ABSTRACT Here, we describe the molecular and immunological characterization of the bdr gene family of Borrelia turicatae , a relapsing-fever spirochete. Nine bdr alleles belonging to two different subfamilies were sequenced and localized to linear plasmids. Anti-Bdr antiserum was generated and used to analyze Bdr expression in pre- and postinfection isogenic populations. The analyses presented here provide a detailed characterization of the Bdr proteins in a relapsing-fever spirochete species, enhancing our understanding of these proteins at the genus-wide level.
Azoles are a class of antimicrobial drugs used clinically to treat yeast and fungal infections. Against pathogenic yeast and fungi, azoles act by inhibiting the activity of the cytochrome P450 Cyp51, which is involved in the synthesis of a critical component of the yeast and fungal cell membrane. Azoles have antibacterial activity, including against mycobacteria, but the basis for this activity is not well-understood. We demonstrated that imidazoles are bactericidal to Mycobacterium tuberculosis. A marked increase in reactive oxygen species (ROS) was observed within imidazole-treated M. tuberculosis. The generation of ROS did not appear to be related to the mechanism of killing of imidazoles, as the addition of antioxidants or altered expression of detoxifying enzymes had no effect on growth. We examined the metabolic changes induced by econazole treatment in both wild-type and econazole-resistant mutant strains of M. tuberculosis. Econazole treatment induced changes in carbohydrates, amino acids, and energy metabolism in both strains. Notably, the untreated mutant strain had a metabolic profile similar to the wild-type drug-treated cells, suggesting that adaptation to similar stresses may play a role in econazole resistance.
Current estimates indicate that nearly a third of the world's population is latently infected with Mycobacterium tuberculosis. Reduced oxygen tension and nitric oxide exposure are two conditions encountered by bacilli in vivo that may promote latency. In vitro exposure to hypoxia or nitric oxide results in bacterial stasis with concomitant induction of a 47-gene regulon controlled by the transcription factor DosR. In this report we demonstrate that both the dosS gene adjacent to dosR and another gene, dosT (Rv2027c), encode sensor kinases, each of which can autophosphorylate at a conserved histidine and then transfer phosphate to an aspartate residue of DosR. Mutant bacteria lacking both sensors are unable to activate expression of DosR-regulated genes. These data indicate that DosR/DosS/DosT comprise a two-component signaling system that is required for the M. tuberculosis genetic response to hypoxia and nitric oxide, two conditions that produce reversible growth arrest in vitro and may contribute to latency in vivo. Current estimates indicate that nearly a third of the world's population is latently infected with Mycobacterium tuberculosis. Reduced oxygen tension and nitric oxide exposure are two conditions encountered by bacilli in vivo that may promote latency. In vitro exposure to hypoxia or nitric oxide results in bacterial stasis with concomitant induction of a 47-gene regulon controlled by the transcription factor DosR. In this report we demonstrate that both the dosS gene adjacent to dosR and another gene, dosT (Rv2027c), encode sensor kinases, each of which can autophosphorylate at a conserved histidine and then transfer phosphate to an aspartate residue of DosR. Mutant bacteria lacking both sensors are unable to activate expression of DosR-regulated genes. These data indicate that DosR/DosS/DosT comprise a two-component signaling system that is required for the M. tuberculosis genetic response to hypoxia and nitric oxide, two conditions that produce reversible growth arrest in vitro and may contribute to latency in vivo. Tuberculosis (TB) 1The abbreviations used are: TB, tuberculosis; MTB, Mycobacterium tuberculosis; CAPS, 3-(cyclohexylamino)propanesulfonic acid; MES, 4-morpholineethanesulfonic acid; HRP, horseradish peroxidase.1The abbreviations used are: TB, tuberculosis; MTB, Mycobacterium tuberculosis; CAPS, 3-(cyclohexylamino)propanesulfonic acid; MES, 4-morpholineethanesulfonic acid; HRP, horseradish peroxidase. has placed a heavy burden on the global community for centuries, earning such morbid nicknames as The White Plague and The Captain of the Men of Death (1Dock W. Am. Rev. Tuberc. 1946; 53: 297-305PubMed Google Scholar). The causative agent, Mycobacterium tuberculosis (MTB), kills about 2 million people annually making it a leading cause of infectious death worldwide (2Bloom B.R. Small P.M. N. Engl. J. Med. 1998; 338: 677-678Crossref PubMed Scopus (87) Google Scholar, 3Dye C. Scheele S. Dolin P. Pathania V. Raviglione M.C. J. Am. Med. Assoc. 1999; 282: 677-686Crossref PubMed Scopus (2714) Google Scholar). The success of MTB as a pathogen is closely linked with its capacity to persist for years or decades in humans in the absence of any clinical disease symptoms. Current estimates place the number of people latently infected with MTB at nearly 2 billion, or one-third of the Earth's population (3Dye C. Scheele S. Dolin P. Pathania V. Raviglione M.C. J. Am. Med. Assoc. 1999; 282: 677-686Crossref PubMed Scopus (2714) Google Scholar, 4Enarson D.A. Murray J.F. Rom W.N. Garay S. Tuberculosis. Little, Brown and Co., Boston1996: 55-75Google Scholar). Eradicating this enormous reservoir of latently infected carriers is complicated by several factors, including the availability, cost, and length of drug therapy required for successful treatment of latent TB. Although TB has been studied for centuries, the triggers that promote and maintain latent infections are still obscure. Two conditions frequently associated with latent TB in vivo are reduced oxygen tension and nitric oxide (NO) exposure (5Wayne L.G. Sohaskey C.D. Annu. Rev. Microbiol. 2001; 55: 139-163Crossref PubMed Scopus (643) Google Scholar, 6Nathan C. Shiloh M.U. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8841-8848Crossref PubMed Scopus (1147) Google Scholar). Both of these stimuli can induce reversible bacterial stasis in vitro (7Wayne L.G. Infect. Immun. 1977; 17: 528-530Crossref PubMed Google Scholar, 8Voskuil M.I. Schnappinger D. Harrell M.I. Visconti K.C. Dolganov G. Sherman D.R. Schoolnik G.K. J. Exp. Med. 2003; 198: 705-713Crossref PubMed Scopus (765) Google Scholar), and both are encountered by bacilli in vivo (5Wayne L.G. Sohaskey C.D. Annu. Rev. Microbiol. 2001; 55: 139-163Crossref PubMed Scopus (643) Google Scholar, 9Choi H.S. Rai P.R. Chu H.W. Cool C. Chan E.D. Am. J. Respir. Crit. Care Med. 2002; 166: 178-186Crossref PubMed Scopus (132) Google Scholar, 10Canetti G. The Tubercle Bacillus in the Pulmonary Lesion of Man. Springer Publishing Co., New York1955: 111-126Google Scholar). Further, although MTB requires oxygen for growth, it can survive without oxygen for surprisingly long periods of time (11Corper H.J. Cohn M.L. Am. Rev. Tuberc. 1933; 28: 856-874Google Scholar, 12Wayne L.G. Lin K.Y. Infect. Immun. 1982; 37: 1042-1049Crossref PubMed Google Scholar). Still the evidence linking hypoxia and NO to latent TB in vivo remains circumstantial. Analysis of the MTB response to these factors is needed to define the role they may play in promoting and maintaining TB latency in humans. Previous reports identified a set of 47 MTB genes that are rapidly up-regulated in response to reduced oxygen tension or NO (8Voskuil M.I. Schnappinger D. Harrell M.I. Visconti K.C. Dolganov G. Sherman D.R. Schoolnik G.K. J. Exp. Med. 2003; 198: 705-713Crossref PubMed Scopus (765) Google Scholar, 13Sherman D.R. Voskuil M. Schnappinger D. Liao R. Harrell M.I. Schoolnik G.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7534-7539Crossref PubMed Scopus (633) Google Scholar). Among the MTB genes induced by hypoxia or exposure to NO is the putative two-component regulatory system dosR-dosS (also called devR-devS, Rv3133c/Rv3132c 2Genes are denoted both by name and the designation assigned by the TubercuList Web site: genolist.pasteur.fr/TubercuList/.2Genes are denoted both by name and the designation assigned by the TubercuList Web site: genolist.pasteur.fr/TubercuList/.) (8Voskuil M.I. Schnappinger D. Harrell M.I. Visconti K.C. Dolganov G. Sherman D.R. Schoolnik G.K. J. Exp. Med. 2003; 198: 705-713Crossref PubMed Scopus (765) Google Scholar, 13Sherman D.R. Voskuil M. Schnappinger D. Liao R. Harrell M.I. Schoolnik G.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7534-7539Crossref PubMed Scopus (633) Google Scholar). In bacteria, two-component response regulator systems are an important means by which a variety of environmental signals are transduced into a phenotypic response. These systems typically consist of a membrane-bound sensor kinase and soluble response regulator that is activated by a histidine-aspartate phosphorelay to bind upstream of specific genes and alter their expression (14Hoch J.A. Silhavy T.J. Two-component Signal Transduction. ASM Press, Washington, D. C.1995Crossref Google Scholar). We hypothesized that dosR and dosS may form a signaling system involved in the initial adaptation of bacilli to conditions within the host (13Sherman D.R. Voskuil M. Schnappinger D. Liao R. Harrell M.I. Schoolnik G.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7534-7539Crossref PubMed Scopus (633) Google Scholar). We showed previously that DosR binds upstream of hypoxic response genes (15Park H.D. Guinn K.M. Harrell M.I. Liao R. Voskuil M.I. Tompa M. Schoolnik G.K. Sherman D.R. Mol. Microbiol. 2003; 48: 833-843Crossref PubMed Scopus (565) Google Scholar). Further, nearly all MTB genes rapidly up-regulated in response to low doses of NO (8Voskuil M.I. Schnappinger D. Harrell M.I. Visconti K.C. Dolganov G. Sherman D.R. Schoolnik G.K. J. Exp. Med. 2003; 198: 705-713Crossref PubMed Scopus (765) Google Scholar) or by hypoxia require DosR for their induction (15Park H.D. Guinn K.M. Harrell M.I. Liao R. Voskuil M.I. Tompa M. Schoolnik G.K. Sherman D.R. Mol. Microbiol. 2003; 48: 833-843Crossref PubMed Scopus (565) Google Scholar). In this report, we demonstrate that DosS (Rv3132c) is a functional kinase of the two-component class and that it can transfer phosphate to DosR in vitro. We show that a second putative kinase, DosT (Rv2027c), also phosphorylates DosR. We demonstrate that mutants lacking both DosS and DosT can no longer activate DosR-dependent MTB gene expression in response to reduced oxygen tension. Targeted Gene Inactivation—Inactivation of the dosS gene was performed by gene replacement as described previously (13Sherman D.R. Voskuil M. Schnappinger D. Liao R. Harrell M.I. Schoolnik G.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7534-7539Crossref PubMed Scopus (633) Google Scholar). Inactivation of the dosT gene also was by gene replacement. We amplified a portion of the wild type gene by PCR using mutagenic primers that introduced three in-frame stop codons and a PacI site for screening and eliminated the putative histidine phosphorylation site (primers RPL8 to RPL11; see Table I). The 952-bp PCR product then was cloned into the vector pKO2 containing a hygromycin resistance marker and a sacB counters-election marker. The resulting plasmid then was electroporated into MTB isolates H37Rv and H37Rv:ΔdosS. Homologous recombination and gene replacement was detected stepwise using hygromycin resistance followed by sacB counterselection and loss of hygromycin resistance as described previously (13Sherman D.R. Voskuil M. Schnappinger D. Liao R. Harrell M.I. Schoolnik G.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7534-7539Crossref PubMed Scopus (633) Google Scholar). Gene replacement was confirmed by PCR using mutagenic and wild type dosT-specific primers along with primers specific for sequences flanking the site of integration (RPL12 and RPL13; see Table I) (data not shown). Digestion of the resulting amplicons with PacI also was performed to confirm the PCR screen results (data not shown).Table IPrimer sequences Primers used in the experiments are listed with sequences and a brief description (see text for details). Underlined sequences represent non-native nucleotide additions including ligase independent cloning (LIC) sequences, introduced restriction sites, and artificial ribosome binding sites. Nucleotides in bold represent base substitutions in mutagenic primers.NameSequenceDescriptionRPL85′-gaggagaagcccggtttatgcgacaacggtgctgaccatc-3′5′ dosTRPL95′-ttaattaactacctacctatgcgatccggtcgcgatc-3′5′ mutagenic dosTRPL105′-taggtaggtagttaattaaatccagcggctcttcg-3′3′ mutagenic dosTRPL115′-taataagcttcaggccgctttcggtgatgtc-3′3′ dosTRPL125′-gcggccgggattgccgttga-3′5′ dosT flankRPL135′-ctccggtcggcatgttctcga-3′3′ dosT flankDMR285′-gttgacaagcttaggaggagctatgtgacacaccc-3′5′ dosT with RBSDMR295′-cgtaacggtacccatcaatccatcagcgcagcg-3′3′ dosTDMR0055′-gacgacgacaagatgacacaccctgacagg-3′5′ LIC-dosTDMR0065′-gaggagaagcccggtcatcaatccatcagcgcagcgg-3′3′ LIC-dosTDMR0075′-gacgacgacaagatgacaacagggggcctcgtc-3′5′ LIC-dosSDMR0085′-gaggagaagcccggtttatgcgacaacggtgctgaccatc-3′3′ LIC-dosSDMR0245′-cgtgatctgaaagacaaagtcatccag-3′5′ mutagenic dosTDMR0255′-ctggatgactttgtctttcagatcacg-3′3′ mutagenic dosTDMR0265′-cgtgacctcaaagacaaagtcatccag-3′5′ mutagenic dosSDMR0275′-ctggatgactttgtctttgaggtcacg-3′3′ mutagenic dosSGW15′-ccatggtaaaggtcttcttggtcgatgacc-3′5′ dosRGW25′-ctcgagtggtccatcaccgggtgg-3′3′ dosR Open table in a new tab Luciferase Reporter Assay—H37Rv, H37Rv:ΔdosS, H37Rv:ΔdosT, and H37Rv:ΔdosS:ΔdosT isolates were transformed with the plasmid pMH108 (16Mdluli K. Sherman D.R. Hickey M.J. Kreiswirth B.N. Morris S. Stover C.K. Barry III, C.E. J. Infect. Dis. 1996; 174: 1085-1090Crossref PubMed Scopus (102) Google Scholar, 17Yuan Y. Crane D.D. Simpson R.M. Zhu Y. Hickey M.J. Sherman D.R. Barry III, C.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9578-9583Crossref PubMed Scopus (264) Google Scholar) containing the firefly luciferase reporter gene (luc) under the control of the DosR-activated acr (hspX, Rv2031c) promoter. This plasmid integrates into the L5 phage attachment site within the Gly tRNA locus of the MTB chromosome (18Lee M.H. Pascopella L. Jacobs W.R.J. Hatfull G.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3111-3115Crossref PubMed Scopus (310) Google Scholar). In three separate experiments transformants were grown in 7H9 medium in triplicate to an A600 of 0.3-0.4. 0.5 ml of each culture was removed to 13-ml tubes, which were sealed tightly with rubber septa. 0.2% O2 was infused through an 18-gauge needle for 30 s, and tubes then were incubated on a rotator for 2 h at 37 °C. Promoter activity was measured by combining a 100-μl aliquot of MTB isolate culture with 100 μl of luciferase assay reagent (Promega) in the wells of a 96-well plate, and incubation was continued for 5 min at room temperature. Luciferase activity was measured using a TD2020 luminometer. dosT Complementation—For complementation studies, a pMH108-based construct was developed in which dosT is driven by mycobacterial optimal promoter (19George K.M. Yuan Y. Sherman D.R. Barry III, C.E. J. Biol. Chem. 1995; 270: 27292-27298Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). The dosT upstream region was amplified using primers DMR28 and DMR29 (see Table I). This construct was electroporated into H37Rv, H37Rv:ΔdosS, H37Rv:ΔdosT, and H37Rv:ΔdosS: ΔdosT with the DNA integrating into the MTB chromosome at the L5 phage attachment site. Luciferase reporter activity was measured as indicated above. Cloning and Expression of dosS and dosT Genes—Full-length dosS and dosT were PCR-amplified from H37Rv genomic DNA using the primers DMR005 to DMR008 (see Table I) and were cloned into the pET32 Ek/LIC vector (Novagen). In addition, dosS and dosT mutants encoding amino acid substitutions DosS (H395K/H397K) and DosT (H392K/H394K) were generated by mutagenic fusion PCR with primers DMR024 to DMR027 (see Table I) and were cloned into pET32 Ek/LIC. Recombinant proteins were overexpressed as S-Tag fusion proteins in Escherichia coli BL21(DE3) and localized within inclusion bodies. The proteins were extracted from the inclusion bodies using the protein refolding kit from Novagen. Briefly, E. coli cells were disrupted using the BugBuster protein extraction reagent (Novagen), and inclusion bodies were recovered by centrifugation. After several washes in dilute BugBuster reagent, the inclusion bodies were solubilized in 500 mm CAPS, pH 11.0, supplemented with 0.3% N-lauroylsarcosine. After centrifugation, the supernatant containing the proteins was dialyzed at 4 °C against 20 mm Tris-HCl, pH 8.5, and 0.1 mm dithiothreitol. After several dialysis buffer changes, dithiothreitol was omitted, and final overnight dialysis was performed in the presence of oxidized and reduced glutathione (1 and 0.2 mm, respectively) to promote proper protein folding. Samples were concentrated using Centricon Plus-20 spin filters (Amicon). Cloning and Expression of DosR and DosR (D54E) Mutant—The plasmids pDosR and pDosR (D54E) were obtained by insertion of the wild type DosR and the DosR (D54E) mutant coding sequences (15Park H.D. Guinn K.M. Harrell M.I. Liao R. Voskuil M.I. Tompa M. Schoolnik G.K. Sherman D.R. Mol. Microbiol. 2003; 48: 833-843Crossref PubMed Scopus (565) Google Scholar) into pET-21d(+) vector (Novagen) via NcoI and XhoI sites. DNA was amplified with primers GW1 and GW2 (see Table I). The Rosetta (DE3) (Novagen) and the BL21-Gold(DE3) (Stratagene) E. coli strains were used as hosts for plasmids pDosR and pDosR (D54E), respectively. Cells were grown in LB media with 100 mg/liter ampicillin (with additional 34 mg/liter chloramphenicol for cells containing plasmid pDosR (D54E)) at 37 °C to A600 = 0.6 and then were induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside. Growth was continued for 3 h at 30 °C after induction. The cells were harvested by centrifugation at 5,000 rpm for 20 min at 4 °C and stored at -80 °C before use. DosR and DosR (D54E) Protein Purification—Cell pellets containing wild type DosR and DosR (D54E) mutant were resuspended in 300 mm NaCl, 10% glycerol, 1 mm 2-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride, 20 mm Tris-HCl buffer, pH 8.0, and were lysed by French press. Protamine sulfate was added, and the lysate was incubated on ice for 30 min. The lysate was centrifuged at 20,000 × g for 40 min, and the supernatant was clarified by filtration and then was applied to a nickelnitrilotriacetic acid affinity column. The nonspecifically bound proteins were eluted with 30 mm imidazole in 300 mm NaCl, 10% glycerol, 20 mm Tris-HCl buffer, pH 8.0. DosR and DosR (D54E) mutant were eluted from the column with 200 mm imidazole in the same buffer. Protein fractions were pooled and dialyzed against a buffer containing 20 mm MES buffer, pH 6.0, 50 mm NaCl, 10% glycerol, 1 mm EDTA, and 1 mm tri[2-carboxyethyl]phosphine hydrochloride. The proteins then were purified by cation exchange chromatography using a 20HS column (PerSeptive Biosystems) and were eluted with 50 mm to 1 m NaCl gradient. Peak fractions were combined and concentrated using Amicon Centriplus spin concentrators. In Vitro Phosphorylation Assay—4 μg of the recombinant S-Tag fusion protein DosT or DosT (H392K/H394K) was assayed for the ability to autophosphorylate in a reaction mixture containing 50 μCi [γ-32P]ATP (6000 Ci/mmol, PerkinElmer Life Sciences), 100 mm Tris-HCl, pH 8.0, 5 mm MgCl2, 50 mm KCl2 in a final volume of 20 μl. The reaction was incubated at room temperature for 60 min. 4-μl aliquots were removed from the autophosphorylation reaction at 0 and 60 min and were stopped with the addition of an equal volume of SDS-PAGE (Laemmli) buffer and immediate immersion in a dry ice/EtOH bath. An additional 4-μl aliquot (800 ng) was removed and added to reactions containing 4 μg of either recombinant DosR or DosR (D54E) in the same reaction buffer as above (excluding isotope) for a final volume of 20 μl. 4-μl aliquots were removed for analysis at 0, 30 s, 1 min, 5 min, and 30 min. All aliquots were analyzed by SDS-PAGE and blotted to polyvinylidene difluoride membrane. The membranes were exposed to film overnight at -80 °C. Studies with DosS and DosS (H395K/H397K) were performed in the same manner except that a 10-μl aliquot (2 μg) was carried over from the autophosphorylation reaction to the reaction containing either wild type or mutant DosR, and membranes were exposed to film for 72 h at -80 °C. Western Analysis—All blots were screened first with HRP-conjugated S-protein (Novagen), which binds the S-Tag with high affinity, at a 1:5000 dilution. ECL analysis was performed with exposure times varying from 1 to 10 s. Blots were then stripped in 200 mm glycine, pH 2.5, 0.2% Tween 20 followed by incubation in HRP color substrate to confirm loss of signal. The blots were rescreened with rabbit anti-rDosR polyclonal IgG antibody at a 1:5000 dilution followed by incubation with goat anti-rabbit IgG-HRP-conjugated secondary antibody (Pierce), and ECL analysis was performed again. Electrophoretic Mobility Shift Assay—A double-stranded oligonucleotide containing the palindromic consensus promoter sequence of hypoxic response genes (15Park H.D. Guinn K.M. Harrell M.I. Liao R. Voskuil M.I. Tompa M. Schoolnik G.K. Sherman D.R. Mol. Microbiol. 2003; 48: 833-843Crossref PubMed Scopus (565) Google Scholar) was used as a DNA probe. The binding of DosR and DosR (D54E) was carried out by incubation at room temperature for 30 min in 10 μl of reaction mixtures composed of 12.5 μm DNA, 25 μm protein, 24 mm Tris-HCl buffer, pH 7.5, 20 mm MgCl2 in the presence or absence of the phosphate donor lithium potassium acetyl phosphate (20 mm). Following incubation the entire reaction volume was electrophoresed in a 15% non-denaturing Tris borate EDTA polyacrylamide gel (Bio-Rad). The gel was stained with 1 μg/ml ethidium bromide and visualized with a 312 nm transilluminator (Fisher Scientific). Sensor Kinase Mutagenesis—Two-component response regulators activate gene transcription following phosphorylation by a sensor kinase (14Hoch J.A. Silhavy T.J. Two-component Signal Transduction. ASM Press, Washington, D. C.1995Crossref Google Scholar). When the putative phosphorylation site of the regulator DosR is mutated, it no longer activates transcription of the MTB genes normally induced under hypoxic conditions (15Park H.D. Guinn K.M. Harrell M.I. Liao R. Voskuil M.I. Tompa M. Schoolnik G.K. Sherman D.R. Mol. Microbiol. 2003; 48: 833-843Crossref PubMed Scopus (565) Google Scholar). DosS is an obvious candidate to carry out the phosphorylation and activation of DosR. The dosS gene is adjacent to dosR in the genome, is co-transcribed, and is predicted to encode a sensor kinase (13Sherman D.R. Voskuil M. Schnappinger D. Liao R. Harrell M.I. Schoolnik G.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7534-7539Crossref PubMed Scopus (633) Google Scholar, 20Dasgupta N. Kapur V. Singh K.K. Das T.K. Sachdeva S. Jyothisri K. Tyagi J.S. Tuber. Lung Dis. 2000; 80: 141-159Abstract Full Text PDF PubMed Scopus (167) Google Scholar). However, the role of the putative kinase and its interaction with DosR was unclear as targeted deletion of dosS had little effect on the ability of DosR to activate gene transcription (13Sherman D.R. Voskuil M. Schnappinger D. Liao R. Harrell M.I. Schoolnik G.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7534-7539Crossref PubMed Scopus (633) Google Scholar), and disruption of dosS had relatively little effect on survival under hypoxic conditions (21Boon C. Dick T. J. Bacteriol. 2002; 184: 6760-6767Crossref PubMed Scopus (212) Google Scholar). These results led to speculation that a second putative sensor kinase gene in the MTB genome with homology to DosS, DosT (Rv2027c), may complement the function of DosS (13Sherman D.R. Voskuil M. Schnappinger D. Liao R. Harrell M.I. Schoolnik G.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7534-7539Crossref PubMed Scopus (633) Google Scholar, 20Dasgupta N. Kapur V. Singh K.K. Das T.K. Sachdeva S. Jyothisri K. Tyagi J.S. Tuber. Lung Dis. 2000; 80: 141-159Abstract Full Text PDF PubMed Scopus (167) Google Scholar). To assess the role of the putative histidine sensor kinases DosS and DosT in the phosphorylation of DosR, we targeted these genes for deletion. The dosS gene was inactivated in isolate H37Rv by gene replacement as reported previously (13Sherman D.R. Voskuil M. Schnappinger D. Liao R. Harrell M.I. Schoolnik G.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7534-7539Crossref PubMed Scopus (633) Google Scholar). We then inactivated the dosT gene in both H37Rv and H37Rv: ΔdosS. A mutated copy of dosT was inserted that contained premature stop codons, introduced a PacI restriction site, and lacked the sequence encoding the histidine-containing putative signaling region. Confirmation of the dosT gene replacement was determined by performing PCR on transformants and amplicon digestion with PacI (data not shown). Effects of Sensor Kinase Gene Mutagenesis—One of the MTB genes powerfully induced under hypoxic conditions is acr (also called Rv2031c or hspX) encoding an α-crystallin-like heat shock protein (13Sherman D.R. Voskuil M. Schnappinger D. Liao R. Harrell M.I. Schoolnik G.K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7534-7539Crossref PubMed Scopus (633) Google Scholar, 22Yuan Y. Crane D.D. Barry III, C.E. J. Bacteriol. 1996; 178: 4484-4492Crossref PubMed Scopus (269) Google Scholar). The acr promoter contains two high affinity DosR binding sequences (15Park H.D. Guinn K.M. Harrell M.I. Liao R. Voskuil M.I. Tompa M. Schoolnik G.K. Sherman D.R. Mol. Microbiol. 2003; 48: 833-843Crossref PubMed Scopus (565) Google Scholar, 23Florczyk M.A. McCue L.A. Purkayastha A. Currenti E. Wolin M.J. McDonough K.A. Infect. Immun. 2003; 71: 5332-5343Crossref PubMed Scopus (82) Google Scholar). Mutation of these sequences abolished DosR binding and resulted in dramatically reduced levels of expression (15Park H.D. Guinn K.M. Harrell M.I. Liao R. Voskuil M.I. Tompa M. Schoolnik G.K. Sherman D.R. Mol. Microbiol. 2003; 48: 833-843Crossref PubMed Scopus (565) Google Scholar). To determine the roles of DosS and DosT, both wild type and mutant bacteria were transformed with plasmid pMH108 in which the acr promoter is fused upstream of the firefly luciferase (luc) reporter gene. Isolates were subjected to hypoxic conditions, and levels of luciferase activity were measured (Fig. 1). In the wild type strain H37Rv, luc gene expression was induced about 115-fold (Fig. 1). In both the H37Rv:ΔdosS and H37Rv:ΔdosT mutants luc induction was reduced to 40-45% of wild type values. When both dosS and dosT genes were inactivated, reporter gene expression levels dropped to base-line values, indicating that DosR no longer is able to activate transcription (Fig. 1). These results demonstrate that both DosS and DosT are required for full activation of DosR under conditions of reduced oxygen tension. dosT Complementation Analyses—To determine whether the interruption of reporter gene expression was caused by the targeted disruption of dosS and dosT, we restored a functional dosT gene to the mutants H37Rv:ΔdosS, H37Rv:ΔdosT, and H37Rv:ΔdosS:ΔdosT. The wild type dosT gene was PCR-amplified and cloned into the reporter construct pMH108 under the control of the constitutive mycobacterial optimal promoter. Each MTB isolate was transformed with the pMH108:dosT complementation constructs, incubated under hypoxic conditions, and tested for luciferase activity (Fig. 1). When dosT was reintroduced into each isolate, reporter gene expression was restored to levels comparable with or greater than those observed with wild type H37Rv, indicating that loss of hypoxic responsiveness in the mutants was caused by the lack of an appropriate sensor kinase. In Vitro Phosphorylation Assay—The first steps in two-component signal transduction are generally signal recognition by the sensor kinase followed by dimerization and autophosphorylation at a specific His residue (14Hoch J.A. Silhavy T.J. Two-component Signal Transduction. ASM Press, Washington, D. C.1995Crossref Google Scholar). Once autophosphorylated, the kinase transfers the phosphate to a second protein, the response regulator. We utilized recombinant DosS, DosT, and DosR in an in vitro phosphorylation assay to determine whether they functioned as a two-component system. In addition to testing wild type proteins, mutant DosS and DosT were generated via mutagenic fusion PCR. The mutations encoded amino acid substitutions intended to disrupt the histidine kinase phosphorylation motifs, giving rise to DosS (H395K/H397K) and DosT (H392K/H394K). Recombinant proteins were overexpressed in E. coli as S-Tag fusion proteins. The production of the recombinant S-Tag proteins and their purification from inclusion bodies was verified via SDS-PAGE Coomassie Blue staining and Western analysis with S-protein HRP conjugate (data not shown). In both instances a protein slightly larger than the 75-kDa marker was detected, corresponding to the molecular mass of both DosS and DosT (∼62 kDa) fused to the 16-kDa S-Tag. First we analyzed whether DosT had the ability to autophosphorylate and then transfer the phosphate to DosR. DosT was incubated at room temperature with 50 μCi [γ-32P]ATP, and aliquots of the reaction were removed for analysis at 0 and 60 min. An additional aliquot from the autophosphorylation reaction was removed at 60 min and put into a relay reaction containing purified DosR. Aliquots of the relay reaction were removed at 0, 0.5, 1, 5, and 30 min. The aliquots from both reactions were fractionated by SDS-PAGE, blotted, and visualized by autoradiography (Fig. 2). The DosT (H392K/H394K) mutant also was analyzed for its ability to autophosphorylate in the same manner. The autoradiograph of the blot corresponding to the DosT autophosphorylation/phosphorelay reaction is shown in Fig. 2A. By 60 min a radiolabeled protein was detected consistent with the predicted ∼78-kDa DosT fusion protein size. Upon transfer to the relay reaction, this same protein was observed at time 0, but the radiolabel in this band waned with time until it was barely detectable at 30 min. In contrast, a radiolabeled protein consistent with the 26-kDa size of DosR was barely detectable at time 0 of the phosphorelay reaction yet became more intense as the 30-min mark approached, indicating that phosphotransfer is occurring between the two proteins. To confirm the identity of the differentially labeled proteins, immunoblot analyses were performed with an S-protein HRP conjugate to detect DosT (Fig. 2B) and after stripping with an anti-DosR antibody (Fig. 2C). Reactivity with the anti-DosR antibody was observed only in the phosphorelay lanes with the radiolabeled 26-kDa protein confirming its identity as DosR, whereas reactivity with the S-protein HRP conjugate was observed with the radiolabeled protein at ∼78 kDa confirming it as DosT. The additional bands in Fig. 2 also reacted with the S-protein HRP conjugate indicating that they are truncated forms of DosT that no longer have the ability to transfer phosphate (data not shown). When the DosT (H392K/H394K) mutant replaced the wild type DosT in the assay, there was no detectable autophosphorylation after 60 min and thus no subsequent phosphate transfer to DosR (data not shown). In addition, no labeling was observed in control phosphotransfer reactions from which DosT was omitted (data not shown), demonstrating that labeling of DosR was caused by phosphotransfer from DosT as opposed to DosR autophosphorylation. To determine whether the phosphorelay proceeded in a m
Chromosome segregation is an essential process of cell multiplication. In prokaryotes, segregation starts with the newly replicated sister origins of replication, oriCs, which move apart to defined positions in the cell. We have developed a genetic screen to identify mutants defective in placement of oriC during spore development in the Gram-positive bacterium Bacillus subtilis. In addition to the previously identified proteins Soj and DivIVA, our screen identified several new factors involved in polar recruitment of oriC: a reported regulator of competence ComN, and the regulators of division site selection MinD and MinJ. Previous work implicated Soj as an important regulator of oriC positioning in the cell. Our results suggest a model in which the DivIVA-interacting proteins ComN and MinJ recruit MinD to the cell pole, and that these proteins work upstream of Soj to enable oriC placement. We show that these proteins form a polar complex, which acts in parallel with but distinct from the sporulation-specific RacA pathway of oriC placement, and also functions during vegetative growth. Our study further shows that MinD has two distinct cell cycle roles, in cell division and chromosome segregation, and highlights that cell probably use multiple parallel mechanisms to ensure accurate chromosome segregation.
Abstract Nucleotide binding domain, leucine rich repeat CARD containing protein 3 (NLRC3) is a member of the NLR gene family. Members of this family have been associated with human inflammatory diseases such as Crohn’s disease and cryopyrin-associated periodic syndrome. Although NLRC3 is not associated with human diseases, it is expressed preferentially in the immune system and functions in pathogen detection. NLRC3 is an intracellular protein involved in the sensing of lipopolysaccharide and cytosolic nucleic acids. NLRC3 is hypothesized to act as a negative regulator in response to bacterial and viral infection, suggesting that the vertebrate immune system has evolved specific inhibitors to limit the inflammatory response. We performed an unbiased yeast two-hybrid screen using an amino terminal fragment of NLRC3 to identify putative interacting proteins that might help elucidate the mechanism by which NLRC3 might inhibit inflammatory responses. To this end, we identified several interacting proteins. One protein, in particular, IQGAP1, acts as a scaffold important in regulating the cytoskeleton, cell adhesion and proliferation. Structure function analysis has localized the domains necessary and sufficient for interacting with NLRC3. Confocal microscopy demonstrates that these two proteins co-localize in transformed human epithelial cells. Functionally, in the absence of IQGAP1, human monocytic cells are hyperactive in response to cytosolic nucleotides, phenocopying NLRC3 deficiencies. These data suggest that NLRC3 can interact with novel proteins to facilitate squelching of cellular responses to cytosolic nucleotides.
Next‐generation sequencing accelerates disease gene discovery, especially for orphan diseases, though at present it outpaces functional studies needed to provide ‘proof of causation.’ Working from a small, community‐based, clinical laboratory, the Clinic for Special Children has identified more than 170 allelic variants associated with disability, disease, or untimely death among the Amish and Mennonite (Plain) populations of North America. In collaboration with the Clinic, we developed an HHMI‐funded program that integrates functional studies of novel disease gene alleles into our undergraduate curriculum. Roughly 150 students per year in our introductory cell biology and neuroscience courses clone human disease genes and study the functional impacts of gene variants through expression in mammalian cell culture. We use these authentic research experiences to teach key concepts in cell biology, genetics, and neuroscience. Students later build upon this experience in upper‐level courses in cell biology, neuroscience, genetics, cancer biology, and immunology in which they engage in semester‐long research projects in small teams. Teams conduct functional studies of disease gene variants or study novel cellular or animal models of disease. These experiences engender talented undergraduates to assume greater research responsibility through independent study and summer research projects that transition them into the role of co‐PI for their project. Data published through this project ( e.g., PLoS ONE 7:e28936) have been used by other institutions to establish novel diagnoses, including a high‐profile diagnosis of a critically‐ill, non‐Plain newborn with lethal neonatal seizure‐rigidity syndrome ( Sci Transl Med 4:154ra135). This provides important proof‐of‐concept for integrating novel disease gene functional studies into a carefully structured undergraduate research curriculum. Students in our Public Health program are collaborating with the Clinic to develop handbooks to help parents care for children with special medical needs with the goal of producing one to two high‐quality disease handbooks annually. Our program represents a model for engaging undergraduates in meaningful research at the front lines of biomedical science and public health in ways that directly impact the diagnosis and care of children with rare inherited disorders and promote STEM retention. Support or Funding Information HHMI Undergraduate Science Education Awards 52006294 and 52007538.
There is a pressing need to develop novel anti-tubercular drugs. High-throughput phenotypic screening yields chemical series that inhibit bacterial growth. Target identification for such series is challenging, but necessary for optimization of target engagement and the development of series into clinical drugs. We constructed a library of recombinant Mycobacterium tuberculosis strains each expressing a single protein from an inducible promoter as a tool for target identification. The library of 1733 clones was arrayed in 96-well plates for rapid screening and monitoring growth. The library contains the majority of the annotated essential genes as well as genes involved in cell wall and fatty acid biosynthesis, virulence factors, regulatory proteins, efflux, and respiration pathways. We evaluated the growth kinetics and plasmid stability over three passages for each clone in the library. We determined expression levels (mRNA and/or protein) in 396 selected clones. We screened the entire library and identified the Alr-expressing clone as the only recombinant strain, which grew in the presence of d-cycloserine (DCS). We confirmed that the Alr-expressing clone was resistant to DCS (7-fold shift in minimum inhibitory concentration). The library represents a new tool that can be used to screen for compound resistance and other phenotypes.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)
