Bacterial 'histone-like protein I' (HLP-I) is an outer membrane constituent?

The nucleoid-associated 'histone-like protein I' (HLP-I) protein of E. coli was found to be homologous with the cationic 16-kDa outer membrane protein OmpH of Salmonella typhimurium. Deduced from the nucleotide sequence, the HLP-I protein has 91% identical residues with the OmpH protein. Both proteins have very similar cleavable signal sequences. The nucleotide sequence similarity between the corresponding genes hlpA and ompH is 87%. The ompH gene is located in a gene cluster resembling the hlpA-ORF17 region of E. coli which is close to the Ipx genes involved in the biosynthesis of lipopolysaccharides. The localization of the OmpH/HLP-I protein in the cell is discussed.     

Cloning and characterization of the major outer membrane protein gene (ompH) of Pasteurella multocida X-73.

The major outer membrane protein (OmpH) of Pasteurella multocida X-73 was purified by selective extraction with detergents, followed by size exclusion chromatography. The planar lipid bilayer assay showed that OmpH has pore-forming function. The average single channel conductance in 1.0 M KCl was 0.62 nS. The gene (ompH) encoding OmpH has been isolated and sequenced by construction of a genomic library and PCR techniques. The coding region of this gene is 1,059 bp long. The predicted primary protein is composed of 353 amino acids, with a 20-amino-acid signal peptide. The mature protein is composed of 333 amino acids with a molecular mass of 36.665 kDa. The ompH gene encoding mature protein has been expressed in Escherichia coli by using a regulatable expression system. The ompH gene was distributed among 15 P. multocida serotypes and strain CU. Protection studies showed that OmpH was able to induce homologous protection in chickens. These findings demonstrate that OmpH is a protective outer membrane porin of strain X-73 and is conserved among P. multocida somatic serotypes.     

Primary structure and expression of the Ssc-protein of Salmonella typhimurium

A 1020-bp open reading frame (ORF) was found immediately downstream of the ompH gene of Salmonella typhimurium. This ORF (ORF-36) encodes a moderately hydrophobic protein with 341 amino acid residues (calculated molecular mass, 35,928 Da). The ORF-36 product was detected in minicells. Downstream of ORF-36, another ORF was found. It is highly homologous to the E. coli ORF (ORF-17.4) which precedes the lpx-genes involved in lipid A biosynthesis. ORF-36 is probably analogous to the firA gene of E. coli, the sequence of which has not yet been published. Thus it appears that the enterobacterial ompH and lpx genes are separated only by the ORF-36 and ORF-17.4 genes. We also discuss the data on the function of the ORF-36 protein. On this basis, we suggest that the protein could be called the Ssc protein.     

Construction of genetically modified tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis> cv. Petit Havana) harboring <Emphasis Type="Italic">ompH(A:3)</Emphasis> from <Emphasis Type="Italic">Pasteurella multocida</Emphasis> (A:3)

Fowl cholera, caused by Pasteurella multocida (A:3), is a fearsome disease leading to a nonproductive influence upon poultry industry. It has been known that outer membrane protein H (OmpH) in the bacterium is a strong candidate to bring on the notorious ailment. Genetically modified (GM) tobacco (Nicotiana tabacum cv. Petit Havana) harboring ompH(A:3) was constructed to develop a plant expression system for the protein, OmpH(A:3). Some 987 bp-long (ORF with the stop codon, TAA) of the ompH(A:3) excluding the nucleotide for signal peptide, was amplified by RT-PCR with the gene specific primers and pGEM-T-ompH(A:3) as template DNA. The PCR-amplified DNA was ligated into BamHI/Sacl-cut pBI121 to obtain a recombinant plasmid, pBI121-ompH(A:3). It was then transformed into Agrobacterium tumefaciens (LBA 4404) by liquid nitrogen method to generate a recombinant clone of Agrobacterium LBA4404/pBI121-ompH(A:3). The Agrobacterium LBA4404/pBI121-ompH(A:3) was inoculated into leaf discs of tobacco (2 day old). The gene-transfected leaves were cultured on Murashige-Skoog basal medium containing kanamycin (50 mg/mL) to generate numerous calli, from which some GM tobacco plants were obtained. Transgenicity of the tobacco plant was confirmed by PCR screening along with the DNA sequencing. Also, its expression in the GM-tobacco was examined qualitatively as well as quantitatively by ELISA/Western blot. These results suggest that the genetically modified tobacco plant can be potentially used as a model system to develop plant-based vaccine against the fowl cholera.     

The ompH gene of Yersinia enterocolitica: cloning, sequencing, expression, and comparison with known enterobacterial ompH sequences

We have recently described a previously uncharacterized outer membrane protein of Salmonella typhimurium and Escherichia coli and cloned and sequenced the corresponding gene, the ompH gene, of S. typhimurium (P. Koski, M. Rhen, J. Kantele, and M. Vaara, J. Biol. Chem. 264:18973-18980, 1989). We report here the cloning, sequencing, and expression of the corresponding gene of Yersinia enterocolitica. It is significantly homologous to the ompH genes of E. coli and S. typhimurium (homology percentages, 65 and 64%, respectively), has a promoter region strongly homologous to the E. coli 17-bp class consensus promoter, and encodes a protein consisting of 165 amino acids (22 of which form the signal sequence). The plasmid-borne Y. enterocolitica ompH was found to be expressed both in the E. coli host and in minicells. The isolated outer membrane of Y. enterocolitica was shown to contain OmpH. The homology of the Y. enterocolitica OmpH protein is 66% with E. coli OmpH and 64% with S. typhimurium OmpH. All OmpH proteins have almost identical hydrophobic profiles, charge distributions, and predicted secondary structures. Because yersiniae are considered rather distant relatives of E. coli and S. typhimurium in the Enterobacteriaceae family, these results might indicate that most or all strains of the family Enterobacteriaceae have OmpH proteins remarkably homologous to those now sequenced.     

Heterologous protective immunization elicited in mice by Pasteurella multocida fur ompH

Different strategies have been developed to produce vaccines against Pasteurella multocida. The approach described herein involves overexpression on the bacterial cell surface of Fur-regulated IROMPs (iron-regulated outer-membrane proteins). Accordingly, the ability of fur mutants to promote heterologous protection was examined in a Swiss mouse animal model. Twofur mutants derived from P. multocida were isolated, one of which was also defective in the OmpH protein. In mice challenged with virulent P. multocida, outer-membrane protein (OMP) extracts of fur cells conferred the same protection as obtained with wild-type cells grown in iron-depleted medium. Total protection was achieved with 40 microg of OMP extract from the fur ompH mutant. Mice administered heat-inactivated fur ompH cells were 60% cross-protected. The presence of a galE mutation in these cells did not further increase the protection level. Additionally, cell disruption by sonication provoked a higher level of protection than conferred by heat-treated cells. Taken together, the results showed that P. multocida fur ompH cells offer a simple and suitable approach for cross-protecting animals against infection with P. multocida.     

立氏立克次体外膜蛋白H基因片段的克隆表达与免疫原性分析

目的：克隆表达立氏立克次体（Rickettsia rickettsii）外膜蛋白H基因（ompH）片段并对其进行免疫原性分析。方法：采用PCR技术从立氏立克次体基因组中扩增ompH基因片段,将该基因片段与原核表达载体pET32a连接,构建重组原核表达质粒pET32a/ompH;将pET32a/ompH转入大肠杆菌细胞内,用IPTG诱导转化大肠杆菌表达目的基因。结果：获得长为327bp的ompH基因片段,SDS-PAGE分析发现pET32a/ompH转化菌表达了大小约27kDa蛋白,该蛋白与立氏立克次体免疫豚鼠血清及斑点热患者血清在免疫印迹分析中呈阳性反应,经该重组蛋白免疫血清中和后的立氏立克次体感染VERO活力减低。结论：pET32a/ompH转化的大肠杆菌表达了ompH基因片段,所产生的重组蛋白具有良好的免疫反应性及保护性。     

Outer membrane protein H for protective immunity against Pasteurella multocida

Pasteurella multocida, a Gram-negative facultative anaerobic bacterium, is a causative animal pathogen in porcine atrophic rhinitis and avian fowl cholera. For the development of recombinant subunit vaccine against P. multocida, we cloned and analyzed the gene for outer membrane protein H (ompH) from a native strain of Pasteurella multocida in Korea. The OmpH had significant similarity in both primary and secondary structure with those of other serotypes. The full-length, and three short fragments of ompH were expressed in E. coli and the recombinant OmpH proteins were purified, respectively. The recombinant OmpH proteins were antigenic and detectable with antisera produced by either immunization of commercial vaccine for respiratory disease or formalin-killed cell. Antibodies raised against the full-length OmpH provided strong protection against P. multocida, however, three short fragments of recombinant OmpHs, respectively, showed slightly lower protection in mice challenge. The recombinant OmpH might be a useful vaccine candidate antigen for P. multocida.     

Genetic diversity of the Campylobacter genes coding immunodominant proteins

Three Campylobacter jejuni 72Dz/92 genes (cjaA (ompH1), cjaC (hisJ) and cjaD (omp18)) encoding immunodominant proteins are considered to be potential chicken vaccine candidates. The presence and conservation of cjaA, cjaC and cjaD genes among different Campylobacter clinical isolates were determined. The genes were detected in thirty Campylobacter strains using hybridization as well as Western blot analysis. However, PCR products of the predicted size were amplified only from ten out of thirty examined strains regardless of the employed primer pair. The nucleotide sequence of the C. jejuni 72Dz/92 genes was compared with the nucleotide sequences of their homologs cloned from other Campylobacter strains as well as with the whole genome sequence of C. jejuni NCTC 11168. The examined sequences revealed 0 to 16% divergence. Strain-dependent levels of divergence were observed. The polymorphism detected in cjaC was mainly within the 5' region of the gene, while the nucleotide substitutions in cjaA and cjaD are distributed uniformly along the whole genes. Most of the observed nucleotide substitutions occurred at the third base of the codons. This observation is consistent with the results of Western blot experiments.     

Isolation, cloning, and primary structure of a cationic 16-kDa outer membrane protein of Salmonella typhimurium

By using acid-urea polyacrylamide gel electrophoresis, two cationic proteins were found in the isolated outer membranes of Salmonella typhimurium SH5014. Also, all the other enterobacterial strains studied (five additional strains of S. typhimurium, one strain of Salmonella minnesota, and three strains of Escherichia coli K12) had those proteins. The most abundant (OMB2) was purified in preparative acid-urea polyacrylamide gel electrophoresis and reversed-phase high pressure liquid chromatography (HPLC). It had a molecular mass of 16 kDa, a pI above 10.0, and was rich in arginine and lysine. 72% of the total amino acid sequence was determined by sequencing several HPLC-purified proteolytic fragments and 55 amino acids from the NH2 terminus. Furthermore, we isolated by molecular cloning the corresponding gene, named it ompH, and determined its nucleotide sequence. By combining protein and nucleotide sequence data, we determined the primary structure of the entire OmpH protein. It consists of 141 amino acids, possesses regions very rich in basic amino acids, and has a molecular mass of 15,862 kDa.     

New components of protein folding in extracytoplasmic compartments of Escherichia coli SurA, FkpA and Skp/OmpH

A global search for extracytoplasmic folding catalysts in Escherichia coli was undertaken using different genetic systems that produce unstable or misfolded proteins in the periplasm. The extent of misfolding was monitored by the increased activity of the σ^E regulon that is specifically induced by misfolded proteins in the periplasm. Using multicopy libraries, we cloned two genes, surA and fkpA , that decreased the σ^E-dependent response constitutively induced by misfolded proteins. According to their sequences and their biochemical activities, SurA and FkpA belong to two different peptidyl prolyl isomerase (PPI) families. Interestingly, surA was also selected as a multicopy suppressor of a defined htrM ( rfaD ) null mutation. Such mutants produce a defective lipopolysaccharide that is unable to protect outer membrane proteins from degradation during folding. The SurA multicopy suppression effect in htrM ( rfaD ) mutant bacteria was directly associated with its ability to catalyse the folding of outer membrane proteins immediately after export. Finally, Tn 10 insertions were isolated, which led to an increased activity of the σ^E regulon. Such insertions were mapped to the dsb genes encoding catalysts of the protein disulphide isomerase (PDI) family, as well as to the surA , fkpA and ompH/skp genes. We propose that these three proteins (SurA, FkpA and OmpH/Skp) play an active role either as folding catalysts or as chaperones in extracytoplasmic compartments.     

Genetic characterization of ompH mutants in the deep-sea bacterium Photobacterium sp. strain SS9

OmpH is an outer membrane protein produced by the deep-sea bacterium Photobacterium species strain SS9 in response to elevated hydrostatic pressure. In order to facilitate studies of the function of this protein, a series of OmpH⁺ and OmpH^- strains were obtained from SS9 by Tn5 gene replacement mutagenesis. A previously isolated ompH::lacZ strain and a derivative of this strain harboring a plasmid expressing the wild-type ompH gene were also utilized. The acridine mutagen ICR 191 preferentially inhibited the growth of OmpH⁺ over OmpH^- cells. Indeed, OmpH⁺ cultures treated with the mutagen rapidly accumulated mutants producing reduced levels of OmpH. In addition. OmpH⁺ cells took up the peptide Met-Leu-Phe approximately 15 times more rapidly than OmpH^- cells. The results are consistent with the hypothesis that OmpH functions as a relatively large, nonspecific diffusion channel.Abbreviations OMP Outer membrane protein     

Verification of the PREFAB alignment database

The verification of the PREFAB database containing golden standard protein alignments was performed. It has revealed a significant number of differences between the sequences from PREFAB and PDB databases. It was shown that compared to the sequences given in the PDB database 575 alignments refered to a sequence with a gap; such alignments were excluded. Furthermore, compared to the PDB-sequences a single substitute or the insertions were found for 440 aminoacid sequences from PREFAB database; these sequences were edited. SCOP domain analysis has shown that only 502 alignments in the resulting set contain the sequences from the same family. Finally, eliminating duplicates, we have created a new golden standard alignment database PREFAB-P based on PREFAB; the PREFAB-P database contains 581 alignments.     

Characterization of the gene encoding the immunodominant 35 kDa protein of Mycobacterium leprae

Analysis of the interaction between the host immune system and the intracellular parasite Mycobacterium leprae has identified a 35 kDa protein as a dominant antigen. The native 35 kDa protein was purified from the membrane fraction of M. leprae and termed MMPI (major membrane protein I). As the purified protein was not amenable to N-terminal sequencing, partial proteolysis was used to establish the sequences of 21 peptides. A fragment of the 35 kDa protein-encoding gene was amplified by the polymerase chain reaction from M. leprae chromosomal DNA with oligonucleotide primers derived from internal peptide sequences and the whole gene was subsequently isolated from a M. leprae cosmid library. The nucleotide sequence of the gene revealed an open reading frame of 307 amino acids containing most of the peptide sequences derived from the native 35 kDa protein. The calculated subunit mass was 33.7 kDa, but the native protein exists as a multimer of 950 kDa. Database searches revealed no identity between the 35 kDa antigen and known protein sequences. The gene was expressed in Mycobacterium smegmatis under the control of its own promoter or at a higher level using an‘up-regulated’promoter derived from Mycobacterium fortuitum. The gene product reacted with monoclonal antibodies raised to the native protein. Using the bacterial alkaline phosphatase reporter system, we observed that the 35 kDa protein was unable to be exported across the membrane of recombinant M. smegmatis. The 35 kDa protein-encoding gene is absent from members of the Mycobacterium tuberculosis complex, but homologous sequences were detected in Mycobacterium avium, Mycobacterium haemophilum and M. smegmatis. The avaibility of the recombinant 35 kDa protein will permit dissection of both antibody- and T-cell-mediated immune responses in leprosy patients.     

Verification of the PREFAB alignment database

Verification of the PREFAB database containing golden standard protein alignments was performed. It has revealed a significant number of differences between the sequences from PREFAB and PDB databases. It was shown that, compared with the sequences given in the PDB, 575 alignments referred to a sequence with a gap; such alignments were excluded. Furthermore, compared with the PDB sequences, single substitutions or insertions were found for 440 amino acid sequences from PREFAB; these sequences were edited. SCOP domain analysis has shown that only 502 alignments in the resulting set contain sequences from the same family. Finally, eliminating duplicates, we have created a new golden standard alignment database PREFAB-P based on PREFAB; the PREFAB-P database contains 581 alignments.     

An analysis of the Sargasso Sea resource and the consequences for database composition

Background  

The environmental sequencing of the Sargasso Sea has introduced a huge new resource of genomic information. Unlike the protein sequences held in the current searchable databases, the Sargasso Sea sequences originate from a single marine environment and have been sequenced from species that are not easily obtainable by laboratory cultivation. The resource also contains very many fragments of whole protein sequences, a side effect of the shotgun sequencing method.     

Molecular cloning and sequencing of cDNA encoding the phosphatidylinositol kinase from rat brain.

A complementary DNA (cDNA) clone that encodes phosphatidylinositol 4-kinase (PI 4-kinase) was isolated from a rat brain cDNA library. The deduced amino acid sequence of 697 residues revealed that the protein contains two putative transmembrane sequences and that the N-terminal part of the protein has several sequences representing potential phosphorylation sites for cAMP- and calmodulin-dependent kinase. The C-terminal region is probably a phosphotransferase domain homologous to the kinase region of protein kinase family proteins. Specific antibody against the protein expressed in Escherichia coli successfully immunoprecipitated rat brain PI 4-kinase. The messenger RNA for PI 4-kinase was found predominantly in brain and rat neural cell lines. This PI kinase may play a specific role in neural signal transduction.     

Purification and characterization of the R64 shufflon-specific recombinase

The shufflon, a multiple DNA inversion system in plasmid R64, consists of four invertible DNA segments which are separated and flanked by seven 19-bp repeat sequences. The product of a site-specific recombinase gene, rci, promotes site-specific recombination between any two of the inverted 19-bp repeat sequences of the shufflon. To analyze the molecular mechanism of this recombination reaction, Rci protein was overproduced and purified. The purified Rci protein promoted the in vitro recombination reaction between the inverted 19-bp repeats of supercoiled DNA of a plasmid carrying segment A of the R64 shufflon. The recombination reaction was enhanced by the bacterial host factor HU. Gel electrophoretic analysis indicated that the Rci protein specifically binds to the DNA segments carrying the 19-bp sequences. The binding affinity of the Rci protein to the four shufflon segments as well as four synthetic 19-bp sequences differed greatly: among the four 19-bp repeat sequences, the repeat-a and -d sequences displayed higher affinity to Rci protein. These results suggest that the differences in the affinity of Rci protein for the 19-bp repeat sequences determine the inversion frequencies of the four segments.     

Application of InterPro for the functional classification of the proteins of fish origin in SWISS-PROT and TrEMBL

InterPro (http://www.ebi.ac.uk/interpro/) is an integrated documentation resource for protein families, domains and sites, developed initially as a means of rationalizing the complementary efforts of the PROSITE, PRINTS, Pfam and ProDom database projects. It is a useful resource that aids the functional classification of proteins. Almost 90% of the actinopterygii protein sequences from SWISS-PROT and TrEMBL can be classified using InterPro. Over 30% of the actinopterygii protein sequences currently in SWISS-PROT and TrEMBL are of mitochondrial origin, the majority of which belong to the cytochrome b/b6 family. InterPro also gives insights into the domain composition of the classified proteins and has applications in the functional classification of newly determined sequences lacking biochemical characterization, and in comparative genome analysis. A comparison of the actinopterygii protein sequences against the sequences of other eukaryotes confirms the high representation of eukaryotic protein kinase in the organisms studied. The comparisons also show that, based on InterPro families, the trans-species evolution of MHC class I and II molecules in mammals and teleost fish can be recognized.