ompA Sequences Research Articles

Sensitivity of Chlamydia suis to cathelicidin peptides

Nine Chlamydia suis isolates, obtained from pigs with conjunctivitis, were molecularly characterized by ompA sequencing and their in vitro susceptibility to six cathelicidin peptides (SMAP-29, BAC-7, BMAP-27, BMAP-27, BMAP-28, PG-1, LL-37) determined in cell culture. SMAP-29 was the most active peptide, reducing the intracellular inclusion number by ≥50% at a concentration of 10 μg/ml (3 μM) in six of the nine isolates tested. Three molecularly identical isolates were insensitive at a concentration as high as 80 μg/ml (25 μM). Of the remaining cathelicidin peptides tested, BAC-7 and BMAP-27 were active against six C. suis isolates at a concentration of 80 μg/ml (25 and 26 μM, respectively). Cathelicidins LL-37 and PG-1 did not show any anti-chlamydial activity at 80 μg/ml.

The Presence of N-Terminal Secretion Signal Sequences Leads to Strong Stimulation of the Total Expression Levels of Three Tested Medically Important Proteins during High-Cell-Density Cultivations of Escherichia coli

Genetic optimizations to achieve high-level production of three different proteins of medical importance for humans, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon alpha 2b (IFN-alpha2b), and single-chain antibody variable fragment (scFv-phOx), were investigated during high-cell-density cultivations of Escherichia coli. All three proteins were poorly expressed when put under control of the strong Pm/xylS promoter/regulator system, but high volumetric yields of GM-CSF and scFv-phOx (up to 1.7 and 2.3 g/liter, respectively) were achieved when the respective genes were fused to a translocation signal sequence. The choice of signal sequence, pelB, ompA, or synthetic signal sequence CSP, displayed a high and specific impact on the total expression levels for these two proteins. Data obtained by quantitative PCR confirmed relatively high in vivo transcript levels without using a fused signal sequence, suggesting that the signal sequences mainly stimulate translation. IFN-alpha2b expression remained poor even when fused to a signal sequence, and an alternative IFN-alpha2b coding sequence that was optimized for effective expression in Escherichia coli was therefore synthesized. The total expression level of this optimized gene remained low, while high-level production (0.6 g/liter) was achieved when the gene was fused to a signal sequence. Together, our results demonstrate a critical role of signal sequences for achieving industrial level expression of three human proteins in E. coli under the conditions tested, and this effect has to our knowledge not previously been systematically investigated.

Development of a Chlamydophila psittaci species-specific and genotype-specific real-time PCR

A Chlamydophila psittaci species-specific real-time PCR targeting the rDNA ribosomal spacer was developed as well as a genotype-specific real-time PCR targeting the Cp. psittaci outer membrane protein A (ompA) gene. The SYBR Green-based species-specific real-time PCR detected Cp. psittaci genotypes A to F, and the recently discovered E/B genotype. The genotype-specific real-time PCR could easily distinguish genotypes C, D, F by use of TaqMan probes. Genotypes A, B and E could not be distinguished from each other by simply using TaqMan probes. For this purpose, non-fluorescent competitor oligonucleotides, had to be used next to the TaqMan probes. Genotype E/B could only be detected by use of a minor groove binder (MGB) probe. Both real-time PCR assays allowed reproducible, sensitive (10 rDNA or ompA copies/microL DNA extract) and specific detection of Cp. psittaci DNA. The genotype-specific real-time PCR was compared to ompA sequencing and ompA restriction fragment length polymorphism (RFLP) analysis using five Cp. psittaci field isolates (99, 61/8, 7344/2, 8615/1 and 7778B15) each consisting of two different genotypes. The currently developed real-time PCR assays were used in a case study on a veterinary school and a turkey farm. In the veterinary school, Cp. psittaci genotypes D, E/B and F infection were detected in all five groups of turkeys, and one veterinarian who was taking care of all these turkeys. On the turkey farm, the presence of two Cp. psittaci genotype B infection waves was demonstrated in one randomly selected turkey, the first wave at the age of 6 weeks, and the second at the age of 12 weeks.

Structural Determinants of Transmembrane β-Barrels.

The recognition of β-barrel membrane proteins based on their sequence is more challenging than the recognition of α-helical membrane proteins. This goal could benefit from a better understanding of the physical determinants of transmembrane β-barrel structure. To that end, we first extend the IMM1 implicit membrane model in a way that allows the modeling of membrane proteins with an internal aqueous pore. The new model (IMM1-pore) gives stable molecular dynamics trajectories for three β-barrel membrane proteins of different sizes and negative water-to-membrane transfer energies of reasonable magnitude. It also discriminates the correct fold for a pair of 10-stranded and 12-stranded transmembrane β-barrels. We then consider a pair of β-barrel proteins: OmpA, which is a membrane β-barrel with hydrophobic residues on the exterior and polar residues in the interior, and retinol binding protein, which is a water soluble protein with polar residues on the exterior and hydrophobic residues in the interior. By threading the sequence of one onto the structure of the other we make two pairs of structures for each sequence, one native and the other a decoy, and evaluate their energy. The energy function discriminates the correct structure. By decomposing the energy into residue contributions we examine which features of each sequence make it fold into one or the other structure. It is found that for the OmpA sequence the largest contribution to stability comes from interactions between polar residues in the interior of the barrel. The major factor that prevents the retinol binding protein sequence from adopting a transmembrane fold is the presence of polar/charged residues at the edges of the putative transmembrane β-strands as well as the less favorable interior polar residue interactions. These results could help design simplified scoring functions for fold recognition and structure prediction of transmembrane β-barrels.

Genetic variation of the ompA and 16S rRNA genes of Riemerella anatipestifer

The genetic diversity of the 16S rRNA and ompA genes of Riemerella anatipestifer was investigated. A 16S rRNA gene-based PCR was able to amplify all 18 Taiwanese strains and 10 reference strains. The identity of 16S rRNA sequence of these strains and seven other sequences retrieved from GenBank was 95.0–100.0%. The percentage identity of the ompA sequence of the 15 Taiwanese strains and eight reference strains amplified in this study and two other sequences retrieved from GenBank was 88.1–100.0%. Phylogenetic analysis based on the 16S rRNA gene showed that all the R. anatipestifer strains fell into a single cluster. It is concluded that the 16S rRNA gene-based PCR is suitable for the screening of R. anatipestifer infections. Phylogenetic analysis of the ompA of R. anatipestifer resulted in three different clusters, while seven clusters were found when the derived amino acid sequence was the basis of analysis. No apparent cluster was found using the criteria of host, isolate serotype, the year or location of isolation.

Structure--function studies of the recombinant protein bioemulsifier AlnA.

Acinetobacter radioresistens KA53 produces an extracellular bioemulsifier, referred to as alasan. The surface active component of alasan is a 35.77 kDa protein AlnA. Although AlnA and the Escherichia coli outer membrane protein A (OmpA) have a high amino acid sequence homology, E. coli OmpA has no emulsifying activity. Comparison of the amino acid sequences of AlnA and E. coli OmpA revealed four hydrophobic regions in AlnA that were absent in E. coli OmpA. Deletions and substitutions (with the homologous OmpA sequence) were constructed in each of the four hydrophobic regions of AlnA by site-directed polymerase chain reaction (PCR) mutagenesis, using the overlap PCR method. Analysis of the emulsifying activities of the mutated AlnA molecules demonstrated that all four hydrophobic regions were necessary for emulsifying activity. However, most of the inactive mutated proteins still adhered avidly to hexadecane. These findings indicate that in addition to binding to hydrocarbons, the protein emulsifier must form a specific structure on the surface of the hydrocarbon that prevents coalescence of oil droplets. This is the first structure-function study of a protein bioemulsifer.

OmpA genotypic evidence for persistent ocular Chlamydia trachomatis infection in Tanzanian village women

BACKGROUND. Trachoma is still a significant problem in the developing world. Adult women are at higher risk of developing scarring and trichiasis, the potentially blinding sequelae, compared to men. In part, the higher risk may be due to more frequent infections in women because of their frequent contact with children, the main reservoir of C. trachomatis infection. However, other factors associated with infection, particularly constant infection, in adult women need to be identified. METHODS. A group of 118 women who were infected with C. trachomatis and 118 women who were not infected, but of similar age and trachoma status, were identified in 1996 from a population-based sample of women age 16 and older from eleven villages in Kongwa, Tanzania. This group of 236 was re-contacted three years later to ascertain trachoma status and determine infection status using polymerase chain reaction-enzyme immunoassay (PCR-EIA). Positive samples at both time points were examined for serovar and genotype shift, using ompA sequencing information. RESULTS. Of the original 236 women, 165 (70%) completed exams in 1999. Fifty-eight (35%) of the 165 women were excluded from this analysis because they received antibiotic treatment for trachoma in the six months prior to the second exam. Infection at baseline was the most important predictor of infection three years later (Age-adjusted odds ratio (95% confidence interval) 6.6 (1.8–24.4)). A total of 17 women (16%) were infected at the two examinations, and of the 15 for whom genotyping could be done, 11 (73%) were infected with the same ompA genotype at both time points. Chronically infected women were more likely to have trichiasis, scarring, and active trachoma at baseline than those never infected or infected only once. Only 41% of the chronically infected women were living in houses with infected pre-school children, but 24% were in houses with no children. Four of ten women with trichiasis developed incident corneal opacity over the three years. CONCLUSIONS. The data provide evidence for persistence of infection in a sub-group of women. The strongest predictor of infection at follow-up was baseline infection, and most were infected with the same genotype at both time points. For women with persistent infection, at least half were either not living with children or not living with infected children, suggesting that continual re-exposure from a close family member was less likely. Chronic infection is likely related to both exposure and immunological factors, and these need to be further identified. Inclusion of women in community-based treatment programs, regardless of whether a child is present in the house, is likely to be important in preventing the progression of inflammatory trachoma and scarring to trichiasis.

SRP-dependent co-translational targeting and SecA-dependent translocation analyzed as individual steps in the export of a bacterial protein.

Recently it has been recognized that the signal recognition particle (SRP) of Escherichia coli represents a specific targeting device for hydrophobic inner membrane proteins. It has remained unclear, however, whether the bacterial SRP functions in concert with SecA, which is required for the translocation of secretory proteins across the inner membrane. Here, we have analyzed a hybrid protein constructed by fusing the signal anchor sequence of an SRP-dependent inner membrane protein (MtlA) to the mature part of an exclusively SecA-requiring secretory protein (OmpA). We show that the signal anchor sequence of MtlA confers the novel properties onto nascent chains of OmpA of being co-translationally recognized and targeted to SecY by SRP. Once targeted to SecY, ribosome-associated nascent chains of the hybrid protein, however, remain untranslocated unless SecA is present. These results indicate that SRP and SecA cooperate in a sequential, non-overlapping manner in the topogenesis of those membrane proteins which, in addition to a signal anchor sequence, harbor a substantial hydrophilic domain to be translocated into the periplasm.

Short report: isolation and identification of two spotted fever group rickettsial strains from patients in Catalonia, Spain.

Two rickettsial strains, 16B (previously isolated) and FB1, were isolated from blood from patients with Mediterranean spotted fever in Catalonia, Spain. These are the only 2 human rickettsial isolates of the spotted fever group obtained so far in Spain. These strains were identified by the polymerase chain reaction and sequence analysis of a fragment of the outer membrane protein A (ompA) gene. The partial ompA sequence was found to be 100% identical with that of Rickettsia conorii (Malish 7 strain) for both strains. These results confirm the presence of R. conorii in Catalonia, despite the fact that in a previous study, no R. conorii were isolated, but a new rickettsial strain of the spotted fever group (Bar29) was isolated from dog ticks (Rhipicephalus sanguineus) in Catalonia. Further studies are necessary to get a better knowledge of the epidemiology of rickettsiae in Catalonia.

NewRickettsiaein Ticks Collected in Territories of the Former Soviet Union

Dermacentor nuttallii from Siberia, Rhipicephalus sanguineus from Crimea, and Rh. pumilio from the Astrakhan region were infected with Rickettsia sibirica (12%), R. conorii (8%), and the Astrakhan fever agent (3%), respectively. Three new Rickettsiae of the R. massiliae genogroup were identified in ticks by 16S rDNA, gltA, and ompA sequencing.

Expression of ribonucleolytic toxin restrictocin in Escherichia coli: purification and characterization

Restrictocin is a toxin produced by the fungus Aspergillus restrictus. The DNA coding for restrictocin was isolated from the host by polymerase chain reaction and cloned into a T7 promoter-based expression vector. The protein was overproduced in Escherichia coli and remained insoluble in the cell in the form of inclusion bodies. Recombinant restrictocin was purified in large amounts, by a simple denaturation-renaturation protocol involving a redox system, with typical yields of 45 mg/l of original culture. Restrictocin could be secreted into the bacterial medium using ompA, pelB and LTB signal sequences. Among the three signal sequences, ompA was found to be the most efficient in secreting the recombinant protein. The protein secreted into the extracellular medium was properly processed as evident by the amino-terminal sequencing. Recombinant restrictocin was readily purified to homogeneity from either the medium or inclusion bodies by simple chromatographic techniques and was found to be functionally as active as the native fungal protein in inhibiting the eukaryotic translation.

Cloning and nucleotide sequence of the gene coding for the major 25-kilodalton outer membrane protein of Brucella abortus.

The cloning and sequencing of the Brucella abortus major 25-kDa outer membrane protein (OMP) is reported. The 25-kDa (group 3) OMP has been proposed, on the basis of amino acid composition, to be the counterpart of OmpA (D. R. Verstraete, M. T. Creasy, N. T. Caveney, C. L. Baldwin, M. W. Blab, and A. J. Winter, Infect. Immun. 35:979-989, 1982). However, the amino acid sequence predicted from the cloned B. abortus gene did not reveal significant homology with either OmpA sequences from different members of the family Enterobacteriaceae or other known protein sequences.

Serum resistance of Neisseria gonorrhoeae. Does it thwart the inflammatory response and facilitate the transmission of infection?

N. gonorrhoeae differentially subvert the effectiveness of complement (C) and alter the inflammatory responses elicited in human infection. Disseminated (DGI) isolates typically resist killing by normal serum (are serum-resistant), inactivate more C3b (to iC3b preferentially bound via amide linkages), generate less C5a, and result in less inflammation at local sites. Pelvic inflammatory disease isolates are serum-sensitive, inactivate less C3b (while maintaining active C3b via stable amide linkages), generate more C5a, and result in more inflammation at local sites. Sialylation of SS gonococci, presumed to occur in vivo, converts them to serum-resistant, but it does not change the patterns of C3b inactivation and therefore may not affect local inflammation. IgG antibody directed against gonococcal reduction modifiable protein (Rmp) blocks C-mediated killing of N. gonorrhoeae. Anti-Rmp blocking antibodies may harbor specificity for OmpA sequences shared with other neisserial species or Enterobacteriaceae or may be directed against unique Rmp upstream cysteine loop specific sequences, or both. Preexisting antibodies directed against Rmp facilitate transmission of gonococcal infection to exposed women; exclusion of highly immunogenic Rmp antigens from vaccine candidates may be important.

Expression and secretion of staphylococcal nuclease in yeast: effects of amino-terminal sequences

Staphylococcus aureus nuclease A hybrid genes, encoding proteins OmpA-nuclease, lipo-nuclease and Pin-nuclease, were cloned downstream of the yeast GAL10 inducible promoter. OmpA-nuclease and lipo-nuclease contain the mature staphylococcal nuclease sequence preceded by the Escherichia coli OmpA and lipoprotein signal sequences, respectively, whereas Pin-nuclease lacks a defined signal sequence at its amino terminus. We found that: (a) the nuclease gene products synthesized in yeast are active, but they do not affect cell growth; (b) OmpA-nuclease and lipo-nuclease are partially processed and constitute approximately 1.0-1.5% of the yeast cell protein; (c) OmpA and lipoprotein signal sequences function similarly in secretion, allowing 35-40% of the processed nuclease to be translocated into the yeast periplasm; and (d) Pin-nuclease, which lacks hydrophobic sequences at its amino-terminus, is accumulated at a level tenfold lower than the hybrid proteins that do contain signal sequences. Nevertheless, 50% of the enzyme activity of Pin-nuclease in yeast is localized in the periplasmic space.

A novel expression vector for high-level synthesis and secretion of foreign proteins in Escherichia coli: overproduction of bovine pancreatic phospholipase A 2

A novel expression plasmid (pTO-N) has been constructed that allows for the production of large quantities of foreign proteins (or fragments thereof) in an unfused state. The vector has a strong and tightly regulated T7 10 promoter and the ompA Shine-Dalgarno (SD) sequence, followed by the ompA sequence and a cloning linker region. The mRNAs produced by the vector are protected by secondary structures at both ends of the mRNAs. The OmpA signal peptide directed the synthesized proteins into the periplasmic space of Escherichia coli. Phospholipase A 2 and prophospholipase A 2 from bovine pancreas have been produced to a high level by using this expression vector. One additional feature, which is essential for the stable maintenance of the plasmid in the E. coli expression host, BL21(DE3)[pLysS], is the shortened distance between the 5′ secondary structure sequence (immediately following the gene 10 promoter) and the SD sequence. This vector could be particularly useful for synthesis of toxins in E. coli.

Export of a protein into the outer membrane of Escherichia coli K12. Stable incorporation of the OmpA protein requires less than 193 amino-terminal amino-acid residues.

The cloned ompA gene encoding the major outer membrane protein OmpA of Escherichia coli has been shortened in vitro by exonuclease digestion from the end corresponding to the CO2H terminus of the protein. Nine derivatives were identified which still possessed substantial parts of the ompA gene and one was constructed which had suffered a small deletion early in the gene. Gene fragments encoding NH2-terminal OmpA sequences of 45, 133, 193, and 227 residues of the 325 amino acids of OmpA were examined in detail at the DNA level and for OmpA protein fragments synthesized. The latter two fragments were incorporated into the outer membrane and all known functions of the OmpA protein were expressed whereas the fragment with 133 OmpA-specific residues was not stably incorporated into this membrane. In all cases where OmpA functions were observed, an OmpA-specific polypeptide of Mr 24 000 was found in cell envelopes, regardless of the size of the residual ompA sequences and of the fused coding sequences in the vector DNA. Pulse-label experiments revealed larger initial translation products, most of which were degraded to the protein of Mr 24000. The 133-residue OmpA fragment was also detected but proved to be entirely unstable. It is argued that the OmpA protein consists of two domains and that the NH2-terminal moiety from residues 1 to about 180 represents the membrane domain of the polypeptide. Therefore, the loss of about 50, possibly less, CO2H-terminal residues from this domain suffices to interfere with stable incorporation into the outer membrane.