Bacterial Porin Disrupts Mitochondrial Membrane Potential and Sensitizes Host Cells to Apoptosis

The bacterial PorB porin, an ATP-binding β-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (ΔΨ_m). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of β-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of ΔΨ_m. The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce ΔΨ_m loss and apoptosis, demonstrating that dissipation of ΔΨ_m is a requirement for cell death caused by neisserial infection.     

Crystallographic analysis of Neisseria meningitidis PorB extracellular loops potentially implicated in TLR2 recognition

Among all Neisseriae species, Neisseria meningitidis and Neisseria gonorrhoeae are the only human pathogens, causative agents of bacterial meningitis and gonorrhoea, respectively. PorB, a pan-Neisseriae trimeric porin that mediates diffusive transport of essential molecules across the bacterial outer membrane, is also known to activate host innate immunity via Toll-like receptor 2 (TLR2)-mediated signaling. The molecular mechanism of PorB binding to TLR2 is not known, but it has been hypothesized that electrostatic interactions contribute to ligand/receptor binding. Strain-specific sequence variability in the surface-exposed loops of PorB which are potentially implicated in TLR2 binding, may explain the difference in TLR2-mediated cell activation in vitro by PorB homologs from the commensal Neisseriae lactamica and the pathogen N. meningitidis. Here, we report a comparative structural analysis of PorB from N. meningitidis serogroup B strain 8765 (63% sequence homology with PorB from N. meningitidis serogroup W135) and a mutant in which amino acid substitutions in the extracellular loop 7 lead to significantly reduced TLR2-dependent activity in vitro. We observe that this mutation both alters the loop conformation and causes dramatic changes of electrostatic surface charge, both of which may affect TLR2 recognition and signaling.     

Interspecies recombination In nature: a meningococcus that has acquired a gonococcal PIB porin

A. vaginal isolate of Neisseria has been reported to resemble Neisseria meningitidis in biochemical characteristics but to react with serological reagents that are specific to the PI porin from Neisseria gonorrhoeae. We have confirmed that this isolate has the biochemical attributes of a meningococcus and have shown that it clusters among meningococcal Isolates on a dendrogram based on isoenzyme variation within housekeeping enzymes from populations of N. meningitidis and N. gonorrhoeae. Furthermore, the sequences of the fbp and adk genes were typical of those of N. meningitidis and were distinct from those of N. gonorrhoeae. However, the porB gene was very similar to the por genes of N. gonorrhoeae isolates that express the PIB class of outer-membrane porin (differing from one gonococcal por allele at only a single nucleotide site), and was clearly distinct from the porB genes of N. meningitidis. The isolate therefore appears to be a typical meningococcus, except that its porB gene has been replaced with the por gene from a gonococcus.     

Heterogeneity in non‐epitope loop sequence and outer membrane protein complexes alters antibody binding to the major porin protein PorB in serogroup B Neisseria meningitidis

PorB is a well‐characterized outer membrane protein that is common among Neisseria species and is required for survival. A vaccine candidate, PorB induces antibody responses that are directed against six variable surface‐exposed loops that differ in sequence depending on serotype. Although Neisseria meningitidis is naturally competent and porB genetic mosaicism provides evidence for strong positive selection, the sequences of PorB serotypes commonly associated with invasive disease are often conserved, calling into question the interaction of specific PorB loop sequences in immune engagement. In this report, we provide evidence that antibody binding to a PorB epitope can be altered by sequence mutations in non‐epitope loops. Through the construction of hybrid PorB types and PorB molecular dynamics simulations, we demonstrate that loops both adjacent and non‐adjacent to the epitope loop can enhance or diminish antibody binding, a phenotype that correlates with serum bactericidal activity. We further examine the interaction of PorB with outer membrane‐associated proteins, including PorA and RmpM. Deletion of these proteins alters the composition of PorB‐containing native complexes and reduces antibody binding and serum killing relative to the parental strain, suggesting that both intramolecular and intermolecular PorB interactions contribute to host adaptive immune evasion.     

The class 3 outer membrane protein (PorB) of Neisseria meningitidis: gene sequence and homology to the gonococcal porin PIA

The class 3 protein (PorB) is an important component of the meningococcal outer membrane. The structural gene (porB) encoding the class 3 protein has been cloned using primers suitable for the amplification of the corresponding chromosomal fragment by the polymerase chain reaction (PCR). The complete nucleotide sequence was determined and predicts a mature protein of 310 amino acids, preceded by a signal peptide of 19 residues. The predicted protein sequence of the class 3 protein exhibits essential structural homology to the gonococcal porin PIA. The class 3 protein encoding gene was expressed in Escherichia coli under the control of an inducible promoter.     

A putative alpha-helical porin from Corynebacterium glutamicum

The cell wall of Corynebacterium glutamicum contains a mycolic acid layer, which is a protective nonpolar barrier similar to the outer membrane of Gram-negative bacteria. The exchange of material across this barrier requires porins. Porin B (PorB) is one of them. Recombinant PorB has been produced in Escherichia coli, purified, crystallized and analyzed by X-ray diffraction, yielding 16 independent molecular structures in four different crystal forms at resolutions up to 1.8 Å. All 16 molecules have the same globular core, which consists of 70 residues forming four α-helices tied together by a disulfide bridge. The 16 structures vary greatly with respect to the 29 residues in the N- and C-terminal extensions. Since corynebacteria belong to the group of mycolata that includes some prominent human pathogens, the observed structure may be of medical relevance. Due to the clearly established solid structure of the core, the native porin has to be oligomeric, and the reported structure is one of the subunits. An α-helical porin in a bacterial outer envelope is surprising because all presently known structures of such porins consist of β-barrels. Since none of the four crystal packing arrangements was compatible with an oligomeric membrane channel, we constructed a model of such an oligomer that was consistent with all available data of native PorB. The proposed model is based on the required polar interior and nonpolar exterior of the porin, on a recurring crystal packing contact around a 2-fold axis, on the assumption of a simple C_n symmetry (a symmetric arrangement around an n-fold axis), on the experimentally established electric conductivity and anion selectivity and on the generally observed shape of porin channels.     

Mutations Affecting the α Subunit of Bordetella pertussis RNA Polymerase Suppress Growth Inhibition Conferred by Short C-Terminal Deletions of the Response Regulator BvgA

The effects of short deletions of the C terminus of the BvgA response regulator protein of the BvgAS two-component system were examined in Bordetella pertussis. When present as a single copy in the chromosome, deletions removing as few as two amino acids conferred a completely Bvg⁻ phenotype. When provided in trans, on the broad-host-range plasmid pRK290, under the control of the native bvgAS promoter, deletions of two or three amino acids conferred a profound growth inhibition which was dependent on the integrity and activity of the wild-type chromosomal bvgAS locus. It is proposed that this phenotype was the result of an inappropriate interaction of the mutant BvgA protein with the RNA polymerase enzyme, specifically the α subunit. Mutant strains in which this growth inhibition was relieved were isolated and characterized. Although most of the suppressor mutations affected either the mutant plasmid copy or the wild-type chromosomal bvg locus, three mutations which affected the α subunit of B. pertussis RNA polymerase were also isolated. Two of these resulted in increased levels of the α subunit, and one caused a substitution of glycine for the aspartic acid residue at position 171, in the N-terminal domain. All three mutations also resulted in a differential phenotype in that expression of fha was essentially normal, but expression of ptx was greatly reduced.     

Cloning and Characterization of a Rhizobium leguminosarum Gene Encoding a Bacteriocin with Similarities to RTX Toxins

A 3-kb region containing the determinant for bacteriocin activity from Rhizobium leguminosarum 248 was isolated and characterized by Tn5 insertional mutagenesis and DNA sequencing. Southern hybridizations showed that this bacteriocin was encoded on the plasmid pRL1JI and that homologous loci were not found in other unrelated R. leguminosarum strains. Tn5 insertional mutagenesis showed that mutations in the C-terminal half of the bacteriocin open reading frame apparently did not abolish bacteriocin activity. Analysis of the deduced amino acid sequence revealed that, similarly to RTX proteins (such as hemolysin and leukotoxin), this protein contains a characteristic nonapeptide repeated up to 18 times within the protein. In addition, a novel 19- to 25-amino-acid motif that occurred every 130 amino acids was detected. Bacteriocin bioactivity was correlated with the presence of a protein of approximately 100 kDa in the culture supernatants, and the bacteriocin bioactivity demonstrated a calcium dependence in both R. leguminosarum and Sinorhizobium meliloti. A mutant of strain 248 unable to produce this bacteriocin was found to have a statistically significant reduction in competitiveness for nodule occupancy compared to two test strains in coinoculation assays. However, this strain was unable to compete any more successfully with a third test strain, 3841, than was wild-type 248.     

Influence of Culture Conditions on Expression of the 40-Kilodalton Porin Protein of Vibrio anguillarum Serotype O2

Vibrio anguillarum serotype O2 strains express a 40-kDa outer membrane porin protein. Immunoblot analysis revealed that antigenic determinants of the V. anguillarum O2 40-kDa porin were conserved within bacterial species of the genus Vibrio. The relative amounts of the V. anguillarum O2 40-kDa porin were enhanced by growth of V. anguillarum O2 in CM9 medium containing 5 to 10% sucrose or 0.1 to 0.5 M NaCl. In contrast, the levels of the porin were significantly reduced when cells were grown at 37°C, and a novel 60-kDa protein was also observed. However, the osmolarity or ionic concentration of the growth medium did not influence expression of the 60-kDa protein. Growth in medium containing greater than 0.6 mM EDTA reduced production of the V. anguillarum O2 40-kDa porin and enhanced levels of a novel 19-kDa protein. Thus, expression of the V. anguillarum O2 40-kDa porin was osmoregulated and possibly coregulated by temperature. The N-terminal amino acid sequence of the V. anguillarum O2 40-kDa protein and the effect of environmental factors on the cellular levels of the porin suggested that the V. anguillarum O2 40-kDa porin was functionally similar to the OmpC porin of Escherichia coli. However, pore conductance assays revealed that the V. anguillarum O2 40-kDa porin was a general diffusion porin with a pore size in the range of that of the OmpF porin of E. coli.     

Biochemical and Genetic Characterization of Mundticin KS, an Antilisterial Peptide Produced by Enterococcus mundtii NFRI 7393

Mundticin KS, a bacteriocin produced by Enterococcus mundtii NFRI 7393 isolated from grass silage in Thailand, is active against closely related lactic acid bacteria and the food-borne pathogen Listeria monocytogenes. In this study, biochemical and genetic characterization of mundticin KS was done. Mundticin KS was purified to homogeneity by ammonium sulfate precipitation, sequential ion-exchange chromatography, and solid-phase extraction. The gene cluster (mun locus) for mundticin KS production was cloned, and DNA sequencing revealed that the mun locus consists of three genes, designated munA, munB, and munC. The munA gene encodes a 58-amino-acid mundticin KS precursor, munB encodes a protein of 674 amino acids involved in translocation and processing of the bacteriocin, and munC encodes a mundticin KS immunity protein of 98 amino acids. Amino acid and nucleotide sequencing revealed the complete, unambiguous primary structure of mundticin KS; mundticin KS comprises a 43-amino-acid peptide with an amino acid sequence similar to that of mundticin ATO6 produced by E. mundtii ATO6. Mundticin KS and mundticin ATO6 are distinguished by the inversion of the last two amino acids at their respective C termini. These two mundticins were expressed in Escherichia coli as recombinant peptides and found to be different in activity against certain Lactobacillus strains, such as Lactobacillus plantarum and Lactobacillus curvatus. Mundticin KS was successfully expressed by transformation with the recombinant plasmid containing the mun locus in heterogeneous hosts such as E. faecium, L. curvatus, and Lactococcus lactis. Based on our results, the mun locus is located on a 50-kb plasmid, pML1, of E. mundtii NFRI 7393.     

Analysis of outer membrane porin complexes of Neisseria meningitidis in wild‐type and specific knock‐out mutant strains

The structure of the porin complexes of Neisseria meningitidis was assessed in the vaccine strain H44/76 and its homologous mutants lacking the main porins (PorA and PorB) and other outer membrane (OM) components (RmpM and FetA). The analysis using 1‐D blue native (BN) electrophoresis, 2‐D BN/SDS‐PAGE and 2‐D diagonal electrophoresis, followed by LC/MS‐MS (for 1‐D gels) or MALDI‐TOF (for 2‐D gels) revealed at least six porin complexes in the wild‐type strain with molecular masses (MW) ranging from 145 to 195 kDa and variable composition: The two higher MW complexes are formed by PorA, PorB and RmpM, the following three are formed by PorA and PorB, and the lower MW one is formed by only PorB. Complexes in the mutants lacking either PorA, PorB or RmpM, but not those in the mutant lacking FetA, were alterered respect to those in the wild‐type strain. The most evident alteration was seen in the mutant lacking PorB, in which PorA formed only a high MW complex (≈?800 kDa). Our results suggest that PorA and PorB could form a ‘basic’ template for the transportation systems in the OM of the meningococci. Other proteins (such as RmpM) could be transiently associated to the porin complexes, depending on the specific tranport needs at different stages of the meningococcal life cycle, resulting in a dynamic net of pores of variable composition.     

Monoclonal antibodies specific to porin of Haemophilus influenzae type b: localization of their cognate epitopes and tests of their biological activities

The major outer membrane protein of Haemophilus influenzae type b (Hib) is porin (Mr 38,000, 341 amino acids). To identify antigenic determinants on Hib porin that might be exposed at the bacterial cell surface, seven mouse monoclonal anti-Hib porin antibodies were generated. The monoclonal antibodies were tested for their binding to intact cells by flow cytometry; all but one bound to the cell surface. Digestions of Hib porin with cyanogen bromide, hydroxylamine or trypsin generated fragments, the identities of which were confirmed by microsequencing of the amino termini. Following electrophoresis and immunoblotting of the fragments, the specificities of the monoclonal antibodies for their cognate sequences were determined. The porin gene ompP2 was expressed in the baculovirus expression vector system; the recombinant porin was recognized by all of the monoclonal antibodies. Deletions were created by omega mutagenesis of ompP2, generating proteins truncated after amino acids 139, 174, 182, and 264. These deletion proteins were tested for reactivities with the monoclonal antibodies, thereby establishing the boundaries of three antigenic determinants that were recognized by the monoclonals: domain (i), amino acids 104-139; domain (ii) amino acids 162-174; and domain (iii), amino acids 267-341. The biological activities of monoclonal antibodies that were representative of these three classes were tested for their bactericidal activity in complement-mediated lysis of whole cells. The monoclonal antibodies were also tested for their immunoprotective properties in the infant rat model of bacteraemia. Although the monoclonal antibodies were surface-binding, they were neither bactericidal nor protective.     

Loss-of-function mutations identified in the Helical domain of the G protein α-subunit,Gα2, of Dictyostelium discoideum

The guanine nucleotide binding regulatory proteins (G proteins) play essential roles in a wide variety of physiological processes, such as vision, hormone responses, olfaction, immune response, and development. The heterotrimeric G proteins consist of α-, β-, and γ-subunits and act as molecular switches to relay information from transmembrane receptors to intracellular effectors. The switch mechanism is a function of the inherent GTPase activity of the α-subunit. The α-subunit is comprised of two domains, the GTPase domain and the Helical domain. The GTPase domain performs all of the known α-subunit functions while little is know about the role of the Helical domain. To gain a better understanding of α-subunit function, we performed a screen for loss-of-function mutations, using the Gα2-subunit of Dictyostelium. Gα2 is essential for the developmental life cycle of Dictyostelium. It is known that the loss of Gα2 function results in a failure of cells to enter the developmental phase, producing a visibly abnormal phenotype. This allows the easy identification of amino acids essential to Gα2 function. A library of random point mutations in the gα2 cDNA was constructed using low fidelity polymerase chain reaction (PCR). The library was then expressed in a gα2 null cell line and screened for loss-of-function mutations. Mutations were identified in isolated clones by sequencing the gα2 insert. To date, sixteen single amino acids changes have been identified in Gα2 which result in loss-of-function. Of particular interest are seven mutations found in the Helical domain of the α-subunit. These loss-of-function mutations in the α-subunit Helical domain may provide important insight into its function.     

Helicobacter pylori porCDAB and oorDABC Genes Encode Distinct Pyruvate:Flavodoxin and 2-Oxoglutarate:Acceptor Oxidoreductases Which Mediate Electron Transport to NADP

Helicobacter pylori, a major cause of human gastric disease, is a microaerophilic bacterium that contains neither pyruvate nor 2-oxoglutarate dehydrogenase activity. Previous studies (N. J. Hughes, P. A. Chalk, C. L. Clayton, and D. J. Kelly, J. Bacteriol. 177:3953–3959, 1995) have indicated that the major routes for the generation of acetyl coenzyme A (acetyl-CoA) and succinyl-CoA are via pyruvate:flavodoxin oxidoreductase (POR) and 2-oxoglutarate:acceptor oxidoreductase (OOR), respectively. The purified POR is a heterotetrameric protein, with subunits of 48 (PorA), 36 (PorB), 24 (PorC), and 14 (PorD) kDa. In this study OOR has been purified, and it is similarly composed of polypeptides of 43 (OorA), 33 (OorB), 24 (OorC), and 10 (OorD) kDa. Both POR and OOR are oxygen labile and are likely to be major contributors to the microaerophilic phenotype of H. pylori. Unlike POR, OOR was unable to use a previously identified flavodoxin (FldA) as an electron acceptor. Although the purified enzymes were unable to reduce NAD(P), electrons from both pyruvate and 2-oxoglutarate could reduce NADP in cell extracts, consistent with a role for these oxidoreductases in the provision of NADPH as a respiratory electron donor. The H. pylori por, oor, and fldA genes were cloned and sequenced. The deduced por gene products showed significant sequence similarity to archaeal four-subunit 2-oxoacid:acceptor oxidoreductases. However, the amino acid sequences of OorA and -B were more closely related to that of the two-subunit POR of the aerobic halophile Halobacterium halobium. Both porD and oorD encode integral ferredoxin-like subunits. POR and OOR are probably essential enzymes in H. pylori, as insertion inactivation of porB and oorA appeared to be lethal.     

三角鲂和长春鳊肌肉营养成分分析与品质评价

用常规方法测定、分析了三角鲂(Megalobrama tarminalis)和长春鳊(Parabramis pekinensis肌肉中营养成分组成与含量.结果显示,三角鲂肌肉蛋白质、脂肪含量分别为18.19％和3.06％,长春鳊肌肉蛋白质、脂肪含量分别为19.38％和2.89％.三角鲂和长春鳊肌肉中均检测出18种氨基酸,其中包括了8种人体必需氨基酸.三角鲂肌肉中氨基酸总量为76.27％,其中,8种人体必需氨基酸含量为32.17％,占氨基酸总量的42.18％;长春鳊肌肉中氨基酸总量为77.60％,其中,8种人体必需氨基酸含量为31.70％,占氨基酸总量的40.85％.必需氨基酸的构成比例基本符合FAO/WHO的标准.三角鲂肌肉中限制性氨基酸主要为甲硫氨酸加胱氨酸,必需氨基酸指数为63.55,4种呈味氨基酸为氨基酸总量的32.81％;长春鳊肌肉中限制性氨基酸主要为色氨酸,必需氨基酸指数为66.81,4种呈味氨基酸为氨基酸总量的33.80％.脂肪酸中二十碳五烯酸(EPA)与二十二碳六烯酸(DHA)含量均较高,三角鲂为7.96％,长春鳊为3.11％.矿物元素比值合理.以上分析表明,三角鲂和长春鳊均为营养价值、经济价值都较高的优质鱼类,相比而言,三角鲂肌肉脂肪、脂肪酸含量和质量更优,而长春鳊肌肉在蛋白质、氨基酸组成与含量方面更优.     

Conditions affecting protein synthesis in amphibian oocytes.

The endogenous protein synthesis of Xenopus laevis and Calyptocephalella caudiverbera oocytes was studied by measuring the incorporation into acid-precipitable material of radioactive amino acids placed in the extracellular medium. Large differences of incorporation into protein were observed by using different labeled amino acids. For example, it was found that radioactive aspartic acid or glutamic acid was very poorly incorporated at concentrations under 0.1 mm. These differences are due to differences in uptake constants and in the internal pools of free amino acids which are very large for the acidic amino acids. Both types of oocytes behaved similarly with respect to magnesium ion concentration, temperature optimum and inhibitors of protein synthesis. They differed however in sensitivity to pH since Xenopus laevis oocyte protein synthesis was twofold higher at pH 8.5 than at pH 7 while Calyptocephalella caudiverbera oocytes showed no difference. Isolation of oocyte germinal vesicles allowed a study of the entrance of newly synthesized protein into the cell nuclei.     

Identification of a cation transport pathway in Neisseria meningitidis PorB

Neisseria meningitidis is the main causative agent of bacterial meningitis. In its outer membrane, the trimeric Neisserial porin PorB is responsible for the diffusive transport of essential hydrophilic solutes across the bilayer. Previous molecular dynamics simulations based on the recent crystal structure of PorB have suggested the presence of distinct solute translocation pathways through this channel. Although PorB has been electrophysiologically characterized as anion‐selective, cation translocation through nucleotide‐bound PorB during pathogenesis is thought to be instrumental for host cell death. As a result, we were particularly interested in further characterizing cation transport through the pore. We combined a structural approach with additional computational analysis. Here, we present two crystal structures of PorB at 2.1 and 2.65 Å resolution. The new structures display additional electron densities around the protruding loop 3 (L3) inside the pore. We show that these electron densities can be identified as monovalent cations, in our case Cs⁺, which are tightly bound to the inner channel. Molecular dynamics simulations reveal further ion interactions and the free energy landscape for ions inside PorB. Our results suggest that the crystallographically identified locations of Cs⁺ form a cation transport pathway inside the pore. This finding suggests how positively charged ions are translocated through PorB when the channel is inserted into mitochondrial membranes during Neisserial infection, a process which is considered to dissipate the mitochondrial transmembrane potential gradient and thereby induce apoptosis. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Effect of growth conditions on accumulation of internal nitrate,ammonium, amino acids,and protein in three marine diatoms

The capacity of marine phytoplankton to change their cellular content of nitrate, ammonium, amino acids, and protein in response to different growth conditions was systematically investigated. Cellular concentrations of these compounds were measured in N-starved, N-deficient, and N-sufficient Skeletonema costatum (Grev.) Cleve and in N-deficient Chaetoceros debilis Cleve and Thalassiosira gravida Cleve, both before and after the addition of a pulse of nitrogen.N-sufficient Skeletonema costatum contains high concentrations of protein, large persistent pools of amino acids, and, if it is growing on nitrate, sizeable amounts of nitrate. As it becomes N-starved, the total cellular nitrogen decreases, the internal nitrate and amino acids become entirely depleted, and the protein content is drastically reduced. After nitrogen additions to N-deficient and N-starved cultures, transient pools of unassimilated nitrogen form which can account for a large fraction of newly taken up nitrogen. The size and kind of pool which accumulates is determined by the preconditioning of the cells, the nitrogen compound which is added, and the species identity. The pools which form in S. costatum indicate that nitrate reduction is the slowest step in nitrogen assimilation, the synthesis of protein from amino acids is the next slowest, and the incorporation of ammonium into amino acid is the fastest. However, the rate limiting steps may vary between diatom species.For the first time, measurements of the variation in cellular nitrogen compounds over a wide range of environmental conditions reveal the ability of some phytoplankton to buffer the effects of a changing, and sometimes growth-limiting, nitrogen supply. They accomplish this by utilizing stored internal nitrogen for growth when the external supply is low and by quickly storing unassimilated nitrogen when the external supply is suddenly increased beyond their ability to immediately assimilate it. The accumulation of large pools of unassimilated nitrogen compounds can explain the often observed difference between nitrogen uptake rates and growth rates.