.org/
Dow
nloaded from
TABLE 2 Genes significantly upregulated by exposure of P. gingivalis to nitric oxidea
Locusb Common name Mf Fldc Pd Repeate
PG0893 Prismane protein 3.800083 13.929606 0.003953 4PG0456 PHP N-terminal domain protein 2.562149 5.905868 0.000084 4PG1358 Acetyltransferase, GNAT family 2.337527 5.054354 0.000479 4PG1142 Exopolysaccharide synthesis protein 2.216705 4.648305 0.038262 4PG1441 Lysozyme-related protein 2.154463 4.452030 0.000705 4PG1130 TPR domain protein 2.154308 4.451550 0.000027 4PG1118 ClpB protein 2.149263 4.436011 0.013444 4PG0982 TPR domain protein 2.146700 4.428137 0.000842 4PG1513 Phosphoribosyltransferase 2.091644 4.262334 0.000960 4PG1089 DNA-binding regulator RprY 2.067106 4.190453 0.000983 4PG1203 Transcriptional regulator, putative 2.039493 4.111011 0.000053 4PG1033 Conserved hypothetical protein 2.028274 4.079166 0.004690 4PG1205 DNA-binding protein, histone-like 2.017790 4.049630 0.000717 4PG2213 Nitrite reductase-related protein 1.992824 3.980155 0.002534 4PG0234 Immunoreactive 23-kDa PG66 1.893655 3.715754 0.000044 4PG0840 Hypothetical protein 1.876870 3.672774 0.000290 4PG0192 Cationic outer membrane protein 1.860632 3.631666 0.000391 4PG0726 Lipoprotein, putative 1.852133 3.610336 0.000782 4PG0173 Transcriptional regulator, putative 1.852074 3.610188 0.000198 4PG0521 Chaperonin, 10 kDa 1.805918 3.496516 0.000447 4PG2135 Lipoprotein, putative 1.800741 3.483992 0.035156 3PG0037 Ribosomal protein L19 1.800722 3.483945 0.007767 4PG1501 Transcriptional regulator, TetR family 1.800294 3.482913 0.017777 3PG1431 DNA-binding regulator, LuxR family 1.771290 3.413590 0.003835 4PG1432 Sensor histidine kinase 1.766057 3.401230 0.005609 4PG2056 Transposase, ISPg2-related 1.758423 3.383282 0.000442 4PG0817 Integrase, truncation 1.744717 3.351291 0.012031 4PG0395 DNA-directed RNA polymerase, beta 1.722192 3.299372 0.013181 4PG0850 DNA binding protein, excisionase 1.709125 3.269625 0.011821 3PG1180 Membrane protein, putative 1.698288 3.245157 0.003909 4PG0162 RNA polymerase sigma-70, ECF 1.696737 3.241670 0.003283 4PG1140 Glycosyl transferase, group 2 family 1.692872 3.232997 0.005320 4PG0411 Hemagglutinin, putative 1.683408 3.211858 0.000131 4PG1858 Flavodoxin 1.677289 3.198263 0.030572 4PG1551 HmuY protein 1.667009 3.175555 0.006028 4PG0385 Ribosomal protein S21 1.663097 3.166955 0.004649 4PG0910 FHA domain protein 1.652527 3.143838 0.001831 4PG1821 Cytochrome c nitrite reductase, NrfH 1.647664 3.133260 0.000084 4PG0801 Poly(A) polymerase family protein 1.637456 3.111166 0.001452 4PG0436 Capsular polysaccharide transport protein 1.623879 3.082027 0.000325 4PG0121 DNA-binding protein HU 1.607604 3.047453 0.000200 4PG0807 NusB family protein 1.588143 3.006620 0.000052 4PG0873 Mobilizable transposon, TnpC protein 1.581083 2.991943 0.028997 4PG0487 ISPg4, transposase 1.562312 2.953267 0.000015 4PG1445 RteC protein, truncation 1.561100 2.950787 0.012181 4PG1471 Conserved hypothetical protein 1.556188 2.940757 0.000325 4PG2105 Lipoprotein, putative 1.550548 2.929283 0.002336 4PG0045 Heat shock protein HtpG 1.544451 2.916931 0.002594 4PG0111 Capsular polysaccharide biosynthesis 1.517216 2.862382 0.000103 4PG0836 Integrase, truncation 1.496088 2.820769 0.006799 3PG0275 Thioredoxin family protein 1.484867 2.798914 0.000108 4PG0754 DNA topoisomerase I 1.483458 2.796181 0.008538 4PG2117 Ribosomal protein S16 1.479544 2.788606 0.006323 4PG0090 Dps family protein 1.479230 2.787999 0.001504 4PG0968 Mrr restriction system protein 1.459893 2.750879 0.001419 4PG1907 ISPg3, transposase, interruption 1.440110 2.713415 0.003599 4PG1548 Thiol protease-hemagglutinin PrtT 1.421396 2.678446 0.001450 4PG0415 Peptidyl-prolyl cis-trans isomerase 1.416012 2.668469 0.000244 4PG0912 Polysaccharide transport protein 1.409927 2.657237 0.000032 4PG1775 GrpE protein 1.400450 2.639839 0.001569 4PG1048 N-Acetylmuramoyl-L-alanine amidase 1.398556 2.636375 0.004470 4PG2014 CRISPR-associated protein Cas1 1.381516 2.605421 0.000035 4
(Continued on following page)
Identification and Role of HcpR in P. gingivalis
September 2012 Volume 80 Number 9 iai.asm.org 3323
on May 21, 2021 by guest
http://iai.asm.org/
Dow
nloaded from
overlap between other nitrite and GSNO-regulated genes. Up-regulated by GSNO were ones coding for reactive oxygen detoxi-fication proteins, such as PG0195, coding for rubrerythrin,PG0275 and PG0616, encoding putative thioredoxins, andPG0090, coding for Dps (Table 2; see Table S4 in the supplementalmaterial) (54, 55). The upregulation of rbr expression is consistentwith the protective role of rubrerythrin against reactive nitrogenspecies (39).
Several genes coding for regulators, such as PG1203, PG1205,PG0173, PG1431-2, PG0162, PG0121, PG0465, and PG1240, wereupregulated. Also, a large portion of the upregulated genes in-cluded ones coding for hypothetical proteins, indicating thatnovel aspects may be detected when considering nitric oxide pro-tection mechanisms in P. gingivalis. Interestingly, upregulatedgenes included PG1551, coding for HmuY protein, which hasbeen shown to mediate hemin uptake in P. gingivalis (33, 43, 50),and PG2213, encoding putative nitrite reductase (Oralgen). Inaddition, the gene coding for fumarate reductase (PG1614-15)was downregulated (see Table S4 in the supplemental material).This result is similar to what was observed after challenge withnitrite. Furthermore, several rRNA-encoding genes and an alkylhydroperoxide reductase-encoding gene (PG0619) were down-regulated (see Table S4).
The microarray results were verified and extended by using theqRT-PCR results (Table 3). Significant upregulation (over 150-fold) of PG0893 (hcp) encoding prismane (hydroxylamine reduc-tase) was observed when this assay was used. This was by far themost upregulated gene after exposure to both nitrite and GSNO.In addition, we tested gene expression in P. gingivalis grown inmycoplasma broth as well as in BHI supplemented with hemin,and under both conditions we noted significant upregulation ofPG0893 (hcp) (Table 3).
Sequence and structure of P. gingivalis HcpR. The 684-bpPG1053 (here designated the hcpR gene) is the last gene located inthe six-gene genomic locus coding for cell envelope biogenesisproteins (Fig. 2A). The first gene of the locus, amiA, codes for apeptidoglycan biosynthesis protein (N-acetylmuramoyl-L-ala-nine amidase); two genes, PG1049 and PG1050, encode proteinswith unknown function; and PG1051 codes for O-antigen poly-merase. The last two genes in the locus code for regulatory pro-teins. PG1052, a 354-bp gene, codes for a 118-amino-acid protein
with similarity to MerR-like regulators (Fig. 2A). Residues 11 to106 of the protein encoded by PG1052 are 33% similar to thenegative regulator of heat shock response (HspR) protein, whichis a regulator from Mycobacterium tuberculosis (H37RV) (12, 53).PG1053 codes for an Fnr-like regulator named here HcpR. Com-parison of the P. gingivalis hcpR genomic locus to hcpR-likegenomic loci in other bacteria showed that the P. gingivalis locus isdistinct in that it does not include the hcp gene (data not shown).Sequence comparisons of the P. gingivalis HcpR to other proteinswere done using an NCBI BLAST search. The highest similaritywas to a putative transcriptional regulator from P. endodontalis(68% identity), and the similarity dropped drastically for putativeregulators from other members of the order Bacteroidales (41%for Bacteroides coprosuis and 36% for both Parabacteroides johnso-nii and Prevotella multiformis) (see Table S5 in the supplementalmaterial). Also, similarity was detected with proteins of knownfunction. For example, DnrS, a regulator from Pseudomonasstutzeri, was 24% similar for residues 28 to 225 (57); Fnr-like pro-tein from Lactococcus lactis was 24% similar for residues 29 to 224(22); a Crp-like transcriptional regulator from Thermatoga mari-tima (putative HcpR) was 28% similar for residues 51 to 220 (52).Comparison of the latter protein sequences with P. gingivalisHcpR is shown in Fig. 2B. We noted differences in two DNAspecificity-conferring amino acids, 180 and 181 (residues under-lined in Fig. 2B). These amino acids bind DNA; R180 correlateswith G3, and Q181 correlates with G6 in HcpR.
The 684-bp hcpR gene codes for a 228-amino-acid protein (Fig.2). We built a model of full-length HcpR using Dnr from Pseu-domonas aeruginosa (structure 3dkw) as a template (21). Severalfunctional regions, as identified using bioinformatics analysis, in-cluded amino acids 25 to 135, which code for the CAP-ED super-family region that encompasses the ligand binding site and theflexible hinge region; the region including amino acids 170 to 210codes for a helix-turn-helix (HTH) family, which is an HTHDNA-binding site (Fig. 2C). The region between the ligand-bind-ing domain and the HTH domain comprises a dimerization do-main (also known as a dimerization helix). Next, we compared thepredicted structure of P. gingivalis HcpR with that of P. aeruginosaDnr (Fig. 2D). Most of the protein could be overlaid with thestructure of Dnr; however, differences in the HTH domain and theligand-binding domain were noted, indicating that the DNA-
TABLE 2 (Continued)
Locusb Common name Mf Fldc Pd Repeate
PG0465 Ferric uptake transcriptional regulator 1.373162 2.590376 0.020403 4PG1049 Conserved hypothetical protein 1.364268 2.574456 0.000523 4PG1617 Hypothetical protein 1.358899 2.564894 0.000008 4PG0829 Hypothetical protein 1.352715 2.553923 0.006952 4PG1240 Transcriptional regulator, TetR family 1.350249 2.549562 0.000342 4PG1149 Glycosyl transferase, group 1 family 1.339523 2.530677 0.001612 4PG0742 Antigen PgaA 1.335788 2.524132 0.001708 4PG1688 Transcription elongation factor GreA 1.289511 2.444451 0.003854 4PG0115 Hexapeptide transferase 1.283816 2.434821 0.004863 4a Significantly upregulated genes encoding hypothetical proteins are excluded from this table. PHP, polymerase/histidinol phosphatase; GNAT, acetyl-coenzme Asynthetase/acetyltransferase; TPR, tetratricopeptide repeat; ECF, extracytoplasmic function; CRISPR, clustered regularly interspaced short palindromic repeats.b Locus, Gene ID according to JCVI/NCBI.c Fld, ratio of transcript level in the presence of GSNO to transcript level in the absence of GSNO.d A P value of at least �0.05 was used to select the regulated genes.e Number of replicates used in the analysis.f M � log(transcript level in the presence of nitrosative stress [GSNO]/transcript level in the absence of nitrosative stress).
Lewis et al.
3324 iai.asm.org Infection and Immunity
on May 21, 2021 by guest
http://iai.asm.org/
Dow
nloaded from
binding sites may differ in the case of P. gingivalis HcpR comparedto P. aeruginosa Dnr. Also, the differences in the ligand-bindingdomain may indicate differences in the nature of the substrate towhich the regulator responds. In addition, we compared the pre-dicted structure of P. gingivalis HcpR with that of a putative HcpRfrom T. maritima (the structure of the C-terminal truncatedTM1171 is available; the disordered HTH domain was cleaved topromote crystallization of the protein) (52) (results not shown).Again, extensive structural similarity between the two proteinstructures was noted; however, differences in the ligand-bindingdomain were observed, indicating that the substrate specificity forboth regulators may differ.
Construction and preliminary characterization of P. gingi-valis hcpR mutant strain. The 684-bp hcpR gene encodes a 26-kDa protein. In this study, we disrupted the hcpR gene at the SspIsite located 291 bp within the gene (Fig. 2A). The mutant strain,designated V2807, grew in a manner similar to that of the parentalW83 strain in BHI medium. In addition, it formed black-pig-mented colonies when grown on blood agar plates (results notshown). The growth characteristics of the mutant and parental P.gingivalis strains were investigated under anaerobic and aerobic(6% oxygen) conditions. This oxygen concentration was used asP. gingivalis does not grow under higher oxygen concentrations(32). Growth studies were done in mycoplasma broth withoutcysteine. The growth characteristics of the parental and mutantstrains were indistinguishable under anaerobic conditions (datanot shown). Also, under aerobic conditions both strains grew in asimilar manner (data not shown). Thus, we concluded that HcpRis not required for P. gingivalis growth under either aerobic oranaerobic conditions.
Growth of P. gingivalis HcpR mutant is impaired by nitrite.HcpR-like regulators are implicated in bacterial sensitivity to ni-trosative stress (9). To gain insight into the role of P. gingivalisHcpR in response to nitrosative stress, growth studies were doneusing broth cultures. Our studies regarding the response of theparental strain to nitrate and nitrite verified the results we ob-tained earlier, and they showed that P. gingivalis is highly tolerantto nitrate (grows with 40 mM nitrate) and also could grow in thepresence of 1 mM nitrite (Fig. 3A). The V2807 strain also grew inthe presence of 40 mM nitrate (Fig. 3B). However, significantgrowth reduction was observed when 0.2 mM nitrite was used(P � 0.05, W83 versus V2807 under 0.2 mM nitrite, Student’s ttest). Furthermore, V2807 did not grow in the presence of 1 mMnitrite (Fig. 3B) (*, P � 0.002, W83 versus V2807 under 1 mMnitrite, Student’s t test). Similar results were obtained using a diskdiffusion assay; while no growth inhibition was observed for thewild-type W83 strain using 2 M nitrite, a significant zone of inhi-bition was noted for the V2807 strain (data not shown). This resultsignificantly differed from the growth inhibition observed in thepresence of 1% peroxide, where a very sharp and clear inhibitionzone of similar size was observed for the two strains examined,indicating that HcpR plays no role in the response of P. gingivalisto peroxide (data not shown). These results indicate that HcpRplays a role in adaptation to nitrite but is not required for toleranceto oxidative stress.
To further verify the role of HcpR in P. gingivalis growth in thepresence of nitrite, we constructed the hcpR mutant revertantstrain, V2835. Growth studies were done to examine the suscep-tibility of the strains to nitrate and nitrite. Significant growth in-hibition was noted for the V2807 strain grown in medium supple-
TA
BLE
3C
omparison
ofgen
eexpression
inP
.gingivalisstrain
sin
response
ton
itrosativestress
usin
gqR
T-P
CR
Gen
e
Gen
eexpression
un
dercon
dition
W83
�n
itrite/W
83�
nitrite
a
W83
�n
itrite/W
83�
nitrite
b
W83
�G
SNO
/W
83�
GSN
Oc
V2807
�n
itrite/V
2807�
nitrite
a
V2807
�n
itrite/V
2807�
nitrite
b
V2807
�G
SNO
/V
2807�
GSN
Oc
V2807
�n
itrite/W
83�
nitrite
V2807
�G
SNO
/W
83�
GSN
Oc
V2835
�n
itrite/V
2835�
nitrite
V2798
�n
itrite/V
2798�
nitrite
PG
0893(hcp)
171�
2.327.5
�0.4
226�
3.10.6
�0.1
0.53�
0.01.1
�0.1
9.2�
10�
4�
0.0*
6.6�
10�
4�
0.0153.2
�29
71.1�
4.9P
G1820
(nrfA)
0.81�
0.10.30
�0.0
1.2�
0.20.25
�0.0
1.11�
0.10.38
�0.0
0.09�
0.00.36
�0.0
0.58�
0.20.82
�0.3
PG
16140.91
�0.1
0.10�
0.00.57
�0.1
2.33�
0.20.89
�0.1
1.03�
0.14.20
�0.3
1.57�
0.20.5
�0.01
1.22�
0.3P
G2213
0.96�
0.00.89
�0.0
5.57�
0.40.32
�0.0
0.92�
0.10.87
�0.0
0.11�
0.0a
0.26�
0.0N
Dd
ND
PG
1551(hm
uY)
0.79�
0.10.02
�0.0
2.38�
0.20.42
�0.1
0.66�
0.00.97
�0.1
0.01�
0.00.07
�0.0
ND
ND
PG
0195(rbr)
0.56�
0.2N
D3.63
�0.2
0.28�
0.0N
D1.39
�0.0
0.37�
0.10.68
�0.0
ND
ND
PG
0619(ahpF)
0.8�
0.070.03
�0.0
0.4�
0.00.3
�0.0
0.04�
0.00.25
�0.0
0.1�
0.00.43
�0.0
1.1�
0.060.95
�0.01
PG
10531.2
�0.1
ND
1.3�
0.2N
DN
DN
DN
DN
DN
DN
DP
G1340
1.2�
0.2N
DN
DN
DN
DN
DN
DN
DN
DN
DP
G1863
3.52�
0.2N
D0.156
�0.01
0.08�
0.01N
D0.375
�0.04
0.018�
0.0010.90
�0.1
ND
ND
PG
14313.38
�0.4
ND
2.7�
0.20.23
�0.03
ND
1.34�
0.10.04
�0.003
1.27�
0.2N
D3.84
�0.3
PG
10891.68
�0.2
ND
10.3�
0.10.25
�0.06
ND
1.19�
0.080.064
�0.002
0.29�
0.01N
DN
DP
G1181
2.6�
0.4N
D0.3
�0.001
0.37�
0.09N
D0.85
�0.04
1.1�
10�
3�
0.1�
10�
3
0.23�
0.02N
D2.21
�0.02
PG
12402.12
�0.7
ND
114.96�
0.90.24
�0.03
ND
0.65�
0.10.086
�0.1
1.36�
0.1N
DN
Da
Bacterialstrain
sw
eregrow
nin
mycoplasm
abroth
.Experim
entalcu
ltures
were
treatedw
ith0.2
mM
NaN
O2
for60
min
.b
Bacterialstrain
sw
eregrow
nin
BH
Isu
pplemen
tedw
ithh
emin
.Experim
entalcu
ltures
were
treatedw
ith0.2
mM
NaN
O2
for60
min
.c
Bacterialstrain
sw
eregrow
nin
mycoplasm
abroth
.Experim
entalcu
ltures
were
treatedw
ith20
nM
GSN
Ofor
60m
in.
dN
D,n
otdeterm
ined.
Identification and Role of HcpR in P. gingivalis
September 2012 Volume 80 Number 9 iai.asm.org 3325
on May 21, 2021 by guest
http://iai.asm.org/
Dow
nloaded from
mented with 1 mM nitrite, compared to the parental W83 strain(data not shown). However, restoration of the hcpR gene in V2835reduced the nitrite inhibition to the level seen in the parental strain,thus confirming that the nitrite susceptibility observed in V2807 isdue to disruption of the hcpR gene. Similarly low levels of growthinhibition were observed for all strains tested when mycoplasma me-dium was supplemented with 40 mM nitrate (data not shown).
The studies described above were done using mycoplasmabroth without hemin to simulate conditions in healthy sites, suchas nonbleeding periodontal pockets. However, P. gingivalis alsoencounters environments rich in hemin sources, such as bleedingperiodontal pockets, and thus we also examined the role of heminon bacterial susceptibility to nitrite and nitrate. When high heminconcentrations (5 �g/ml) were present, both strains were able togrow with 40 mM nitrate or 1 mM nitrite (results not shown).However, reduced growth of the mutant strain V2807 comparedto the W83 strain was observed when higher concentrations ofnitrite were used (2 mM or higher) (Fig. 3C and D). Growth of theV2835 strain containing restored hcpR gene with both 2 mM and4 mM nitrite was similar to that of the parental W83 strain (Fig. 3Cand D).
HcpR is required for P. gingivalis growth with nitric oxide.As nitrite can be readily generated from nitric oxide as well as totest the susceptibility of the HcpR mutant to other types of nitro-
sative stress encountered under physiological conditions, we com-pared the susceptibility of both strains to nitrite and GSNO. Asshown in Fig. 4, the V2807 strain is more susceptible not only tonitrite but also to GSNO (*, P � 0.001, W83 versus V2807 under30 nM GSNO, Student’s t test).
HcpR regulates hcp expression. In order to determine whichgenes are regulated by HcpR, we compared transcript levels fromthe parental W83 strain and the mutant V2807 strain grown inmycoplasma media with and without nitrosative stress for severalgenes regulated upon exposure to nitrite or GSNO (Tables 1 and2). As shown in Table 3, the major gene regulated by HcpR washcp. qRT-PCR analysis showed that hcp was drastically upregu-lated by nitrite and GSNO exposure; however, no such upregula-tion was detected in the HcpR-deficient V2807 strain (Table 3).The induction of expression of hcp was also observed in the HcpRrevertant strain, V2835, thus verifying that the reduced expressionof hcp in V2807 is indeed due to the mutation in the hcpR gene.Finally, we also tested the effect of OxyR mutation using strainV2798 (see Table S1 in the supplemental material) on expressionof hcp; again nitrite exposure resulted in significant overexpres-sion of the gene, indicating that OxyR plays no role in regulationof hcp in P. gingivalis. These results indicate that HcpR upregulateshcp expression upon exposure to nitrite. Other genes with reducedexpression included PG0616, PG1820, PG2213, and PG1551 (Table
FIG 2 Bioinformatics analysis of HcpR. (A) Genomic locus of hcpR. Genes and their orientations are denoted by arrows. IGR, intergenic region. (B) Comparisonof amino acid sequences of P. gingivalis HcpR (HcpR), Thermatoga maritima CRP-like regulator TM1171 (TM1171), Lactococcus lactis Fnr-like regulatorCAB53581 (LAFNR), and Pseudomonas stutzeri DNR CAB40908 (PSDNR). Amino acids identical in other proteins to that found in HcpR are indicated in red.Two amino acids in the helix-turn-helix (HTH) confer specificity of the protein binding to DNA targets (R180 correlates with G3 and Q181 with G6 in HcpR). (C)Structural model of P. gingivalis HcpR. Amino acids 25 to 135 form a CAP motif (ligand-binding motif) and are shown in green, and the HTH region(DNA-binding domain; residues 170 to 210) is designated in yellow. The two domains are separated by a dimerization helix. (D) Structural overlay of HcpR(blue) with PADNR (3dkw) (orange).
Lewis et al.
3326 iai.asm.org Infection and Immunity
on May 21, 2021 by guest
http://iai.asm.org/
Dow
nloaded from
3). The reduced gene expression indicates that HcpR may also be anactivator of these genes in the presence of nitrite. All regulators de-tected as upregulated in our array study were also upregulated usingqRT-PCR analysis (Table 3), indicating that some of this regulation ofthe above-described gene may be indirect. Interestingly, PG1181, re-ported to be the most upregulated gene in the presence of nitric oxide(7), was slightly downregulated in our study.
To examine the molecular basis of this regulation, we scruti-nized the DNA sequence of the hcp promoter sequence for theHcpR-binding site. The hcp gene is flanked by intergenic se-quences (IGS) and is not part of an operon (Fig. 5A). This wasfurther verified by determination of the hcp transcriptional startsite using high-throughput sequencing of the P. gingivalis tran-scriptome (Fig. 5B and data not shown). The transcriptional start
FIG 3 Roles of HcpR and hemin on P. gingivalis growth in the presence of nitrate or nitrite. P. gingivalis strains were anaerobically grown to midlogarithmic phasein mycoplasma broth without hemin. The cultures were then diluted to an OD660 of 0.1 in mycoplasma minus hemin (�Hm) (A and B) and mycoplasmacontaining 5 �g/ml hemin (�Hm) (C and D). Various concentrations of nitrite or nitrate (A) were then added to the cultures. Unsupplemented cultures servedas controls. The cultures were anaerobically grown for an additional 25 h at 37°C. Means and standard deviations from the experiment performed in triplicateare shown (n � 3; results are � standard error of the mean). *, P � 0.002, W83 versus V2807 under 1 mM nitrite (Student’s t test). Parental W83 strain (A) andHcpR-deficient mutant V2807 strain (B) grown in low hemin conditions (�Hm). W83, V2807, and V2835 (complemented HcpR mutant strain) grown in thepresence of high hemin concentrations (� Hm) with 2 mM (C) and 4 mM (D) nitrite. n � 3; results are � standard error of the mean.
FIG 4 Role of HcpR on P. gingivalis growth in the presence of nitric oxide. P. gingivalis strains were anaerobically grown to midlogarithmic phase in mycoplasmabroth without hemin. The cultures were then diluted to an OD660 of 0.1 in mycoplasma minus hemin, and 1 mM nitrite or 30 nM GSNO was then added to thecultures. Unsupplemented cultures served as controls. The cultures were grown for an additional 18 h at 37°C under anaerobic conditions. Means and standarddeviations from the experiment performed in triplicate are shown (n � 3; results are � standard error of the mean). Parental W83 strain (A), HcpR-deficientmutant strain (B). *, P � 0.001, W83 versus V2807 under 30 nM GSNO (Student’s t test).
Identification and Role of HcpR in P. gingivalis
September 2012 Volume 80 Number 9 iai.asm.org 3327
on May 21, 2021 by guest
http://iai.asm.org/
Dow
nloaded from
site was observed to be located 29 bp upstream of the hcp transla-tional start site (Fig. 5B and data not shown). qRT-PCR withprimers originating within the predicted transcript (1R and 2R)showed the presence of a template whereas no product was de-tected using a primer upstream of the transcriptional start site(3R) (results not shown), thus further reinforcing the results ob-tained from the transcriptome analysis. We identified a direct re-peat sequence, characteristic of the Fnr binding site (TGTCGCnnnnGCGACA), which was 81 bp upstream of the translationalstart site of hcp (Fig. 5B). To verify binding of HcpR to the pre-dicted DNA sequence, we performed an electrophoretic mobilityshift assay with both E. coli cell lysates and purified rHcpR (datanot shown). As shown in Fig. 5C, reduced migration of a DNAfragment containing the HcpR binding site was observed in E. colicell lysates expressing HcpR (lane 2). No reduced mobility was
observed in the lane containing E. coli lysate grown in the absenceof IPTG (Fig. 5C, lane 3). Furthermore, the purified rHcpR boundthe hcp promoter DNA. The binding was observed only whenrHcpR reconstituted with hemin was used (lanes 6 to 9). Also, thebinding was reduced by unlabeled hcp promoter DNA (lanes 7 and9), indicating that the binding is specific. These results indicatethat HcpR binds to the hcp promoter sequence and directly regu-lates hcp expression.
P. gingivalis hcpR mutant has decreased survival in hostcells. P. gingivalis adheres, invades, and survives for prolongedperiods of time in a variety of host cells. Thus, we investigated therole of HcpR in the survival of P. gingivalis in host cells using aHUVEC and HOK survival assay as described by Ueshima et al.(55). As shown in Table 4, the parental strain W83 was recoveredfrom HUVECs following total microbial challenge (total interac-
FIG 5 P. gingivalis HcpR regulates hcp expression. (A) Organization of the hcp (PG0893) locus. Intergenic sequences (IGS) flank the hcp gene. The HcpR bindingsite is shown as an orange oval. (B) Bioinformatics analysis of the hcp promoter. The HcpR binding site is shown in orange. The transcriptional start site (ts) islocated 29 bp upstream of the translational start site (Met). The primer positions used for the generation of the EMSA probe are shown in italics and areunderlined. Positions of primers used for qRT-PCR designated 1R, 2R, and 3R are shown in green, and their direction is indicated by an arrow. (C) EMSA. Lysatesprepared from IPTG induced E. coli-pET30 hcpR (lane 2, �i) and IPTG-uninduced E. coli-pET30 hcpR (lane 3,�u), and increasing amounts of rHcpR (lanes 5to 9) were incubated with a 200-bp DNA fragment containing the hcp promoter (DNA). Reaction mixtures containing DNA only were run as reference controls(lanes 1 and 4). Unlabeled hcp promoter DNA (UDNA) was used as a specific competitor (lanes 7 and 9) (10-fold excess over labeled DNA). rHcpR without (lane5) and reconstituted with (lanes 6 to 9) hemin was used. Lane 5 contained 30 �g of rHcpR, lanes 6 and 7 contained 10 �g of rHcpR, and lanes 8 and 9 had 30 �gof rHcpR.
TABLE 4 Role of HcpR in the interaction of P. gingivalis with eukaryotic cellsa
StrainTotal interaction withHUVECsb
No. of cells internalizedc by:
HUVECs HOK
P. gingivalis W83 (wild type) 5,633 � 170.04 1,070 � 131.00 290.25 � 33.93P. gingivalis V2807 (HcpR-deficient mutant) 0.0 � 0.0 63 � 5.0 4.50 � 2.65P. gingivalis V2835 (HcpR-revertant strain) ND 390 � 31.5 NDa The numbers shown are from an experiment performed in triplicate. ND, not determined.b The number of intracellular bacteria after the infection was determined using the antibiotic protection assay. The number of surviving cells was determined as for total interaction.c The number of total bacteria interacting with the host cells was enumerated by plating eukaryotic cell lysates on blood agar plates and determining the CFU following 8 days ofincubation under anaerobic conditions.
Lewis et al.
3328 iai.asm.org Infection and Immunity
on May 21, 2021 by guest
http://iai.asm.org/
Dow
nloaded from
tion) and from the intracellular environment (intracellular sur-vival). However, we recovered no live V2807 from HUVECs.Next, we examined the ability of the bacterium to survive withHOKs. While live bacteria were recovered from cells challengedwith wild-type W83 P. gingivalis, drastically reduced numbers ofmicrobial cells were recovered when the cells were challenged withV2807. Finally, we also tested the ability of the revertant V2835strain to survive with HUVECs. We observed that the ability ofV2835 to survive with the eukaryotic cells was comparable to thatof W83, thus verifying that the reduced survival of V2807 de-scribed above is due to mutation in HcpR (Table 4). These resultsindicate that HcpR plays a significant role in sustaining bacterialcell viability when bacteria are exposed to eukaryotic cells.
DISCUSSION
We show that P. gingivalis is capable of withstanding high concentra-tions of nitrite. It can grow with nitrite concentrations as high as 2mM, the level found in the oral cavity (34). Previous work has shownthe sensitivity of oral bacteria to acidified nitrite, including the abilityof P. gingivalis to grow with 0.2 mM nitrite (1). Knowledge of theadaptive potential to nitrite has practical applications for nitrate-richfoods, such as vegetables (spinach, beetroot, and lettuce) or fruits,which are readily available and are recommended for daily intake.Believed to be rich sources of nutrients, these foods may also have thepotential to reduce pathogenic bacterial growth by providing largeamounts of nitrate that could be metabolized to toxic nitrite andconsequently promote oral health.
In addition, P. gingivalis can withstand nitric oxide exposure.Thus, it can survive the innate immune response, which is especiallyelevated in inflamed periodontal pockets. Our data verify the resultsrecently reported by Boutrin et al. (7), who have shown that P. gingi-valis can grow in the presence of low nitric oxide concentrations.
With exposure to nitrite or nitric oxide (GSNO), the hcp gene(PG0893) was the most upregulated in our microarray study. Suchresults indicate that the gene product plays an indispensable rolein the protection of P. gingivalis against nitrosative stress. Indeed,previous studies have shown that hcp is required for bacterialgrowth with nitric oxide (7). The upregulation of hcp was quanti-fied using qRT-PCR to be more than 150-fold higher upon expo-sure to nitrosative stress. This result is much higher than thatobserved in a previous study that used NONOate as a nitric oxidedonor (7). Such significant upregulation of hcp is in agreementwith previous reports demonstrating drastic upregulation of thisgene in other anaerobic and facultatively anaerobic bacteria ex-posed to nitrite (26, 46, 48, 56). In addition, array examinations ofthe response of Desulfovibrio vulgaris to nitrite have shown hcp2 tobe upregulated 254-fold upon exposure to nitrite (26). Further-more, significant upregulation of hcp in E. coli grown anaerobi-cally in the presence of nitrate has been recently reported (48).
Despite the significant upregulation of hcp in response to nitrite,indicating that the gene product plays a major role in nitrosative stressprotection, the mechanisms of gene regulation remained poorlycharacterized for a long time. The NsrR and NorR repressors wereproposed to mediate regulation of hcp in facultative anaerobic bacte-ria (enterobacteria, beta- and alphaproteobacteria) (17) and Vibrion-ales, respectively (46). Very recently, the regulation of hcp in E. coliwas reported to be mediated by OxyR (48). However, we demon-strate that the Fnr-like regulator, designated HcpR, activates hcp ex-pression in the anaerobic bacterium P. gingivalis. Likewise, we ob-serve that the P. gingivalis OxyR plays no role in regulation of the gene.
Such results are in agreement with the previous report of Fnr alsoplaying a role in regulation of hcp in anaerobically grown E. coli (17).Other differences in regulation could be speculated to be based ondifferent metabolisms of the bacteria; P. gingivalis is an anaerobicorganism, and E. coli is a facultatively anaerobic bacterium. Despiteextensive biochemical and structural studies, the physiological role ofHcp is still not well defined (3, 11, 18, 56) and its contribution tomicrobial metabolism is unknown. Its role in nitrosative stress mayindicate that it acts through both common intermediates generatedduring nitrite, nitric oxide, and hydroxylamine metabolism/detoxifi-cation (Fig. 6). Another intriguing finding was the genomic location/organization of the gene coding for Hcp. In facultative anaerobes, hcpis followed by a gene encoding a putative NADH oxidoreductase,while in obligate anaerobes, hcp loci lack the NADH oxidoreductase-coding gene (46, 56). In addition, in some bacteria, such as Desulfo-vibrio vulgaris, two genes code for Hcp, hcp1 and hcp2. While hcp1expression is unaffected by nitrite, there is significant upregulation ofhcp2 in the presence of nitrite (26). These results are consistent withthe different roles of Hcp proteins in various bacteria. Thus, possiblydifferent regulation mechanisms are required to activate expressionof these genes.
HcpR is part of a multigene locus. Two intriguing observationswere noted. First, HcpR is encoded downstream of another regu-lator with similarity to HspR (12). Second, the other upstreamgenes code for cell envelope biogenesis. In addition, Hsp regulatescell surface biogenesis in response to heat (12). Thus, it is possiblethat the P. gingivalis locus is involved in a multifaceted response toenvironmental stress. Furthermore, the genomic locus coding forHcpR differs in P. gingivalis compared to other Bacteroides bacte-ria (data not shown). In Prevotella intermedia 17, HcpR is encodeddownstream of Hcp, whereas in B. fragilis and B. thetaiotamicron itis encoded upstream of the Hcp-coding gene. Interestingly, in T.maritima, a locus encoding functionally similar proteins to thatfound in Bacteroidetes is present (data not shown). As this is anancient anaerobic Gram-negative bacterium (28), it is probablethat the hcp-hcpR locus has been derived from this bacterium.Otherwise, it may have been acquired from the archaea due to the
FIG 6 Schematic representation of P. gingivalis mechanisms mediating re-sponse to nitrosative stress.
Identification and Role of HcpR in P. gingivalis
September 2012 Volume 80 Number 9 iai.asm.org 3329
on May 21, 2021 by guest
http://iai.asm.org/
Dow
nloaded from
fact that T. maritima has multiple genes of archaeal origin (41).Although many of these genes confer the ability of the bacteriumto live at high temperatures, it is likely that genes required foradaptation to other stress conditions were acquired as well.
Our studies suggest hemin as an important player in defenseagainst nitrosative stress. First, we found upregulation of the genehmuY (PG1551), coding for the major hemin uptake protein (33, 50),in the presence of the nitric oxide-generating agent GSNO. As heminplays an antioxidative role, such upregulation may also be a mecha-nism used by P. gingivalis in defense against nitrosative stress. How-ever, P. gingivalis encodes the nitrite reductase system NrfHA, whichis a multiheme protein system. NrfA is a periplasmic pentaheme cy-tochrome c nitrite reductase that can reduce nitrite, nitric oxide, orhydroxylamine to ammonia, and membrane-anchored NrfH is a tet-raheme cytochrome c menaquinol dehydrogenase (Fig. 6) (37). Thissystem plays a crucial role in defense against nitrite, nitric oxide, andhydroxylamine stress in Wolinella succinogenes (29, 37); thus, it isprobable that NrfHA plays a similar role in P. gingivalis. Lack of he-min would be expected to render the NrfHA system inactive. There-fore, upregulation of the hemin uptake system may be a way to ensuresufficient provision of hemin to keep the system in its active form. Itis likely that the system gets oxidized rapidly as it is located in theperiplasm; thus, high levels of hemin are required to keep it func-tional.
A second connection of hemin to the response to nitrosative stresswas seen when examining regulatory mechanisms; interestingly,HcpR appeared to be an activator of PG1551, although this regulationmay be indirect as no binding of HcpR to DNA containing thePG1551 promoter sequence was observed (results not shown). Hcp isan iron protein, and higher hemin intake may ensure that enoughiron is available to keep the elevated levels of Hcp in its active form. Ofnote, hemin is required for HcpR binding to DNA. As hcpR expres-sion is not affected by nitrosative stress, mechanisms other than tran-scriptional control govern the transition of the regulator to its activeform. We show that hemin is required for HcpR binding to the hcppromoter. Hemin has also been proposed to be involved in the con-version of Dnr to an active DNA-binding form (10). P. gingivalisHcpR is a Dnr-like regulator that also is required for tolerance tonitrosative stress (45). Although it can adopt the typical structure ofFnr-like or Crp-like regulators (20, 23), differences exist in the sub-strate-binding and DNA-binding domains, indicating that it mayregulate different genes and respond to different stimuli. These resultsare consistent with the Dnr-regulating response of genes coding fordenitrification mechanisms (45) and P. gingivalis HcpR primarilyregulating hcp. However, based on the structural similarity betweenDnr and HcpR as well as their roles in response to nitrosative stress, itis probable that the mechanism of activation is similar for both pro-teins. Thus, a higher provision of hemin would also ensure HcpRactivation. The hemin-based activation mechanism of HcpR is con-sistent with the protein lacking the conserved cysteines required forbinding of the Fe-S cluster, as seen in the Fnr-like regulators, and alsoruling out Cys switch or Cys nitrosylation, which are the molecularbasis for activation of the OxyR regulator (48). Finally, in silico anal-ysis has shown that the Dnr-like regulator may regulate hcp expres-sion in A. ferrooxidans and Thermochromatium tepidum (46), thusfurther bridging the gap between the roles, and likely molecularmechanism, of the two regulators.
We show that the growth of the P. gingivalis HcpR mutantV2807 in the presence of nitrite and GSNO is reduced relative tothe parental strain. The ability of P. gingivalis to adhere, invade,
survive, and multiply in a variety of host cells is well documented(13, 62). As release of oxidative and nitrosative reactive species ispart of the immune defense against invading pathogens, P. gingi-valis would be expected to have protective mechanisms againstsuch stress. Our data indicate that HcpR is required for survival inthe presence of nitrosative stress. We also showed that survival ofV2807 in host cells is drastically reduced. The nearly completekilling of the mutant bacteria by the host cells indicates that nitro-sative stress is the major effector against P. gingivalis. These stud-ies, combined with the inhibition of growth of the strain in thepresence of nitrite, which is characteristic of the oral cavity, estab-lish the biological significance of the regulator HcpR.
In summary, this work identifies HcpR as a novel regulator ofP. gingivalis adaptation to nitrosative stress. In addition, it verifiesthe role of the HcpR family of regulators in the regulation of hcpexpression. The important role of HcpR in nitrosative stress war-rants further investigation of the protein, including its regulatorymechanism, through biochemical and structural studies.
ACKNOWLEDGMENTS
This research was supported by USPHS grants 5K22DE14180,R01DE016124 and R01DE018039 from the National Institute of Dentaland Craniofacial Research awarded to Janina P. Lewis.
The determination of the genomic sequence of P. gingivalis was carriedout collaboratively by The Institute for Genomic Research (TIGR) andThe Forsyth Dental Center database with support from NIDCR. The genomicsequence of P. gingivalis W83 was obtained from JCVI (http://www.jcvi.org)and the Los Alamos Oral Pathogen Sequence Database (http://www.oralgen.lanl.gov). We thank Anuya Paranjapee for help with the EMSA studies,Karina Martinez for carrying out the qRT-PCR analyses, and Hiroshi Mi-yazaki for his help preparing cell cultures for this study.
REFERENCES1. Allaker RP, Silva Mendez LS, Hardie JM, Benjamin N. 2001. Antimi-
crobial effect of acidified nitrite on periodontal bacteria. Oral Microbiol.Immunol. 16:253–256.
2. Altschul SF, et al. 1997. Gapped BLAST and PSI-BLAST: a new generation ofprotein database search programs. Nucleic Acids Res. 25:3389–3402.
3. Aragao D, Mitchell EP, Frazao CF, Carrondo MA, Lindley PF. 2008.Structural and functional relationships in the hybrid cluster protein fam-ily: structure of the anaerobically purified hybrid cluster protein fromDesulfovibrio vulgaris at 1.35 A resolution. Acta Crystallogr. D Biol. Crys-tallogr. 64:665– 674.
4. Arnold K, Bordoli L, Kopp J, Schwede T. 2006. The SWISS-MODELworkspace: a web-based environment for protein structure homologymodelling. Bioinformatics 22:195–201.
5. Beck JD, Offenbacher S, Williams R, Gibbs P, Garcia R. 1998. Periodontitis:a risk factor for coronary heart disease? Ann. Periodontol. 3:127–141.
6. Bordoli L, et al. 2009. Protein structure homology modeling usingSWISS-MODEL workspace. Nat. Protoc. 4:1–13.
7. Boutrin MC, Wang C, Aruni W, Li X, Fletcher HM. 2012. Nitric oxidestress resistance in Porphyromonas gingivalis is mediated by a putativehydroxylamine reductase. J. Bacteriol. 194:1582–1592.
8. Cabello P, et al. 2004. Hydroxylamine assimilation by Rhodobacter cap-sulatus E1F1. requirement of the hcp gene (hybrid cluster protein) locatedin the nitrate assimilation nas gene region for hydroxylamine reduction. J.Biol. Chem. 279:45485– 45494.
9. Cadby IT, Busby SJ, Cole JA. 2011. An HcpR homologue from Desulfo-vibrio desulfuricans and its possible role in nitrate reduction and nitrosa-tive stress. Biochem. Soc. Trans. 39:224 –229.
10. Castiglione N, Rinaldo S, Giardina G, Cutruzzola F. 2009. The tran-scription factor DNR from Pseudomonas aeruginosa specifically requiresnitric oxide and haem for the activation of a target promoter in Esche-richia coli. Microbiology 155:2838 –2844.
11. Cooper SJ, Garner CD, Hagen WR, Lindley PF, Bailey S. 2000. Hybrid-cluster protein (HCP) from Desulfiovibrio vulgaris (Hildenborough) at 1.6A resolution. Biochemistry 39:15044 –15054.
Lewis et al.
3330 iai.asm.org Infection and Immunity
on May 21, 2021 by guest
http://iai.asm.org/
Dow
nloaded from
12. Das Gupta T, Bandyopadhyay B, Das Gupta SK. 2008. Modulation ofDNA-binding activity of Mycobacterium tuberculosis HspR by chaperones.Microbiology 154:484 – 490.
13. Dorn BR, Burks JN, Seifert KN, Progulske-Fox A. 2000. Invasion ofendothelial and epithelial cells by strains of Porphyromonas gingivalis.FEMS Microbiol. Lett. 187:139 –144.
14. Dorn BR, Dunn WA, Jr, Progulske-Fox A. 2001. Porphyromonas gingi-valis traffics to autophagosomes in human coronary artery endothelialcells. Infect. Immun. 69:5698 –5708.
15. Ezzo PJ, Cutler CW. 2003. Microorganisms as risk indicators for peri-odontal disease. Periodontol. 2000 32:24 –35.
16. Fang FC. 1997. Perspectives series: host/pathogen interactions. Mecha-nisms of nitric oxide-related antimicrobial activity. J. Clin. Invest. 99:2818 –2825.
17. Filenko N, et al. 2007. The NsrR regulon of Escherichia coli K-12 includesgenes encoding the hybrid cluster protein and the periplasmic, respiratorynitrite reductase. J. Bacteriol. 189:4410 – 4417.
18. Filenko NA, Browning DF, Cole JA. 2005. Transcriptional regulation ofa hybrid cluster (prismane) protein. Biochem. Soc. Trans. 33:195–197.
19. Fletcher HM, et al. 1995. Virulence of a Porphyromonas gingivalis W83mutant defective in the prtH gene. Infect. Immun. 63:1521–1528.
20. Giardina G, Rinaldo S, Castiglione N, Caruso M, Cutruzzola F. 2009. Adramatic conformational rearrangement is necessary for the activation ofDNR from Pseudomonas aeruginosa. Crystal structure of wild-type DNR.Proteins 77:174 –180.
21. Giardina G, et al. 2008. NO sensing in Pseudomonas aeruginosa: structureof the transcriptional regulator DNR. J. Mol. Biol. 378:1002–1015.
22. Gostick DO, et al. 1999. Two operons that encode FNR-like proteins inLactococcus lactis. Mol. Microbiol. 31:1523–1535.
23. Green J, Scott C, Guest JR. 2001. Functional versatility in the CRP-FNRsuperfamily of transcription factors: FNR and FLP. Adv. Microb. Physiol.44:1–34.
24. Hajishengallis G, et al. 2011. Low-abundance biofilm species orchestratesinflammatory periodontal disease through the commensal microbiotaand complement. Cell Host Microbe 10:497–506.
25. Hammes WP. 2012. Metabolism of nitrate in fermented meats: the char-acteristic feature of a specific group of fermented foods. Food Microbiol.29:151–156.
26. Haveman SA, Greene EA, Stilwell CP, Voordouw JK, Voordouw G.2004. Physiological and gene expression analysis of inhibition of Desulfo-vibrio vulgaris Hildenborough by nitrite. J. Bacteriol. 186:7944 –7950.
27. He J, et al. 2006. Role of Porphyromonas gingivalis FeoB2 in metal uptakeand oxidative stress protection. Infect. Immun. 74:4214 – 4223.
28. Huber R, et al. 1986. Thermatoga maritima sp. nov. represents a newgenus of unique extremely thermophilic eubacteria growing up to 90oC.Arch. Microbiol. 144:324 –333.
29. Kern M, Volz J, Simon J. 2011. The oxidative and nitrosative stressdefence network of Wolinella succinogenes: cytochrome c nitrite reductasemediates the stress response to nitrite, nitric oxide, hydroxylamine andhydrogen peroxide. Environ. Microbiol. 13:2478 –2494.
30. Kiley PJ, Beinert H. 1998. Oxygen sensing by the global regulator, FNR:the role of the iron-sulfur cluster. FEMS Microbiol. Rev. 22:341–352.
31. Lamont RJ, Jenkinson HF. 1998. Life below the gum line: pathogenicmechanisms of Porphyromonas gingivalis. Microbiol. Mol. Biol. Rev. 62:1244 –1263.
32. Lewis JP, Iyer D, Anaya-Bergman C. 2009. Adaptation of Porphyromonasgingivalis to microaerophilic conditions involves increased consumption offormate and reduced utilization of lactate. Microbiology 155:3758–3774.
33. Lewis JP, Plata K, Yu F, Rosato A, Anaya C. 2006. Transcriptionalorganization, regulation and role of the Porphyromonas gingivalis W83hmu haemin-uptake locus. Microbiology 152:3367–3382.
34. Lundberg JO, Weitzberg E, Cole JA, Benjamin N. 2004. Nitrate, bacteriaand human health. Nat. Rev. Microbiol. 2:593– 602.
35. MacMicking JD, et al. 1995. Altered responses to bacterial infection andendotoxic shock in mice lacking inducible nitric oxide synthase. Cell 81:641– 650.
36. Mazoch J, Kucera I. 2002. Control of gene expression by FNR-like proteins infacultatively anaerobic bacteria. Folia Microbiol. (Praha) 47:95–103.
37. Meuric V, Rouillon A, Chandad F, Bonnaure-Mallet M. 2010. Putativerespiratory chain of Porphyromonas gingivalis. Future Microbiol. 5:717–734.
38. Munch R, et al. 2005. Virtual Footprint and PRODORIC: an integrative
framework for regulon prediction in prokaryotes. Bioinformatics 21:4187– 4189.
39. Mydel P, et al. 2006. Roles of the host oxidative immune response andbacterial antioxidant rubrerythrin during Porphyromonas gingivalis infec-tion. PLoS Pathog. 2:e76. doi:10.1371/journal.ppat.0020076.
40. Nathan C. 1995. Natural resistance and nitric oxide. Cell 82:873– 876.41. Nelson KE, et al. 1999. Evidence for lateral gene transfer between Archaea
and bacteria from genome sequence of Thermotoga maritima. Nature 399:323–329.
42. Nelson KE, et al. 2003. Complete genome sequence of the oral pathogenicbacterium Porphyromonas gingivalis strain W83. J. Bacteriol. 185:5591–5601.
43. Olczak T, Sroka A, Potempa J, Olczak M. 2008. Porphyromonas gingivalisHmuY and HmuR: further characterization of a novel mechanism ofheme utilization. Arch. Microbiol. 189:197–210.
44. Palmerini CA, Palombari R, Perito S, Arienti G. 2003. NO synthesis inhuman saliva. Free Radic. Res. 37:29 –31.
45. Rinaldo S, Giardina G, Brunori M, Cutruzzola F. 2006. N-oxide sensingand denitrification: the DNR transcription factors. Biochem. Soc. Trans.34:185–187.
46. Rodionov DA, Dubchak IL, Arkin AP, Alm EJ, Gelfand MS. 2005.Dissimilatory metabolism of nitrogen oxides in bacteria: comparative re-construction of transcriptional networks. PLoS Comput. Biol. 1:e55. doi:10.1371/journal.pcbi.0010055.
47. Schwede T, Kopp J, Guex N, Peitsch MC. 2003. SWISS-MODEL: anautomated protein homology-modeling server. Nucleic Acids Res. 31:3381–3385.
48. Seth D, Hausladen A, Wang YJ, Stamler JS. 2012. Endogenous proteinS-Nitrosylation in E. coli: regulation by OxyR. Science 336:470 – 473.
49. Singh A, et al. 2011. The capsule of Porphyromonas gingivalis leads to areduction in the host inflammatory response, evasion of phagocytosis, andincrease in virulence. Infect. Immun. 79:4533– 4542.
50. Smalley JW, et al. 2011. HmuY haemophore and gingipain proteasesconstitute a unique syntrophic system of haem acquisition by Porphy-romonas gingivalis. PLoS One 6:e17182. doi:10.1371/journal-.pone.0017182.
51. Spiro S. 2006. Nitric oxide-sensing mechanisms in Escherichia coli.Biochem. Soc. Trans. 34:200 –202.
52. Spraggon G, et al. 2004. On the use of DXMS to produce more crystal-lizable proteins: structures of the T. maritima proteins TM0160 andTM1171. Protein Sci. 13:3187–3199.
53. Stewart GR, et al. 2002. Dissection of the heat-shock response in Myco-bacterium tuberculosis using mutants and microarrays. Microbiology 148:3129 –3138.
54. Sztukowska M, Bugno M, Potempa J, Travis J, Kurtz DM, Jr. 2002. Roleof rubrerythrin in the oxidative stress response of Porphyromonas gingiva-lis. Mol. Microbiol. 44:479 – 488.
55. Ueshima J, et al. 2003. Purification, gene cloning, gene expression, andmutants of Dps from the obligate anaerobe Porphyromonas gingivalis. In-fect. Immun. 71:1170 –1178.
56. van den Berg WA, Hagen WR, van Dongen WM. 2000. The hybrid-cluster protein (‘prismane protein’) from Escherichia coli. Characteriza-tion of the hybrid-cluster protein, redox properties of the [2Fe-2S] and[4Fe-2S–2O] clusters and identification of an associated NADH oxi-doreductase containing FAD and [2Fe-2S]. Eur. J. Biochem. 267:666 –676.
57. Vollack KU, Hartig E, Korner H, Zumft WG. 1999. Multiple transcrip-tion factors of the FNR family in denitrifying Pseudomonas stutzeri: char-acterization of four fnr-like genes, regulatory responses and cognate met-abolic processes. Mol. Microbiol. 31:1681–1694.
58. Wolfe MT, Heo J, Garavelli JS, Ludden PW. 2002. Hydroxylaminereductase activity of the hybrid cluster protein from Escherichia coli. J.Bacteriol. 184:5898 –5902.
59. Xia DS, Liu Y, Zhang CM, Yang SH, Wang SL. 2006. Antimicrobialeffect of acidified nitrate and nitrite on six common oral pathogens invitro. Chin Med. J. (Engl.) 119:1904 –1909.
60. Xu J, Verstraete W. 2001. Evaluation of nitric oxide production by lac-tobacilli. Appl. Microbiol. Biotechnol. 56:504 –507.
61. Yanamandra SS, Sarrafee SS, Anaya-Bergman C, Jones K, Lewis JP.2012. Role of the Porphyromonas gingivalis extracytoplasmic functionsigma factor, SigH. Mol. Oral Microbiol. 27:202–219.
62. Yilmaz O. 2008. The chronicles of Porphyromonas gingivalis: the micro-bium, the human oral epithelium and their interplay. Microbiology 154:2897–2903.
Identification and Role of HcpR in P. gingivalis
September 2012 Volume 80 Number 9 iai.asm.org 3331
on May 21, 2021 by guest
http://iai.asm.org/
Dow
nloaded from
