scholarly journals Proteomic Identification of a Novel Anaplasma phagocytophilum DNA Binding Protein That Regulates a Putative Transcription Factor

2007 ◽  
Vol 189 (13) ◽  
pp. 4880-4886 ◽  
Author(s):  
Xueqi Wang ◽  
Takane Kikuchi ◽  
Yasuko Rikihisa
Keyword(s):  
Transcription Factor ◽  
Outer Membrane ◽  
Anaplasma Phagocytophilum ◽  
Promoter Region ◽  
Outer Membrane Proteins ◽  
Hypothetical Protein ◽  
Etiologic Agent ◽  
Major Protein ◽  
Mobility Shift ◽  
Putative Transcription Factor

ABSTRACT Anaplasma phagocytophilum, the etiologic agent of human granulocytic anaplasmosis, is an obligatory intracellular bacterium. Little is known about the gene regulatory mechanisms for this bacterium. A gene encoding a putative transcription factor, tr1, upstream of three tandem genes encoding outer membrane proteins, including the major outer membrane protein P44, is driven by a strong promoter. In the present study, gel mobility shift assays revealed the presence of A. phagocytophilum proteins that interact with the promoter region of tr1. These proteins interacting with the tr1 promoter region were purified by biotin-labeled DNA affinity chromatography from a large amount of host cell-free bacteria. Mass spectrometry identified the major protein as an A. phagocytophilum 12.5-kDa hypothetical protein, which was named ApxR. In a DNase I protection assay, recombinant ApxR (rApxR) bound cooperatively to four 24- or 25-bp sites within 235 bp upstream of tr1: regions III and IV proximal to tr1 had higher affinity than regions I and II did. Deletion assays showed that regions III and IV were essential for rApxR binding, whereas regions I and II upstream of regions III and IV were not. The primary cis-acting region was region IV, since region IV alone was sufficient for rApxR to strongly transactivate the downstream gene in a lacZ reporter assay. Addition of regions I, II, and III did not enhance transactivation. These results show that ApxR is a novel transcriptional regulator that directly regulates tr1.

Regulation of yeast COX6 by the general transcription factor ABF1 and separate HAP2- and heme-responsive elements

1992 ◽  
Vol 12 (5) ◽  
pp. 2302-2314
Author(s):  
J D Trawick ◽  
N Kraut ◽  
F R Simon ◽  
R O Poyton
Keyword(s):  
Transcription Factor ◽  
Carbon Source ◽  
Protein Binding ◽  
Glucose Repression ◽  
Protein Complexes ◽  
Carbon Sources ◽  
Major Protein ◽  
Mobility Shift ◽  
Gel Mobility Shift ◽  
In Cells

Transcription of the Saccharomyces cerevisiae COX6 gene is regulated by heme and carbon source. It is also affected by the HAP2/3/4 transcription factor complex and by SNF1 and SSN6. Previously, we have shown that most of this regulation is mediated through UAS6, an 84-bp upstream activation segment of the COX6 promoter. In this study, by using linker scanning mutagenesis and protein binding assays, we have identified three elements within UAS6 and one element downstream of it that are important. Two of these, HDS1 (heme-dependent site 1; between -269 and -251 bp) and HDS2 (between -228 and -220 bp), mediate regulation of COX6 by heme. Both act negatively. The other two elements, domain 2 (between -279 and -269 bp) and domain 1 (between -302 and -281 bp), act positively. Domain 2 is required for optimal transcription in cells grown in repressing but not derepressing carbon sources. Domain 1 is essential for transcription per se in cells grown on repressing carbon sources, is required for optimal transcription in cells grown on a derepressing carbon source, is sufficient for glucose repression-derepression, and is the element of UAS6 at which HAP2 affects COX6 transcription. This element contains the major protein binding sites within UAS6. It has consensus binding sequences for ABF1 and HAP2. Gel mobility shift experiments show that domain 1 binds ABF1 and forms different numbers of DNA-protein complexes in extracts from cells grown in repressing or derepressing carbon sources. In contrast, gel mobility shift experiments have failed to reveal that HAP2 or HAP3 binds to domain 1 or that hap3 mutations affect the complexes bound to it. Together, these findings permit the following conclusions: COX6 transcription is regulated both positively and negatively; heme and carbon source exert their effects through different sites; domain 1 is absolutely essential for transcription on repressing carbon sources; ABF1 is a major component in the regulation of COX6 transcription; and the HAP2/3/4 complex most likely affects COX6 transcription indirectly.

Outer Membrane Protein Composition in Disease Isolates of Haemophilus influenzae : Pathogenic and Epidemiological Implications

1980 ◽  
Vol 30 (3) ◽  
pp. 709-717
Author(s):  
Marilyn R. Loeb ◽  
David H. Smith
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Haemophilus Influenzae ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Outer Membrane Proteins ◽  
Protein Composition ◽  
The Other ◽  
Major Protein ◽  
Single Strain

The outer membrane protein composition of 50 disease isolates of Haemophilus influenzae has been determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All strains, including 28 strains of serotype b , one strain each of serotypes a, c, d, e , and f , and 17 untypable strains, had an outer membrane protein composition typical of gram-negative bacteria, i.e., these membranes contained two to three dozen proteins with four to six proteins accounting for most of their protein content. Variation in the mobility of these major outer membrane proteins from strain to strain was common but not universal; the observed patterns provided useful data and new insight into the epidemiology of type b disease. The basic findings can be summarized as follows: (i) All 50 strains possessed three proteins (one minor and two major) each having identical mobilities. The other proteins, both major and minor, varied in mobility. (ii) All type b strains possessed a fourth (major) protein of identical mobility. (iii) The 28 type b strains, on the basis of the mobility of the six major outer membrane proteins, could be divided into eight subtypes. Of all the other strains examined, both typable and untypable, only the serotype a strain belonged to one of these subtypes. (iv) The untypable strains showed considerable variation in the mobilities of their major outer membrane proteins. Of these 17 strains, 13 had an additional major outer membrane protein not present in encapsulated strains. (v) The outer membrane protein composition of a single strain remained unchanged after many passages on solid media, but varied with the growth phase. (vi) The outer membrane protein composition of isolates obtained from nine patients during an epidemic of type b meningitis varied, indicating that a single strain was not responsible for the epidemic. At least five different strains were responsible for these nine cases. (vii) Identical outer membrane protein compositions were observed in the following: in a type b strain and a mutant of this strain deficient in capsule production, indicating that the level of capsule synthesis is not obviously related to outer membrane protein composition; in type b strains isolated from different anatomic sites of patients acutely ill with meningitis, indicating that the strain associated with bacteremia is the same as that isolated from the cerebrospinal fluid; in type b strains isolated from siblings who contracted meningitis at about the same time, indicating infection with the same strain; and in type b strains isolated from the initial and repeat infection of a single patient, suggesting that reinfection was due to the same strain.

scholarly journals A Disulfide Oxidoreductase (CHU_1165) Is Essential for Cellulose Degradation by Affecting Outer Membrane Proteins in Cytophaga hutchinsonii

2020 ◽  
Vol 86 (8) ◽  
Author(s):  
Dong Zhao ◽  
Ying Wang ◽  
Sen Wang ◽  
Weican Zhang ◽  
Qingsheng Qi ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Disulfide Bond ◽  
Cellulose Degradation ◽  
Outer Membrane Proteins ◽  
Hypothetical Protein ◽  
Pleiotropic Effect ◽  
Content Type ◽  
Cytophaga Hutchinsonii ◽  
Disulfide Oxidoreductase

ABSTRACT Cytophaga hutchinsonii cells can bind to the surface of insoluble cellulose and degrade it by utilizing a novel cell contact-dependent mechanism, in which the outer membrane proteins may play important roles. In this study, the deletion of a gene locus, chu_1165, which encodes a hypothetical protein with 32% identity with TlpB, a disulfide oxidoreductase in Flavobacterium psychrophilum, caused a complete cellulolytic defect in C. hutchinsonii. Further study showed that cells of the Δ1165 strain could not bind to cellulose, and the levels of many outer membrane proteins that can bind to cellulose were significantly decreased. The N-terminal region of CHU_1165 is anchored to the cytoplasmic membrane with five predicted transmembrane helices, and the C-terminal region is predicted to stretch to the periplasm and has a similar thioredoxin (Trx) fold containing a Cys-X-X-Cys motif that is conserved in disulfide oxidoreductases. Recombinant CHU_1165His containing the Cys-X-X-Cys motif was able to reduce the disulfide bonds of insulin in vitro. Site-directed mutation showed that the cysteines in the Cys-X-X-Cys motif and at residues 106 and 108 were indispensable for the function of CHU_1165. Western blotting showed that CHU_1165 was in an oxidized state in vivo, suggesting that it may act as an oxidase to catalyze disulfide bond formation. However, many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and mutation of the cysteine in these proteins did not affect cellulose degradation, indicating that CHU_1165 may have an indirect or pleiotropic effect on the function of these outer membrane proteins. IMPORTANCE Cytophaga hutchinsonii can rapidly digest cellulose in a contact-dependent manner, in which the outer membrane proteins may play important roles. In this study, a hypothetical protein, CHU_1165, characterized as a disulfide oxidoreductase, is essential for cellulose degradation by affecting the cellulose binding ability of many outer membrane proteins in C. hutchinsonii. Disulfide oxidoreductases are involved in disulfide bond formation. However, our studies show that many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and mutation of cysteine did not affect their function, indicating that CHU_1165 did not facilitate the formation of a disulfide bond in these proteins. It may have an indirect or pleiotropic effect on the function of these outer membrane proteins. Our study provides an orientation for exploring the proteins that assist in the appropriate conformation of many outer membrane proteins essential for cellulose degradation, which is important for exploring the novel mechanism of cellulose degradation in C. hutchinsonii.

scholarly journals Anaplasma phagocytophilum Has a Functional msp2 Gene That Is Distinct from p44

2004 ◽  
Vol 72 (7) ◽  
pp. 3883-3889 ◽  
Author(s):  
Quan Lin ◽  
Yasuko Rikihisa ◽  
Suleyman Felek ◽  
Xueqi Wang ◽  
Robert F. Massung ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Anaplasma Phagocytophilum ◽  
Ixodes Scapularis ◽  
Outer Membrane Proteins ◽  
Gene Families ◽  
Single Copy ◽  
The United States ◽  
Granulocytic Anaplasmosis ◽  
The United Kingdom ◽  
Membrane Protein Gene

ABSTRACT The msp2 and p44 genes encode polymorphic major outer membrane proteins that are considered unique to the intraerythrocytic agent of Anaplasma marginale and the intragranulocytic agent of Anaplasma phagocytophilum, respectively. In the present study, however, we found an msp2 gene in A. phagocytophilum that was remarkably conserved among A. phagocytophilum strains from human granulocytic anaplasmosis (HGA) patients, ticks, and a horse from various regions in the United States, but the gene was different in a sheep isolate from the United Kingdom. The msp2 gene in the A. phagocytophilum strain HZ genome was a single-copy gene and was located downstream of two Ehrlichia chaffeensis omp-1 homologs and a decarboxylase gene (ubiD). The msp2 gene was expressed by A. phagocytophilum in the blood from HGA patients NY36 and NY37 and by A. phagocytophilum isolates from these patients cultured in HL-60 cells at 37°C. The msp2 gene was also expressed in a DBA/2 mouse infected by attaching ticks infected with strain NTN-1 and in a horse experimentally infected by attaching strain HZ-infected ticks. However, the transcript of the msp2 gene was undetectable in A. phagocytophilum strain HZ in SCID mice and Ixodes scapularis ticks infected with strain NTN-1. These results indicate that msp2 is functional in various strains of A. phagocytophilum, and relative expression ratios of msp2 to p44 vary in different infected hosts. These findings may be important in understanding roles that Msp2 proteins play in granulocytic ehrlichia infection and evolution of the polymorphic major outer membrane protein gene families in Anaplasma species.

scholarly journals Regulation of yeast COX6 by the general transcription factor ABF1 and separate HAP2- and heme-responsive elements.

1992 ◽  
Vol 12 (5) ◽  
pp. 2302-2314 ◽  
Author(s):  
J D Trawick ◽  
N Kraut ◽  
F R Simon ◽  
R O Poyton
Keyword(s):  
Transcription Factor ◽  
Carbon Source ◽  
Protein Binding ◽  
Glucose Repression ◽  
Protein Complexes ◽  
Carbon Sources ◽  
Major Protein ◽  
Mobility Shift ◽  
Gel Mobility Shift ◽  
In Cells

Transcription of the Saccharomyces cerevisiae COX6 gene is regulated by heme and carbon source. It is also affected by the HAP2/3/4 transcription factor complex and by SNF1 and SSN6. Previously, we have shown that most of this regulation is mediated through UAS6, an 84-bp upstream activation segment of the COX6 promoter. In this study, by using linker scanning mutagenesis and protein binding assays, we have identified three elements within UAS6 and one element downstream of it that are important. Two of these, HDS1 (heme-dependent site 1; between -269 and -251 bp) and HDS2 (between -228 and -220 bp), mediate regulation of COX6 by heme. Both act negatively. The other two elements, domain 2 (between -279 and -269 bp) and domain 1 (between -302 and -281 bp), act positively. Domain 2 is required for optimal transcription in cells grown in repressing but not derepressing carbon sources. Domain 1 is essential for transcription per se in cells grown on repressing carbon sources, is required for optimal transcription in cells grown on a derepressing carbon source, is sufficient for glucose repression-derepression, and is the element of UAS6 at which HAP2 affects COX6 transcription. This element contains the major protein binding sites within UAS6. It has consensus binding sequences for ABF1 and HAP2. Gel mobility shift experiments show that domain 1 binds ABF1 and forms different numbers of DNA-protein complexes in extracts from cells grown in repressing or derepressing carbon sources. In contrast, gel mobility shift experiments have failed to reveal that HAP2 or HAP3 binds to domain 1 or that hap3 mutations affect the complexes bound to it. Together, these findings permit the following conclusions: COX6 transcription is regulated both positively and negatively; heme and carbon source exert their effects through different sites; domain 1 is absolutely essential for transcription on repressing carbon sources; ABF1 is a major component in the regulation of COX6 transcription; and the HAP2/3/4 complex most likely affects COX6 transcription indirectly.

scholarly journals LipL21 Is a Novel Surface-Exposed Lipoprotein of Pathogenic Leptospira Species

2003 ◽  
Vol 71 (5) ◽  
pp. 2414-2421 ◽  
Author(s):  
Paul A. Cullen ◽  
David A. Haake ◽  
Dieter M. Bulach ◽  
Richard L. Zuerner ◽  
Ben Adler
Keyword(s):  
Outer Membrane ◽  
Global Analysis ◽  
Outer Membrane Proteins ◽  
Sucrose Density Gradient ◽  
Etiologic Agent ◽  
Signal Peptidase ◽  
Gene Encoding ◽  
Pathogenic Leptospira ◽  
Outer Membrane Lipoprotein ◽  
Triton X

ABSTRACT Leptospira is the etiologic agent of leptospirosis, a bacterial zoonosis distributed worldwide. Leptospiral lipopolysaccharide is a protective immunogen, but the extensive serological diversity of leptospires has inspired a search for conserved outer membrane proteins (OMPs) that may stimulate heterologous immunity. Previously, a global analysis of leptospiral OMPs (P. A. Cullen, S. J. Cordwell, D. M. Bulach, D. A. Haake, and B. Adler, Infect. Immun. 70:2311-2318, 2002) identified pL21, a novel 21-kDa protein that is the second most abundant constituent of the Leptospira interrogans serovar Lai outer membrane proteome. In this study, we identified the gene encoding pL21 and found it to encode a putative lipoprotein; accordingly, the protein was renamed LipL21. Southern hybridization analysis revealed the presence of lipL21 in all of the pathogenic species but in none of the saprophytic species examined. Alignment of the LipL21 sequence from six strains of Leptospira revealed 96 to 100% identity. When specific polyclonal antisera to recombinant LipL21 were used, LipL21 was isolated together with other known leptospiral OMPs by both Triton X-114 extraction and sucrose density gradient membrane fractionation. All nine strains of pathogenic leptospires investigated by Western blotting, whether culture attenuated or virulent, were found to express LipL21. In contrast, the expression of LipL21 or an antigenically related protein could not be detected in nonpathogenic L. biflexa. Infected hamster sera and two of eight human leptospirosis sera tested were found to react with recombinant LipL21. Native LipL21 was found to incorporate tritiated palmitic acid, consistent with the prediction of a lipoprotein signal peptidase cleavage site. Biotinylation of the leptospiral surface resulted in selective labeling of LipL21 and the previously known OMPs LipL32 and LipL41. These findings show that LipL21 is a surface-exposed, abundant outer membrane lipoprotein that is expressed during infection and conserved among pathogenic Leptospira species.

scholarly journals High affinity of Skp to OmpC revealed by single-molecule detection

2020 ◽  
Author(s):  
Sichen Pan ◽  
Chen Yang ◽  
Xin Sheng Zhao
Keyword(s):  
Outer Membrane ◽  
Single Molecule ◽  
Protein C ◽  
Outer Membrane Proteins ◽  
Correlation Spectroscopy ◽  
Major Protein ◽  
Gram Negative Bacteria ◽  
Fluorescence Correlation ◽  
High Affinity ◽  
Quality Control Mechanism

AbstractOuter membrane proteins (OMPs) are essential to Gram-negative bacteria, and they need molecular chaperones to prevent from aggregation in periplasm during the OMPs biogenesis. Seventeen kilodalton protein (Skp) is the major protein for this purpose. Here we used singlemolecule detection (SMD) to study the stoichiometry modulation of Skp in binding with outer membrane protein C (OmpC) from Escherichia coli. To accomplish our task, we developed the tool of portion selectively chosen fluorescence correlation spectroscopy (pscFCS). We found that Skp binds OmpC with high affinity. The half concentration for Skp to form homotrimer Skp3 (C1/2) was measured to be 250 nM. Under the Skp concentrations far below C1/2 OmpC can recruit Skp monomers to form OmpC·Skp3. The affinity of the process is in picomolar range, indicating that the trimerization of Skp in OmpC·Skp3 complex is induced by OmpC-Skp interaction even though free Skp3 is rarely present. In the concentration range that Skp3 is the predominant form, OmpC may directly interact with Skp3. Under micro-molar concentrations of Skp, the formation of OmpC·(Skp3)2 was observed. Our results suggest that the fine-tuned modulation of Skp composition stoichiometry plays an important role in the safe-guarding and quality control mechanism of OMPs in the periplasm.

scholarly journals Identification of Vibrio parahaemolyticus and Vibrio spp. Specific Outer Membrane Proteins by Reverse Vaccinology and Surface Proteome

2021 ◽  
Vol 11 ◽  
Author(s):  
Wenbin Wang ◽  
Jianxin Liu ◽  
Shanshan Guo ◽  
Lei Liu ◽  
Qianyun Yuan ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Vibrio Parahaemolyticus ◽  
Outer Membrane Proteins ◽  
Subcellular Location ◽  
Hypothetical Protein ◽  
High Adhesion ◽  
Proteomic Study ◽  
Α Helix ◽  
Surface Proteome

The discovery of outer membrane proteins (OMPs) with desirable specificity and surface availability is a fundamental challenge to develop accurate immunodiagnostic assay and multivalent vaccine of pathogenic Vibrio species in food and aquaculture. Herein 101 OMPs were systemically screened from 4,831 non-redundant proteins of Vibrio parahaemolyticus by bioinformatical predication of signaling peptides, transmembrane (TM) α-helix, and subcellular location. The sequence homology analysis with 32 species of Vibrio spp. and all the non-Vibrio strains revealed that 15 OMPs were conserved in at least 23 Vibrio species, including BamA (VP2310), GspD (VP0133), Tolc (VP0425), OmpK (VP2362), OmpW (VPA0096), LptD (VP0339), Pal (VP1061), flagellar L-ring protein (VP0782), flagellar protein MotY (VP2111), hypothetical protein (VP1713), fimbrial assembly protein (VP2746), VacJ lipoprotein (VP2214), agglutination protein (VP1634), and lipoprotein (VP1267), Chitobiase (VP0755); high adhesion probability of flgH, LptD, OmpK, and OmpW indicated they were potential multivalent Vibrio vaccine candidates. V. parahaemolyticus OMPs were found to share high homology with at least one or two Vibrio species, 19 OMPs including OmpA like protein (VPA073), CsuD (VPA1504), and MtrC (VP1220) were found relatively specific to V. parahaemolyticus. The surface proteomic study by enzymatical shaving the cells showed the capsular polysaccharides most likely limited the protease action, while the glycosidases improved the availability of OMPs to trypsin. The OmpA (VPA1186, VPA0248, VP0764), Omp (VPA0166), OmpU (VP2467), BamA (VP2310), TolC (VP0425), GspD (VP0133), OmpK (VP2362), lpp (VPA1469), Pal (VP1061), agglutination protein (VP1634), and putative iron (III) compound receptor (VPA1435) have better availability on the cell surface.

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