15-kDa Polypeptide Research Articles

Reconstitution of ATPase Activity from Individual Subunits of the Clathrin-coated Vesicle Proton Pump THE REQUIREMENT AND EFFECT OF THREE SMALL SUBUNITS

The vacuolar-type proton pump of clathrin-coated vesicles is composed of two general domains, a peripheral, catalytic sector (VC) and a transmembranous proton channel (VB). In its native form, the enzyme can hydrolyze both MgATP and CaATP, whereas VC, when separated from VB, loses its MgATPase activity and switches to a state that can hydrolyze only CaATP. Further dissociation of VC results in subcomplexes that are depleted of one or more subunits and lack ATPase activity altogether. Reconstitution of recombinant subunits to these biochemically prepared subcomplexes has demonstrated the necessity of polypeptides of 70, 58, 40, and 33 kDa (subunits A, B, C, and E, respectively) for CaATPase activity of the VC complex. The current studies demonstrate that mixtures of these four recombinant subunits cannot support CaATPase activity in the absence of a biochemically prepared subcomplex. Investigation of the other components required for ATPase activity has led to the identification of three additional polypeptides present in preparations of VC, with apparent molecular masses of 15, 14, and 10 kDa. Each of these proteins was found to activate ATPase activity of mixtures of subunits A, B, C, and E. In addition, ATPase reconstituted from these individual subunits hydrolyses ATP, not only in the presence of Ca2+ but also in the presence of Mg2+. Investigation of the individual properties of these three subunits revealed that the 10-kDa polypeptide is subunit F, as determined by immunoblot analysis. This subunit had no effect on MgATPase activity of VC but stimulated CaATPase activity 6-fold in the presence of subunit D. Under optimal conditions the 14-kDa component resulted in a 10-fold stimulation and the 15-kDa component a 20-fold stimulation of MgATPase activity; based on this observation, the 14- and 15-kDa polypeptides were named subunits G and H, respectively. In addition, proton pumping activity was reconstituted through the reassembly of subunits A-H with VB and SFD, a previously described pump component composed of polypeptides of 50 and 57 kDa (Xie, X.-S, Crider, B.P., Ma, Y. M., and Stone, D. K. (1994) J. Biol. Chem. 269, 25809-25815). Together, these experiments completely define the catalytic center of the vacuolar proton pump of clathrin-coated vesicles.

Isolation, characterisation and application of a new antifungal protein from broccoli seeds – New food preservative with great potential

The worldwide problem of food loss and recent outbreaks of food-related diseases caused by microbial contamination has highlighted the urgent need of efficient preservation methods. Plants of Brassicaceae familyare particularly rich in compounds with antiviral, antimicrobial and anticancer properties. Herein, a water-soluble extract of broccoli (Brassica oleracea var. italica) seeds showed strong antifungal activity against fungal food contaminants. A 15-kDa polypeptide was selectively extracted with ammonium sulphate precipitation, ion-exchange and size exclusion chromatography Circular dichroism spectroscopy and analysis of broccoli seed transcriptome revealed high similarity to the previously isolated rapessed storage protein napins. This paper is the first report of the isolation and characterization of napin-like proteins from broccoli seedsIn addition, apH-dependent approach was used to increase the yield of extraction with a minimal use of chemicals. The potential of the broccoli napin, termed BoNap, as a novel antifungal agent was shown in in vitro assays against the spores of several fungal contaminants. BoNap prevented the germination of spores of Fusarium culmorum and Penicillium expansum, with a MIC value of 2.31 μM. In addition, the napin-like protein presented an arsenal of mechanisms for the protection of the seeds, including permeabilization of mycelium membrane and trypsin-inhibitory properties. Essential characteristics of heat stability and non-cytotoxicity towards mammalian cells highlight the promises of this compound in the area of food safety and crop protection. As shown here with a successful reduction of fungal contamination of cereals, the discovery of a readily extractable plant protein could contribute to its application as a natural stable preservative and to limit the global issue of food waste.

Identification and Characterization of an Inner Ear-Expressed Human Melanoma Inhibitory Activity (MIA)-like Gene (MIAL) with a Frequent Polymorphism That Abolishes Translation

To discover new cochlea-specific genes as candidate genes for nonsyndromic hearing impairment, we searched in The Institute of Genome Research database for expressed sequence tags isolated from the cochlea only. This led to the cloning and characterization of a human gene named melanoma inhibitory activity-like (MIAL; HGMW-approved symbol OTOR alias MIAL) gene. In situ hybridization revealed MIAL expression in a cell layer beneath the sensory epithelium of cochlea and vestibule of human fetal inner ear. No other human tissue, except fetal brain, showed expression of MIAL when analyzed by in situ hybridization or reverse transcription-polymerase chain reaction. The cDNA of the mouse homologue was also cloned and mapped about 80 cM from the top of mouse chromosome 2. In mouse, Mial was also expressed in the cochlea and the vestibule of the inner ear, as well as in brain, eye, limb, and ovary. Expression in mammalian cell cultures showed that MIAL is translated as an ∼15-kDa polypeptide that is assembled into a covalently linked homodimer, modified by sulfation, and secreted from the cells via the Golgi apparatus. In the human MIAL gene, a frequent polymorphism was discovered in the translation initiation codon (ACG instead of ATG). Of 505 individuals, 48 (9.5%) were ATG/ACG heterozygous and 1 (0.2%) was homozygous for ACG. No MIAL protein was synthesized in cells transfected with cDNA of the ACG allele. The inner ear-restricted expression pattern and the existence of an inactive allele suggest that MIAL may contribute to inner-ear dysfunction in humans.

A novel competence gene, comP, is essential for natural transformation of Acinetobacter sp. strain BD413.

Acinetobacter sp. strain BD413 (= ATCC 33305), a nutritionally versatile bacterium, has an extremely efficient natural transformation system. Here we describe the generation of eight transformation-affected mutants of Acinetobacter sp. strain BD413 by insertional mutagenesis. These mutants were found by Southern blot analysis and complementation studies to result from single nptII marker insertions at different chromosomal loci. DNA binding and uptake studies with one mutant, T205, revealed that the transformation deficiency of this mutant results from a complete lack of DNA binding and, therefore, uptake activity. A novel competence gene essential for natural transformation, named comP, was cloned by complementation of mutant T205. The nucleotide sequence of comP was determined, and its deduced 15-kDa polypeptide displays significant similarities to type IV pilins. Analysis of the ultrastructure of a transformation-deficient comP mutant and the transformation-competent wild-type strain revealed that both are covered with bundle-forming thin fimbriae (3 to 4 nm in diameter) and individual thick fimbriae (6 nm in diameter). These results provide evidence that the pilinlike ComP is unrelated to the piluslike structures of strain BD413. Taking all data into account, we propose that ComP functions as a major subunit of an organelle acting as a channel or pore mediating DNA binding and/or uptake in Acinetobacter sp. strain BD413.

GSP-1 genes are linked to the grain hardness locus (Ha) on wheat chromosome 5D.

An important determinant of wheat grain quality is the hardness of the grain. The trait is controlled by a major locus, Ha, on the short arm of chromosome 5D. Purified starch granules from soft-grained wheats have associated with them 15-kDa polypeptides called grain softness proteins (GSPs) or "friabilins." Genes that encode one family of closely related GSP polypeptides - GSP-1 genes - were mapped using chromosome substitution lines to the group 5 chromosomes. An F2 population segregating for hard and soft alleles at the Ha locus on a near-isogenic background was used in a single-seed study of the inheritance of grain softness and of GSP-1 alleles. Grain softness versus grain hardness was inherited in a 3:1 ratio. The presence versus absence of GSPs in single seed starch preparations was coinherited with grain softness versus hardness. This showed that grain softness is primarily determined by seed, and not by maternal, genotype. In addition, no recombination was detected in 44 F2 plants between GSP-1 restriction fragment length polymorphisms and Ha alleles. Differences between hard and soft wheat grains in membrane structure and lipid extractability have been described and, of the three characterized proteins that are part of the mixture of 15-kDa polypeptides called GSPs, at least two, and probably all three, are proteins that bind polar lipids. The data are interpreted to suggest that the Ha locus may encode one or more members of a large family of lipid-binding proteins.

A Temperature-Sensitive Mutation of the Vaccinia Virus E11 Gene Encoding a 15-kDa Virion Component

Thets49 mutation of vaccinia virus WR was mapped by marker rescue to the E11 gene encoding a 15-kDa polypeptide. During synchronous infection of BSC40 cells with wild-type virus, immunoreactive E11 protein accumulated in parallel with the onset of late protein synthesis. Immunoblotting of extracts of wild-type virions showed that the E11 protein was encapsidated within the virus core. A normal temporal pattern of viral protein synthesis was observed in cells infected withts49 at the nonpermissive temperature (40°) and normal-appearingts49 progeny virions were observed by electron microscopy. Sequencing of the E11 gene ofts49 revealed a single amino acid substitution, Gly(66)Arg, in the mutant E11 polypeptide. The steady-state level of E11 protein duringts49 infection was much lower than that observed during infection with wild-type virus. This was the case at both the permissive and nonpermissive temperatures. We discuss two possible explanations for the thermosensitive growth ofts49: (i) that virus infectivity requires a threshold level of active E11 protein or (ii) that E11 function is conditionally essential.

An isolated reaction center complex from the green sulfur bacterium Chlorobium vibrioforme can photoreduce ferredoxin at high rates

Chlorosome-depleted membranes and a reaction center complex with well-defined subunit composition were prepared from the green sulfur bacterium Chlorobium vibrioforme under anaerobic conditions. The reaction center complex contains a 15-kDa polypeptide with the N-terminal amino acid sequence MEPQLSRPETASNQVR/. This sequence is nearly identical to the N-terminus of the pscD gene product from Chlorobium limicola (Hager-Braun et al. (1995) Biochemistry 34: 9617-9624). In the presence of ferredoxin and ferredoxin:NADP(+) oxidoreductase, the membranes and the isolated reaction center complex photoreduced NADP(+) at rates of 333 and 110 μmol (mg bacteriochlorophyll a)(-1) h(-1), respectively. This shows that the isolated reaction center complex contains all the components essential for steady state electron transport. Midpoint potentials at pH 7.0 of 160 mV for cytochrome c 551 and of 245 mV for P840 were determined by redox titration. Antibodies against cytochrome c 551 inhibit NADP(+) reduction while antibodies against the bacteriochlorophyll a-binding Fenna-Matthews-Olson protein do not.

Characterization of a New Virus from Garlic(Allium sativum L.), Garlic Mite-borne Mosaic Virus.

Flexuous, filamentous virus particles with a length of 700-800nm were detected in garlic (Allium sativum L.) plants showing mosaic symptoms. The virus was sap-transmissible from garlic plants to seven out of 25 plant species tested, inducing local lesions on Chenopodium murale and Gomphrena globosa and a systemic mosaic on garlic. This virus was transmitted by the eriophyid mite, Aceria tulipae Keifer, but not by aphids. Two polypeptides with M.W. 30kDa and 28.5kDa were detected by SDS-PAGE of the purified virus preparation. The virus exhibited no serological relationships with leek yellow stripe, onion yellow dwarf or garlic latent viruses. No cytoplasmic cylindrical inclusions were observed in infected leaf cells of garlic or C. murale. The 3'-terminal nucleotide sequence of 2518bp was determined. It contained three putative open reading frames (ORFs) for 40kDa, 28kDa (a putative coat protein, CP) and 15kDa polypeptides. The arrangement and amino acid sequences of these ORFs show close similarities with those of shallot virus X and garlic virus (GV)-A, -B, -C and -D. The putative CP of the mite-borne virus has 98% amino acid sequence homology with CP of GV-C, but only 60-67% identity with CPs of four other viruses, indicating the classification of the mite-borne virus and GV-C as the same virus. We propose the name garlic mite-borne mosaic virus (GMbMV) for the virus.

A Heme-binding Protein from Hemolymph and Oocytes of the Blood-sucking Insect, Rhodnius prolixus: ISOLATION AND CHARACTERIZATION

A heme-binding protein has been isolated and characterized from both the hemolymph and oocytes of the blood-sucking insect, Rhodnius prolixus. The protein from both sources is identical in most aspects studied. The Rhodnius heme-binding protein (RHBP) is composed of a single 15-kDa polypeptide chain coiled in a highly alpha-helical structure which binds non-covalently one heme/polypeptide chain. This RHBP is not produced by limited degradation of hemoglobin from the vertebrate host, since specific polyclonal antibodies against it do not cross-react with rabbit hemoglobin, and since it differs from hemoglobin in having a distinct amino-acid composition and NH2-terminal sequence. The spectrum of the dithionite-reduced protein has peaks at 426, 530, and 559 nm and resembles that of a b-type cytochrome. RHBP from hemolymph is not saturated with heme and promptly binds heme added to the solution. The oocyte protein, on the other hand, is fully saturated and is not capable of binding additional heme.

Cloning of a wheat 15‐kDa grain softness protein (GSP)

The wheat starch 15-kDa protein (called grain softness protein or GSP) consists of a major polypeptide and several minor polypeptides. An antiserum raised against GSP was used to screen a wheat cDNA library. A cDNA family encoding approximately 15-kDa proteins that included a heptapeptide sequence previously isolated from protease digests of GSP was identified. A partial cDNA was used in a prokaryotic expression system to produce a fusion protein which reacted strongly against the original anti-GSP serum. A new antiserum raised against the fusion protein produced a weak reaction against a 15-kDa polypeptide extracted from wheat seeds. The results suggest that the proteins encoded by the cDNA family form a minor component of the mixture of 15-kDa polypeptides defined as GSP. RNA complementary to the cDNAs could be extracted from both soft and hard wheat grains from about half-way through grain filling. The encoded proteins are novel members of the 2S superfamily of seed proteins, a diverse family of proteins which maintain a characteristic framework of cysteine residues. The deduced proteins show the highest similarity to the oat 16-kDa avenin and to wheat puroindoline (a lipid-binding 15-kDa protein from wheat). Review of previously published data shows that puroindoline is also closely related to the major polypeptide of GSP, suggesting that the lipid-binding properties of GSP polypeptides may influence grain softness.

Influence of intra-amoebic and other growth conditions on the surface properties of Legionella pneumophila.

The surface properties of Legionella pneumophila were examined by analyzing outer membrane (OM) proteins, lipopolysaccharides (LPS), and cellular fatty acids after growth within Acanthamoeba polyphaga and in vitro under various nutrient-depleted conditions. Intra-amoeba-grown legionellae were found to differ in several respects from cells grown in vitro; most notably, they contained a 15-kDa OM protein and a monounsaturated straight-chain fatty acid (18:1(9)). These compounds were also found in abundant quantities in the host amoeba. Immunoblot analysis of intra-amoeba-grown legionellae with antiacanthamoebic serum revealed that both the bacterial whole cells and Sarkosyl-extracted OMs contained amoebic antigens. The findings suggest that the 15-kDa OM protein is likely to be of amoebic origin and associates with the OM of the bacterium. It is proposed that disruption of amoebic membranes, as a result of intra-amoebic infection, may liberate macromolecules, including a 15-kDa polypeptide, a major constituent of the amoebic membrane, which adhere to the surface of the legionellae. Growth under specific nutrient depletions also had a significant effect on the surface composition of L. pneumophila. Cells grown under phosphate depletion were markedly sensitive to protease K digestion and contained lower levels of LPS, as observed by silver staining of the digests on polyacrylamide gels. Intra-amoeba-grown cells contained more bands than the in vitro-grown organisms, reflecting further differences in the nature of the LPS. The whole-cell fatty acids of the phosphate-depleted cells were appreciably different from those of cells grown under other nutritional conditions. We found no evidence for expression of iron-regulated OM proteins under iron depletion.

Pathogenic Entamoeba histolytica: cDNA cloning of a histone H3 with a divergent primary structure

Entamoeba histolytica has an unusual nuclear structure characterized by a low degree of chromatin condensation and the absence of stainable metaphase chromosomes. Although nucleosome-like particles were observed, no information about histones was available so far. In this paper we describe a cDNA clone with significant homology to H3 histones that was isolated from a library of pathogenic E. histolytica. The complete cDNA encodes a 15-kDa polypeptide, which like the histone sequence from Volvox carteri is shorter by one residue than the human homologue. The amino acid sequence has only 69% identity with human H3.3 histone and 67% identity with the human H3.1 histone. This is the highest degree of sequence divergence observed for any eukaryote H3 histone sequence. Our results indicate that this divergence may contribute to the unusual chromatin structure of E. histolytica.

Analyses of genes that encode the 15-kDa zein protein of maize: identification of potential gene regulatory elements

A gene ( gZ15.4.1) encoding the 15-kDa zein polypeptide was isolated from maize cultivar A5707, and its nucleotide sequence was determined. A total of 2085 bp was sequenced, including about 300 bp of 5′-flanking DNA that includes several potential regulatory elements not available in the previously published 15-kDa zein-encoding gene ( gZ15A) sequence. Several nt differences between gZ15.4.1 and gZ15A were observed, which include two single amino acid replacements.

Evidence for an Association of the 15‐kDa Proteolipid of Mediatophore with a 14‐kDa Polypeptide

The present report shows that mediatophore, a nerve terminal membrane protein that translocates acetylcholine on calcium action, forms a complex with a 14-kDa polypeptide. The complex was identified based on the following results. (a) A polyclonal antimediatophore antiserum that immunoprecipitates activity precipitates both the 15- and 14-kDa polypeptides. (b) After HPLC purification of mediatophore, both antigens were found in the same peak. (c) After 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate solubilization of presynaptic membranes or of the purified mediatophore, an immunoaffinity column made with the anti-14-kDa antigen monoclonal antibody retained both the 14-kDa and the 15-kDa polypeptide. Similarly, immunoprecipitation experiments using protein A-coated beads sedimented an immunocomplex in which both antigens were found. (d) The 14-kDa antigen could be localized in the synaptosomal membrane where mediatophore and its 15-kDa component are found.

Identification of the glucocorticoid suppressible mitogen from rat hepatoma cells as an angiogenic platelet-derived growth factor A-chain homodimer.

We have previously shown that BDS.1 rat hepatoma cells are hypersensitive to the antiproliferative effects of glucocorticoids, and secrete a glucocorticoid suppressible mitogenic activity (denoted GSM). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that GSM purified to near homogeneity migrated as a 28-kDa protein under nonreducing conditions and as a single 15-kDa polypeptide in the presence of sulfhydryl reducing agents suggesting a homodimeric structure. Anti-platelet-derived growth factor (PDGF) A-chain specific antibodies selectively immunodepleted the mitogenic activity which can be extracted from nonreducing gels in the 26-30-kDa fraction and, in Western blots, recognized the 15-kDa reduced form of GSM. Western blot analysis further showed that dexamethasone suppressed the level of secreted PDGF A-chain protein in BDS.1 cells but not in glucocorticoid receptor-deficient hepatoma cells. Northern blots revealed that dexamethasone reduced expression of the PDGF A-chain 2.3- and 1.7-kilobase transcripts in proportion to the level of detectable PDGF-AA protein. Similarly to PDGF-AA, the hepatoma cell-derived GSM has a potent angiogenic activity. Taken together, our results demonstrate that the predominant glucocorticoid suppressible mitogen secreted from rat hepatoma cells is a PDGF A-chain homodimer and suggest that in vivo glucocorticoids may potentially regulate hepatoma growth by modulating PDGF-stimulated tumor vascularization.

Sequence analysis, expression, and deletion of a vaccinia virus gene encoding a homolog of profilin, a eukaryotic actin-binding protein

A 4,500-bp BamHI fragment, located within the HindIII A segment of the vaccinia virus genome, was found to contain eight potential coding regions for polypeptides of 78 to 346 amino acids. The open reading frames with 133, 346, and 125 codons were homologous to profilin (an actin-binding protein), 3-beta-hydroxysteroid dehydrogenase, and Cu-Zn superoxide dismutase, respectively. Sequence alignments indicated that the vaccinia virus and mammalian profilins were more closely related to each other than to known profilins of other eukaryotes. The expression and possible role of the profilin homolog in the virus replicative cycle were therefore investigated. Antibody raised to Escherichia coli expressed vaccinia virus profilin was used to demonstrate the synthesis of the 15-kDa polypeptide at late times after vaccinia virus infection of mammalian cells. The protein accumulated in the cytoplasm, but only trace amounts remained associated with highly purified virions. The isolation of vaccinia virus mutants (in strains WR and IHD-J), with nearly the entire profilin gene replaced by the E. coli gpt gene, indicated that the protein is not essential for infectivity. The characteristic vaccinia virus-induced changes in actin fibers, seen by fluorescence microscopy, occurred in cells infected with the mutant. Moreover, the virus-encoded profilin homolog was not required for actin-associated events, including intracellular virus movement to the periphery of the cell, formation of specialized microvilli, or release of mature virions, as shown by electron microscopy and yields of infectious intra- and extracellular virus.

A 25-kDa stretch of the extracellular domain of the human interferon gamma receptor is required for full ligand binding capacity

We investigated which is the shortest fragment of the interferon gamma receptor with ligand binding capacity. A recombinant soluble interferon gamma receptor produced in Escherichia coli was subjected to controlled digestion with several proteolytic enzymes. The fragments generated were assayed by four approaches for interferon gamma binding. A 25-kDa polypeptide comprising residues 6-227 of the mature protein was produced by sequential digestion with trypsin and proteinase K. It was identified as the shortest receptor domain with full interferon gamma binding capacity as judged by ligand blots. The proteolytic fragments were further tested for ligand binding by interferon gamma affinity chromatography. A 15-kDa polypeptide comprising amino acids 94-227 produced by digestion with endoproteinase Glu-C was found to bind with low affinity to immobilized interferon gamma. This fragment, which does not show ligand binding capacity on protein blots, was immunoprecipitated as a complex with interferon gamma by anti-interferon gamma antibodies. It also competed for the binding of radiolabeled interferon gamma to the cell surface receptor when it was assayed as a mixture of the proteolytic products, but not after separation from the cleaved rest of the molecule. The 15-kDa polypeptide probably carries the ligand-binding domain or part of it, but it lacks sequences essential for full interferon gamma binding capacity. The stretch between amino acids 6 and 21 which does not include any disulfide bonds seems to be essential for the receptor to show full activity. The digestion with endoproteinase Glu-C revealed that cysteine residues 60 and 68 of the interferon gamma receptor form a disulfide bond.

Expression of type VIII collagen during morphogenesis of the chicken and mouse heart

The expression of type VIII collagen is restricted, in adult mammals, to specialized extracellular matrices and to a select subset of blood vessels. We have examined the distribution of type VIII collagen in sequential stages of mouse and chicken embryos and found a temporal and spatially restricted pattern of expression during cardiogenesis. Type VIII collagen was first detected by immunocytochemistry on Day 11 in the developing mouse embryo and at stage 19 in the chicken embryo. The distribution of this protein was rapidly modulated during cardiac morphogenesis. Initially (Day 11 in the mouse embryo), type VIII collagen was associated with cardiac myoblasts. From Days 15 to 18, the immunoreactive component was progressively diminished in the myocardium; however, this collagen was observed in the subendocardial layer of the atrioventricular canal and later in the cardiac jelly (or the myocardial basement membrane, an area associated with the formation of cardiac valves). On Day 17, type VIII collagen was also detected in the subendothelium (intima) and tunica media of large vessels. Neonatal and adult hearts contained low to undetectable levels of type VIII collagen. The presence of type VIII collagen was confirmed by immunoblot analysis of heart extracts at different stages of development. A major 185-kDa component, as well as polypeptides of 68 and 15 kDa, reacted with anti-type VIII collagen IgG. Exposure of heart extracts to hyaluronidase or reducing agent eliminated immunoreactivity of the 185-kDa component but not that of the 68- and 15-kDa polypeptides. Type VIII collagen therefore appears to be associated with a hyaluronidase-sensitive component of the extracellular matrix during a temporally restricted stage of embryonic cardiogenesis. The contribution of this collagen to cardiac morphogenesis might reside, in part, in its ability to influence the differentiation of the myocardium and formation of the cardiac valves.

Yersinia pestis pH 6 antigen: genetic, biochemical, and virulence characterization of a protein involved in the pathogenesis of bubonic plague

We studied a protein antigen, designated pH 6 Ag, that has the same regulation of expression as the previously described Yersinia pestis pH 6 Ag. Monospecific antiserum to this antigen recognized several proteins, ranging from 15 to over 75 kilodaltons (kDa), which were strongly expressed when Y. pestis was cultivated at 37 degrees C and pH 6 but were expressed weakly, if at all, at 37 degrees C and pH 8 and at 26 degrees C. The antigen appeared to be composed of aggregates of a 15-kDa subunit. Escherichia coli minicell analysis and Western blotting (immunoblotting) of minicell extracts containing the cloned pH 6 Ag locus revealed that a 1.7-kilobase-pair (kb) region of Y. pestis chromosomal DNA produced 16- and 15-kDa immunoreactive proteins. We used transposon mutagenesis of the pH 6 Ag-coding region to demonstrate that the 16- and 15-kDa polypeptides were produced by the same cistron. The pH 6 Ag structural gene, psaA, was located within a 0.5-kb region of DNA. A Tn10lacZ transposon insertion 1.2 kb upstream of the psaA locus but outside the psaA transcriptional unit caused decreased expression of pH 6 Ag in both E. coli and Y. pestis and defined the psaE locus necessary for maximum pH 6 Ag expression. This locus itself was not regulated by temperature or pH. However, psaA remained responsive to both of these environmental signals in a Y. pestis psaE mutant. Mutation of either psaE or psaA resulted in at least a 100-fold reduction in the intravenous 50% lethal dose of Y. pestis in mice. Accordingly, pH 6 Ag is involved in the pathogenesis of bubonic plague.

Molecular cloning and characterization of the 15-kilodalton major immunogen of Treponema pallidum.

Pathogen-specific membrane immunogens of Treponema pallidum subsp. pallidum (T. pallidum) have been identified previously by phase partitioning with the nonionic detergent Triton X-114. One of these antigens, a 15-kilodalton (kDa) polypeptide, is expressed in relatively small quantities in T. pallidum but is highly immunogenic in both human and experimental syphilis. The native T. pallidum antigen was purified to homogeneity from the mixture of Triton X-114 detergent-phase proteins by chromatofocusing. Recombinant Escherichia coli clones were selected from a T. pallidum genomic DNA library by using monoclonal antibodies specific to the 15-kDa antigen; immunoblotting and minicell analyses confirmed expression of the 15-kDa protein in the transformants. Southern hybridization with a 1.1-kilobase fragment of DNA encoding the 15-kDa-antigen gene indicated that the gene is probably present in a single copy within the genomes of both T. pallidum and T. pallidum subsp. pertenue (the agent of yaws), while it is absent from the genome of the nonpathogenic Treponema phagedenis biotype Reiter. Cell fractionation studies with Triton X-114 demonstrated that the recombinant polypeptide possesses hydrophobic properties similar to those of the native antigen and localized the cloned 15-kDa antigen to the inner membrane of E. coli. Protein processing experiments in minicells revealed that a precursor appears to be processed to the mature 15-kDa polypeptide.