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PubMed2000

Characterization of a zinc-finger protein and its association with apoptosis in prostate cancer cells.

BACKGROUND: The transition from androgen-dependent to androgen-independent prostate cancer is not fully understood but appears to involve multiple genetic changes. We have identified a gene, GC79, that is more highly expressed in androgen-dependent LNCaP-FGC human prostate cancer cells than in androgen-independent LNCaP-LNO human prostate cancer cells. Physiologic levels (0.1 nM:) of androgens repress expression of GC79 messenger RNA (mRNA) in LNCaP-FGC cells. To determine the role of GC79, we cloned its complementary DNA (cDNA) and functionally characterized its product. METHODS: The differentially expressed GC79 gene was cloned from human prostate cDNA libraries, sequenced, and transfected into mammalian cells to study its function. Expression of GC79 was analyzed in various adult and fetal human tissues and in prostate glands of castrated rats. The association of GC79 expression and apoptosis was investigated in COS-1 and LNCaP cells transfected with GC79 cDNA. All statistical tests are two-sided. RESULTS: Sequence analysis indicates that GC79 encodes a large, complex, multitype zinc-finger protein, containing nine C(2)H(2)-type zinc-finger domains, a cysteine-rich region, and a GATA C(4)-type zinc-finger domain. Castration-induced androgen withdrawal increased the expression of GC79 mRNA in the regressing rat ventral prostate, suggesting that the expression of GC79 mRNA is associated with the process of apoptotic cell death in the rat ventral prostate. Transfection and induction of GC79 cDNA in both COS-1 and LNCaP prostate cancer cells led to an apoptotic index that was eightfold higher (P:<.001, two-sided Student's t test) than that observed in uninduced transfected cells. CONCLUSIONS: We have cloned an androgen-repressible gene, GC79, that is potentially involved in apoptosis. This finding may have implications for the development of androgen-independent prostate cancer and, ultimately, for the treatment of prostate cancer.

Amino Acid Sequence↗

PubMed2025

Discovery of Respiratory Pathogens in People Living With HIV in the African Cohort Study.

INTRODUCTION: Respiratory infection outbreaks pose a threat to the readiness of the United States and allied armed forces. Predicting and preventing such outbreaks requires understanding of the epidemiology of potential respiratory pathogens in communities where service members live and work. We conducted pan-viral surveillance of respiratory specimens from people living with HIV or without HIV but under the risk enrolled in the U.S. Military HIV Research Program's African Cohort Study to determine the prevalence and possible clinical presentation of viruses in this population. METHODS: The African Cohort Study is an open-ended prospective cohort study that enrolls people with and without HIV aged ≥15 years at 12 clinical sites in Kenya, Tanzania, Uganda, and Nigeria. The study follows participants every 6 months and collects social, demographic, clinical, and laboratory data. A total of 131 respiratory samples, collected from March 2022 to February 2023 from participants in South Rift Valley Province, Kenya, who had symptoms of respiratory illness or a positive COVID-19 test, were analyzed using a pan-viral hybridization metagenomic next-generation sequencing approach. Libraries were sequenced on the Illumina Next-generation Sequencing System NovaSeq 6000. Sample data were run through several pathogen discovery pipelines. RESULTS: Full genome and partial genome sequences were assembled for several respiratory viruses including SARS-CoV-2, human coronavirus HKU1, human adenovirus 62, human metapneumovirus, human mastadenovirus B (coinfection with SARS-CoV-2), human mastadenovirus C (coinfection with SARS-CoV-2), and human parechovirus 3 (coinfection with adenovirus 62). The results showed that SARS-CoV-2 lineages correspond with lineages circulating during March 2022 to February 2023 and revealed additional viral respiratory pathogens and viruses known to be associated with HIV. CONCLUSIONS: These preliminary results suggest that continued genomic surveillance efforts are needed for data-driven decisions on force health protection, prevention of emerging respiratory infections, and mitigation of impacts on military readiness caused by infectious diseases.

Humans↗

PubMed1991

Identification of a novel murine IAP-promoted placenta-expressed gene.

We have cloned and characterized a novel cellular gene that is promoted by an intracisternal A-particle (IAP) LTR and expressed in the mouse placenta (mouse IAP promoted placental gene, MIPP). A 1067bp cDNA clone containing an IAP LTR U5 region duplicated in its 5' terminus and an ORF coding for a potential 202 amino acids protein was isolated from an 8.5 day old mouse embryo cDNA library. Sequence analysis of the 5' region of a genomic clone revealed the presence of a solo IAP LTR with the same U5 duplication, and primer extension analysis confirmed that transcription of the MIPP gene is under the control of the IAP LTR. Expression of the MIPP gene parallels that of IAP genes in normal mouse tissues with abundant transcripts present in the placenta and also in the myeloma MOPC-315. The MIPP-encoded protein is composed of four 48-amino acid repeat units and shares homology with a vaccinia virus gene product. MIPP-related sequences were also detected in higher eukaryotic genomes including human.

Amino Acid Sequence↗

PubMed1994

Cloning and characterization of a c-myc intron binding protein (MIBP1).

The cDNA for a c-myc intron 1 binding protein 1 (MIBP1) in the rat was isolated from lambda gt11 and lambda ZAPII cDNA libraries. Sequencing of the cDNA clones revealed a long ORF which encoded a putative protein of 2437 amino acid residues. This protein has two widely separated zinc finger regions, each of which carries C2H2 motifs. When expressed in E. coli as a fusion protein, part of the MIBP1 showed sequence-specific binding to the target sequence, i.e., a 9-bp sequence in the rat c-myc intron 1. MIBP1 is most likely the rat counterpart of human MHC binding protein-2 (MBP-2/HIV-EP2), based on the 86% similarity in nucleotide sequence and 93% similarity in amno acid sequence. Northern blotting revealed a high level of MIBP1 mRNA in the brain.

Amino Acid Sequence↗

PubMed1996

High-affinity and specific recognition of human thyroid stimulating hormone (hTSH) by in vitro-selected 2'-amino-modified RNA.

RNA sequences containing 2'-amino pyrimidines that bind with high-affinity to human thyroid stimulating hormone (hTSH) were isolated from a random sequence library by an in vitro selection-amplification procedure. A representative RNA ligand (T-15) has an equilibrium dissociation constant (Kd) of 2.5 nM for its interaction with hTSH and can discriminate between other members of the glycohormone family; no detectable binding was observed at low micromolar concentrations of hCG (human chorionic gonadotropin), while measured Kd values for the interactions with hLH (human leutinizing hormone) and hFSH (human follicle stimulating hormone) were > 1 microM and approximately 0.2 microM, respectively. The detection of hTSH in a dot blot assay with radiolabeled T-15 RNA was demonstrated.

Humans↗

PubMed1997

Methionine aminopeptidase from the hyperthermophilic Archaeon Pyrococcus furiosus: molecular cloning and overexpression in Escherichia coli of the gene, and characteristics of the enzyme.

A gene for a methionine aminopeptidase (MAP; EC 3.4.11.18), which catalyzes the removal of amino-terminal methionine from the growing peptide chain on the ribosome, has been cloned from the hyperthermophilic Archaeon, Pyrococcus furiosus, by a novel method effectively using its cosmid protein library, sequenced and expressed in Escherichia coli. The DNA sequence encodes a protein containing 295 amino acid residues with methionine at the N-terminus. From protein analyses of the recombinant protein expressed in E. coli, by using both amino acid sequence analysis from the N-terminus by automated Edman degradation and analyses of molecular masses of the peptides generated by two enzymatic cleavages performed independently, digestions with lysylendopeptidase and Endoproteinase Asp-N, with ionspray mass spectrometry, the primary structure of the protein has been elucidated to be completely identical with that deduced from its DNA sequence. Comparison of the amino acid sequence of P. furiosus MAP (P.f. MAP) with those of other MAPs from Eukarya and Bacteria showed that the protein has a high degree of sequence homology in the stretches surrounding the five cobalt-binding residues fully preserved in all of MAPs determined so far, but P.f. MAP belongs to Type II because it has an extra long insertion of about 60 amino acid residues between the fourth and fifth cobalt-binding ligands, similar to MAPs from human and rat, and to Met-AP2 from Saccharomyces cerevisiae, in comparison to Type I MAPs from Bacteria. Therefore, P.f. MAP seems to be rather close to those from Eukarya, although it is distinct in lacking the N-terminal extension of about 90-150 residues universally found in MAPs from Eukarya. These findings suggest that P.f. MAP is evolutionally located at the Eukarya-Bacteria boundary. The enzyme expressed in E. coli exhibits a considerable thermostability, with a half-life of approximately 4.5 h at 90 degrees C and an optimum temperature of around 90 degrees C.

Amino Acid Sequence↗

PubMed2000

A pollen-specific and desiccation-associated transcript in Lilium longiflorum during development and stress.

A cDNA clone encoding a desiccation-induced protein (LLA23) has been isolated from the mature pollen of a Lilium longiflorum cDNA library. Sequence analysis revealed significant similarity between the predicted LLA23 polypeptide, particularly at the C-terminal half of the sequence and a group of water-deficit/ripening-induced proteins. The expression of LLA23 gene is pollen-specific and the transcript accumulates only at the later stage of pollen maturation prior to anthesis. Premature drying of developing pollen confirmed that the accumulation of LLA23 transcripts was associated with desiccation. The LLA23 proteins decreased their levels when pollen/pollen tubes grew in the germination buffer. Treatments of pollen with abscisic acid (ABA) and polyethylene glycol (PEG)-8000 during germination greatly retarded the disappearance of LLA23 proteins and mRNAs. The LLA23 transcripts decreased their levels in pollen tubes grown both in vitro and in vivo, but the disappearance of LLA23 transcripts in tube cells grown in vivo was slower than those grown in vitro. In situ localization using anti-chicken immunoglobulin G conjugated with gold particles confirmed that LLA23 was located in the cytoplasm of pollen grains. The protective function of the desiccation-related proteins in the cytoplasm of pollen grains is proposed.

Amino Acid Sequence↗

PubMed2000

Identification of a novel testis-specific leucine-rich protein in humans and mice.

A novel testis-specific protein, termed LRTP, was identified by screening both human and mouse testis and mouse pachytene spermatocyte cDNA libraries. Sequence analyses (GenBank accession number: AF092208) revealed that LRTP contains an amino terminus leucine-rich repeat domain. There are several acidic regions rich in glutamic acid in the C-terminus. The sequence, by GenBank search, shows similarities to LANP and SDS22+, leucine-rich repeat proteins localized to the nucleus and involved in the regulation of protein phosphatases. In mouse, the mRNA is first detected at about Day 14 postpartum, presumably when mid-pachytene spermatocytes are first seen. In situ hybridization confirmed the expression of the LRTP mRNA at this stage of spermatogenesis. Immunohistochemical analysis revealed that the protein is most abundant in the cytoplasm of pachytene and diplotene cells, corresponding to late prophase of meiosis I. Immunohistochemical localization is markedly reduced in secondary spermatocytes, suggesting a functional association of LRTP with meiosis. An LRTP cDNA probe did not bind to mouse ovary RNA in a dot blot assay.

Amino Acid Sequence↗

PubMed2000

Selective Lutheran glycoprotein gene expression at the blood-brain barrier in normal brain and in human brain tumors.

The Lutheran (LU) glycoprotein was shown to be a specific marker of brain capillary endothelium, which forms the blood-brain barrier (BBB) in vivo. A 1.5 kb partial cDNA encoding the bovine LU was isolated from a bovine brain capillary cDNA library. Sequence analysis showed that the bovine and human LU had a 75% and 79% identity in the amino acid and nucleotide sequences, respectively. Northern blot analysis demonstrated a very high level of gene expression of the LU transcript in freshly isolated bovine brain capillaries, but no measurable LU mRNA in whole bovine brain. The high level of LU gene expression was maintained when bovine brain capillary endothelium was grown in tissue culture. Because many BBB specific genes are downregulated in tissue culture and in brain tumors, the expression of the LU mRNA and immunoactive LU protein was investigated in primary and metastatic human brain tumors. Immunocytochemistry of fresh frozen human brain and human brain tumors showed abundant immunostaining of brain capillary endothelium. Northern blot analysis showed the presence of LU transcripts in a panel of primary and metastatic human brain tumors. These studies demonstrated that the LU glycoprotein was a novel new marker of the BBB, and unlike other BBB specific genes, there was a persistent gene expression of the LU glycoprotein both in brain capillary endothelial cells grown in culture and in the endothelium of capillaries perfusing human brain cancer.

Amino Acid Sequence↗

PubMedDate not supplied

Cloning of an interferon-gamma regulated human melanoma-associated antigen: identity to the intercellular adhesion molecule ICAM-1.

The human melanoma-associated antigen identified by the monoclonal antibody (mAb) Me14-D12 is a cell surface protein whose expression is induced by interferon-gamma (IFN-gamma). We have recently reported the molecular cloning of a genomic probe specific for the gene and mRNA of this protein. By screening with the genomic probe, we have now isolated a full length 3.0 kb cDNA from a Raji cell line-derived lambda-gt10 library. Sequence analysis of this cDNA showed a 99.8% homology with the intercellular adhesion molecule-1 (ICAM-1). Mouse Ltk- cells stably transfected with the human cDNA clone were found to express the ICAM-1 antigenic determinants detected by mAb Me14-D12 and a reference anti-ICAM-1 mAb, as judged by surface immunofluorescence. Immunoprecipitation of surface-iodinated proteins with mAb Me14-D12 revealed the presence of a 90 kD molecule with identical mobility to ICAM-1. In addition, mAb Me14-D12 could inhibit the phorbolester-stimulated aggregation of U937 cells. The findings show that the human melanoma-associated Me14-D12 antigen is the adhesion molecule ICAM-1.

Antibodies, Monoclonal↗

PubMed2000

BAG-1, a novel Bcl-2-interacting protein, activates expression of human JC virus.

Transcription of the human polyomavirus JC virus (JCV) genome is regulated by cellular proteins and the large tumour (T) antigen. Earlier studies led to the identification of nuclear factor-1 (NF-1)-binding sites in the JCV enhancer by DNase I protection assays of extracts from retinoic acid (RA)-differentiated P19 embryonal carcinoma (EC) cells. In this study, a cDNA clone that encodes a protein capable of binding to the JCV NF-1 sites was isolated from an RA-differentiated EC cell cDNA library. Sequence analysis revealed that the cDNA isolated was identical to the previously described Bcl-2-interacting protein BAG-1 (Bcl-2-associated athano gene-1). Results from RNA studies indicated that BAG-1 is expressed in several cell types. Co-transfection of a recombinant BAG-1 expression plasmid with JCV promoters indicated that BAG-1 stimulates transcription of the JCV(E) promoter and to a lesser extent the JCV(L) promoter. Mutations in the NF-1 sites in the JCV(E) promoter eliminated the activation by BAG-1. Thus, BAG-1 is a novel transcription factor that may play a role in JCV expression.

Amino Acid Sequence↗

PubMed2001

Acinetobacter baumannii has two genes encoding glutathione-dependent formaldehyde dehydrogenase: evidence for differential regulation in response to iron.

The adhC1 gene from Acinetobacter baumannii 8399, which encodes a glutathione-dependent formaldehyde dehydrogenase (GSH-FDH), was identified and cloned after mapping the insertion site of Tn3-HoHo1 in a recombinant cosmid isolated from a gene library. Sequence analysis showed that this gene encodes a protein exhibiting significant similarity to alcohol dehydrogenases in bacterial, yeast, plant and animal cells. The expression of the adhC1 gene was confirmed by the detection of GSH-FDH enzyme activity in A. baumannii and Escherichia coli cells that expressed the cloned gene. However, the construction and analysis of an A. baumannii 8399 adhC1::Tn3-HoHo1 isogenic derivative revealed the presence of adhC2, a second copy of the gene encoding GSH-FDH activity. Enzyme assays and immunoblot analysis showed that adhC2 encodes a 46.5 kDa protein that is produced in similar amounts under iron-rich and iron-limited conditions. In contrast, the expression of adhC1, which encodes a 45 kDa protein with GSH-FDH activity, is induced under iron limitation and repressed when the cells are cultured in the presence of free inorganic iron. The differential expression of adhC1 is controlled at the transcriptional level and mediated through the Fur iron-repressor protein, which has potential binding sites within the promoter region of this adhC copy. The expression of both adhC copies is significantly enhanced by the presence of sub-inhibitory concentrations of formaldehyde in the culture media. Examination of different A. baumannii isolates indicates that they can be divided into two groups based on the type of GSH-FDH they produce. One group contains only the constitutively expressed 46.5 kDa protein, whilst the other produces this GSH-FDH type in addition to the iron-regulated isoenzyme. Further analysis showed that the presence and expression of the two adhC genes does not confer resistance to exogenous formaldehyde, nor does it enable it to utilize methylated compounds as a sole carbon source when cultured under iron-rich as well as iron-deficient conditions.

Acinetobacter↗

PubMed1999

A novel polymorphic genetic locus in members of the Mycobacterium tuberculosis complex.

It has previously been shown that the PAN promoter from Mycobacterium paratuberculosis can be used as a DNA probe to identify an RFLP between wild-type Mycobacterium bovis and the vaccine strain Mycobacterium bovis BCG. To investigate the genetic basis of this phenomenon, DNA fragments from a New Zealand M. bovis cattle strain and M. bovis BCG Pasteur, containing the PAN-binding region, were isolated from gene libraries, sequenced and characterized. Sequence analysis and comparison with database sequences showed that the PAN region in M. bovis, M. bovis BCG and Mycobacterium tuberculosis is identical and shares 70% similarity to the PAN sequence from M. paratuberculosis. The Shine-Dalgarno sequence and the -10 and -35 promoter regions are conserved between the different species. Analysis of the flanking sequences of the PAN region revealed that less than 1 kb downstream of PAN is a 2405 bp fragment that is present in M. bovis BCG but absent in the M. bovis wild-type strain. The distribution of the 2405 bp fragment in members of the M. tuberculosis complex was investigated and found to be present in 70 out of 70 M. tuberculosis strains, and 7 out of 7 M. bovis BCG daughter strains, 2 out of 2 Mycobacterium africanum strains, 2 out of 2 Mycobacterium microti strains and 7 out of 25 M. bovis strains. This is the first report of a genetic region of M. bovis BCG that is not universally present in M. bovis strains. The fragment does not appear to be present in any mycobacterial species outside the M. tuberculosis complex. It does not possess any characteristics of known transposable elements and the flanking sequences do not have any obvious features to suggest a deletion mechanism. The genetic location of this region is close to the 3' end of the RD1 region of M. bovis and M. tuberculosis. The polymorphic nature of this locus in M. bovis will provide an additional genetic marker for strain differentiation.

Amino Acid Sequence↗

PubMed2004

Cloning and characterization of red clover polyphenol oxidase cDNAs and expression of active protein in Escherichia coli and transgenic alfalfa.

Red clover (Trifolium pratense) leaves contain high levels of polyphenol oxidase (PPO) activity and o-diphenol substrates. Wounding of leaves during harvest and ensiling results in browning of leaf tissues from activity of PPO on the o-diphenols. In association with browning, leaf proteins remain undegraded during ensiling, presumably due to PPO-generated o-quinone inhibition of leaf proteases. We cloned three red clover PPO cDNAs, PPO1, PPO2, and PPO3, from a leaf cDNA library. Sequence comparisons among the three red clover PPO clones indicated they are 87% to 90% identical at the nucleotide level (80%-83% amino acid identity). All three encode proteins predicted to localize to the chloroplast thylakoid lumen. RNA-blotting and immunoblotting experiments indicated PPO1 is expressed primarily in young leaves, PPO2 in flowers and petioles, and PPO3 in leaves and possibly flowers. We expressed mature PPO1 in Escherichia coli. A portion of the expressed protein was soluble and functional in an assay for PPO activity. We also expressed the red clover PPO cDNAs under the control of a constitutive promoter in alfalfa (Medicago sativa). The expressed red clover PPO proteins were active in alfalfa extracts as evidenced by o-diphenol-dependant extract browning and quantitative assays of PPO activity. Proteolysis in leaf extracts of alfalfa expressing red clover PPO1 was dramatically reduced in the presence of an o-diphenol compared to controls. Transgenic alfalfa expressing red clover PPO should prove an excellent model system to further characterize the red clover PPO enzymes and PPO-mediated inhibition of postharvest proteolysis in forage plants.

Amino Acid Sequence↗

PubMed1993

OHP1: a maize basic domain/leucine zipper protein that interacts with opaque2.

OPAQUE2 (O2) is a regulatory gene that predominantly affects the expression of the 22-kD class of zein storage protein genes at the level of transcription. The O2 gene encodes a polypeptide belonging to the basic domain/leucine zipper (bZIP) class of transcriptional regulatory proteins. Our prior analyses have demonstrated that the O2 protein binds 22-kD zein gene promoters as a homodimer in vitro and have also suggested that O2 may bind as a heterodimer in vivo. To identify cDNAs encoding other bZIP motifs that might interact with O2, a portion encoding the bZIP motif from an O2 cDNA was used to screen an endosperm cDNA library. Sequence analysis of one isolated recombinant phage indicated the presence of a bZIP motif similar to O2. The protein product of this partial cDNA, designated OHP1, can bind the O2 target site both as a homodimer and in a heterodimeric complex with O2. Whole genome DNA gel blot analysis of maize recombinant inbreds revealed two strongly hybridizing restriction fragments, neither of which mapped close to any locus known to affect zein expression. RNA gel blot analysis revealed an approximately 1.7-kb transcript that is expressed in all organs examined except the female flower and is also expressed in endosperms homozygous for o2 and other mutations that affect zein expression (opaque7, floury2, and Defective endosperm b-30). Based on these results and previously reported data, we propose models to accommodate OHP1 in the regulation of zein gene expression by O2.

Amino Acid Sequence↗

PubMed1997

The adenylate cyclase gene MAC1 of Magnaporthe grisea controls appressorium formation and other aspects of growth and development.

Magnaporthe grisea, the causal agent of rice blast disease, differentiates a specialized infection structure called an appressorium that is crucial for host plant penetration. Previously, it was found that cAMP regulates appressorium formation. To further understand the cellular mechanisms involved in appressorium formation, we have cloned a gene (MAC1) encoding adenylate cyclase, a membrane-bound enzyme that catalyzes the production of cAMP from ATP, by using a polymerase chain reaction-based strategy. The entire gene was isolated and subcloned from a large insert bacterial artificial chromosome library. Sequence characterization showed that MAC1 has a high degree of identity with other adenylate cyclase genes from several filamentous fungi as well as yeasts. Gene deletion resulted in reduced vegetative growth, conidiation, and conidial germination. Transformants lacking MAC1 were unable to form appressoria on an inductive surface and were unable to penetrate susceptible rice leaves. mac1- transformants were also sterile and produced no perithecia. Appressorium formation was restored in the presence of exogenous cAMP derivatives. These results confirm that cell signaling involving cAMP plays a central role in the development and pathogenicity of M. grisea.

Amino Acid Sequence↗

PubMedDate not supplied

Gene cloning, expression and vaccine testing of Schistosoma japonicum SjFABP.

A 600 bp DNA fragment was amplified by PCR from an adult Schistosoma japonicum cDNA library. Sequence analysis confirmed that this fragment contained an S. japonicum Chinese mainland strain fatty acid binding protein (Sj14FABP) gene. This gene was subsequently expressed in Escherichia coli (E. coli) and in Baculovirus/silkworm systems. The recombinant protein from E. coli was a 41 kDa GST fusion protein (rSj14/GST), which could be purified by glutathione agarose affinity chromatography, with a yield of 25 mg/L E. coli culture. The recombinant protein from the Baculovirus/silkworm system was an 18 kDa fusion protein (rSj14/His), which could be purified by Ni-NTA resin chromatography column with a yield of 3.5 mg per silkworm larva. Both rSj14/GST and rSj14/His could be recognized by S. japonicum-infected mouse sera and anti-rSj14/GST mouse sera in Western blotting. The purified recombinant protein was immunogenic in mice, rats and sheep, and 34.3%, 31.9% and 59.2% worm reductions, respectively, were obtained in vaccinated Kunming mice, Wistar rats and sheep vaccinated with Sj14/GST, compared to non-vaccinated control groups. Worm reductions of 48.8% and 49.0% were recorded in Balb/c mice immunized with Sj14/His, compared to non-vaccinated and BCG-vaccinated groups, respectively. These results indicate that rSj14FABP is a promising candidate vaccine for schistosomiasis japonica, particularly as in the rat and sheep vaccination experiments, no adjuvant was used.

Amino Acid Sequence↗

PubMed2003

A novel type of two-component regulatory system affecting gingipains in Porphyromonas gingivalis.

We surveyed the Porphyromonas gingivalis W83 genome database for homologues of FimS, the first two-component sensor histidine kinase, which could possibly control virulence factors. Including fimS, we found six putative sensor kinase genes in the genome. The gene encoding one of the homologues was cloned from a P. gingivalis plasmid library, sequenced, and analyzed using its mutants. Two gene-disruption mutants were created in strain ATCC 33277 by introducing a drug cassette into the gene. The mutants formed nonpigmented colonies, indicating that they might be defective in proteinase production, a characteristic of this organism. Proteinase activities, measured as arginine- and lysine-specific (Rgp and Kgp gingipains, respectively) activities, of the mutants were almost half those of the parent strain. Unlike the parent and wildtype strains, most of the gingipain activities were detected in the culture supernatant, not in cells, of the mutants. Abnormal production of gingipains was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analyses. These results strongly suggest that this newly-discovered two-component sensor kinase is involved in maturation and proper localization of gingipains to the outer membrane through an unknown mechanism. The gene encoding the sensor histidine kinase was designated gppX, which represents regulation (X) of gingipains and black pigmentation in P. gingivalis.

Adhesins, Bacterial↗