Membrane Subunits Research Articles

In silico identification of outer membrane protein (Omp) and subunit vaccine design against pathogenic Vibrio cholerae

Virulence-related outer membrane proteins (Omps) are expressed in bacteria (Gram-negative) such as V. cholerae and are vital to bacterial invasion in to eukaryotic cell and survival within macrophages that could be best candidate for development of vaccine against V. cholerae. Applying in silico approaches, the 3-D model of the Omp was developed using Swiss model server and validated byProSA and Procheck web server. The continuous stretch of amino acid sequences 26mer: RTRSNSGLLTWGDKQTITLEYGDPAL and 31mer: FFAGGDNNLRGYGYKSISPQDASGALTGAKY having B-cell binding sites were selected from sequence alignment after B cell epitopes prediction by BCPred and AAP prediction modules of BCPreds. Further, the selected antigenic sequences (having B-cell epitopes) were analyzed for T-cell epitopes (MHC I and MHC II alleles binding sequence) by using ProPred 1 and ProPred respectively. The epitope (9mer: YKSISPQDA) that binds to both the MHC classes (MHC I and MHC II) and covers maximum MHC alleles were identified. The identified epitopes can be useful in designing comprehensive peptide vaccine development against V. cholerae by inducing optimal immune response.

Phylogenomic Analysis of Type 1 NADH:Quinone Oxidoreductase.

We performed phylogenomic analysis of the catalytic core of NADH:quinone oxidoreductases of type 1 (NDH-1). Analysis of phylogenetic trees, as constructed for the core subunits of NDH-1, revealed fundamental differences in their topologies. In the case of four putatively homologous ion-carrying membrane subunits, the trees for the NuoH and NuoN subunits contained separate archaeal clades, whereas subunits NuoL and NuoM were characterized by multiple archaeal clades spread among bacterial branches. Large, separate clades, which united sequences belonging to different archaeal subdomains, were also found for cytoplasmic subunits NuoD and NuoB, homologous to the large and small subunits of nickel-iron hydrogenases. A smaller such clade was also shown for subunit NuoC. Based on these data, we suggest that the ancestral NDH-1 complex could be present already at the stage of the Last Universal Cellular Ancestor (LUCA). Ancestral forms of membrane subunits NuoN and NuoH and cytoplasmic subunits NuoD, NuoB, and, perhaps NuoC, may have formed a membrane complex that operated as an ion-translocating membrane hydrogenase. After the complex attained the ability to reduce membrane quinones, gene duplications could yield the subunits NuoL and NuoM, which enabled translocation of additional ions.

Phylogenomic Analysis Identifies a Sodium-Translocating Decarboxylating Oxidoreductase in Thermotogae.

Bacterial sodium-dependent decarboxylases were the first enzymes exemplifying sodium-dependent bioenergetics. These enzyme complexes couple decarboxylation of organic acids with the export of sodium ions via a special membrane subunit. In 711 representative prokaryotic genomes, we have analyzed genomic neighborhoods of the genes that code the membrane subunit of sodium decarboxylases. In representatives of Thermotogae, the operons with the gene of this subunit lack the genes of subunits that perform non-oxidative decarboxylation. Instead, these operons contain the genes of alpha- and delta-subunits of decarboxylating oxidoreductases of alpha-ketoacids. The genes of beta- and gamma-subunits of the decarboxylating oxidoreductases were found within the genomes of respective Thermotogae species as separate, two-gene operons. We suggest that the described two operons code together for sodium-translocating decarboxylating oxidoreductases capable of coupling oxidative decarboxylation of alpha-ketoacids with the export of sodium ions, which is a novel type of bioenergetic coupling.

Extracellular and Luminal pH Regulation by Vacuolar H+-ATPase Isoform Expression and Targeting to the Plasma Membrane and Endosomes

Plasma membrane vacuolar H+-ATPase (V-ATPase) activity of tumor cells is a major factor in control of cytoplasmic and extracellular pH and metastatic potential, but the isoforms involved and the factors governing plasma membrane recruitment remain uncertain. Here, we examined expression, distribution, and activity of V-ATPase isoforms in invasive prostate adenocarcinoma (PC-3) cells. Isoforms 1 and 3 were the most highly expressed forms of membrane subunit a, with a1 and a3 the dominant plasma membrane isoforms. Correlation between plasma membrane V-ATPase activity and invasiveness was limited, but RNAi knockdown of either a isoform did slow cell proliferation and inhibit invasion in vitro. Isoform a1 was recruited to the cell surface from the early endosome-recycling complex pathway, its knockdown arresting transferrin receptor recycling. Isoform a3 was associated with the late endosomal/lysosomal compartment. Both a isoforms associated with accessory protein Ac45, knockdown of which stalled transit of a1 and transferrin-transferrin receptor, decreased proton efflux, and reduced cell growth and invasiveness; this latter effect was at least partly due to decreased delivery of the membrane-bound matrix metalloproteinase MMP-14 to the plasma membrane. These data indicate that in prostatic carcinoma cells, a1 and a3 isoform populations predominate in different compartments where they maintain different luminal pH. Ac45 plays a central role in navigating the V-ATPase to the plasma membrane, and hence it is an important factor in expression of the invasive phenotype.

The N-terminal sequence of the extrinsic PsbP protein modulates the redox potential of Cyt b559 in photosystem II.

The PsbP protein, an extrinsic subunit of photosystem II (PSII) in green plants, is known to induce a conformational change around the catalytic Mn4CaO5 cluster securing the binding of Ca2+ and Cl– in PSII. PsbP has multiple interactions with the membrane subunits of PSII, but how these affect the structure and function of PSII requires clarification. Here, we focus on the interactions between the N-terminal residues of PsbP and the α subunit of Cytochrome (Cyt) b559 (PsbE). A key observation was that a peptide fragment formed of the first N-terminal 15 residues of PsbP, ‘pN15’, was able to convert Cyt b559 into its HP form. Interestingly, addition of pN15 to NaCl-washed PSII membranes decreased PSII’s oxygen-evolving activity, even in the presence of saturating Ca2+ and Cl– ions. In fact, pN15 reversibly inhibited the S1 to S2 transition of the OEC in PSII. These data suggest that pN15 can modulate the redox property of Cyt b559 involved in the side-electron pathway in PSII. This potential change of Cyt b559, in the absence of the C-terminal domain of PsbP, however, would interfere with any electron donation from the Mn4CaO5 cluster, leading to the possibility that multiple interactions of PsbP, binding to PSII, have distinct roles in regulating electron transfer within PSII.

The role of N-glycans and the C-terminal loop of the subunit rBAT in the biogenesis of the cystinuria-associated transporter.

The transport system b(0,+) mediates reabsorption of dibasic amino acids and cystine in the kidney. It is made up of two disulfide-linked membrane subunits: the carrier, b(0,+)AT and the helper, rBAT (related to b(0,+) amino acid transporter). rBAT mutations that impair biogenesis of the transporter cause typeI cystinuria. It has been shown that upon assembly, b(0,+)AT prevents degradation and promotes folding of rBAT; then, rBAT traffics b(0,+)AT from the endoplasmic reticulum (ER) to the plasma membrane. The role of the N-glycans of rBAT and of its C-terminal loop, which has no homology to any other sequence, in biogenesis of system b(0,+) is unknown. In the present study, we studied these points. We first identified the five N-glycans of rBAT. Elimination of the N-glycan Asn(575), but not of the others, delayed transporter maturation, as measured by pulse chase experiments and endoglycosidase H assays. Moreover, a transporter with only the N-glycan Asn(575) displayed similar maturation compared with wild-type, suggesting that this N-glycan was necessary and sufficient to achieve the maximum rate of transporter maturation. Deletion of the rBAT C-terminal disulfide loop (residues 673-685) prevented maturation and prompted degradation of the transporter. Alanine-scanning mutagenesis uncovered loop residues important for stability and/or maturation of system b(0,+). Further, double-mutant cycle analysis showed partial additivity of the effects of the Asn(679) loop residue and the N-glycan Asn(575) on transporter maturation, indicating that they may interact during system b(0,+) biogenesis. These data highlight the important role of the N-glycan Asn(575) and the C-terminal disulfide loop of rBAT in biogenesis of the rBAT-b(0,+)AT heterodimer.

Regulation of the primary quinone binding conformation by the H subunit in reaction centers from Rhodobacter sphaeroides.

Unlike photosystem II (PSII) in higher plants, bacterial photosynthetic reaction centers (bRCs) from Proteobacteria have an additional peripheral membrane subunit "H". The H subunit is necessary for photosynthetic growth, but can be removed chemically in vitro. The remaining LM dimer retains its activity to perform light-induced charge separation. Here we investigate the influence of the H subunit on interactions between the primary semiquinone and the protein matrix, using a combination of site-specific isotope labeling, pulsed electron paramagnetic resonance (EPR), and density functional theory (DFT) calculations. The data reveal substantially weaker binding interactions between the primary semiquinone and the LM dimer than observed for the intact bRC; the amount of electron spin transferred to the nitrogen hydrogen bond donors is significantly reduced, the methoxy groups are more free to rotate, and the spectra indicate a heterogeneous mixture of bound semiquinone states. These results are consistent with a loosening of the primary quinone binding pocket in the absence of the H subunit.

Sepsis-induced selective parvalbumin interneuron phenotype loss and cognitive impairments may be mediated by NADPH oxidase 2 activation in mice.

BackgroundSepsis-associated encephalopathy (SAE) is a diffuse brain dysfunction caused by many pathological events, including neuroinflammation and oxidative stress damage. Increasing evidence suggests that parvalbumin (PV) interneurons play a key role in the cognitive process, whereas the dysfunction of these interneurons has been implicated in a number of major psychiatric disorders. Here, we aimed to investigate whether enhanced inflammation and oxidative stress-mediated PV interneuron phenotype loss plays a role in sepsis-induced cognitive impairments.MethodsMale C57BL/6 mice were subjected to cecal ligation and puncture or sham operation. For the interventional study, the animals were chronically treated with a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, apocynin, at 5 mg/kg. The mice were euthanized at the indicated time points, and the brain tissues were harvested for determination of the PV, membrane subunit of NADPH oxidase gp91phox, and markers of oxidative stress (4-hydroxynonenal and malondialdehyde) and inflammation (tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-6, and IL-10). A separate cohort of animals was used to evaluate the behavioral alterations by the open field and fear conditioning tests. Primary hippocampal neuronal cultures were used to investigate the mechanisms underlying the dysfunction of PV interneurons.ResultsSepsis resulted in cognitive impairments, which was accompanied by selective phenotype loss of PV interneurons and increased gp91phox, 4-hydroxynonenal, malondialdehyde, IL-1β, and IL-6 expressions. Notably, these abnormalities could be rescued by apocynin treatment.ConclusionSelective phenotype loss of PV interneurons, as a result of NADPH oxidase 2 (Nox2) activation, might partly contribute to cognitive impairments in a mouse model of SAE.

GluN2B-containing NMDA receptors are upregulated in plasma membranes by the sphingosine-1-phosphate analog FTY720P

Sphingosine-1-phosphate (S1P) is a ceramide derivative serving not only as a regulator of immune properties but also as a modulator of brain functions. To better understand the mechanism underlying the effects of S1P on brain functions, we investigated the potential impact of S1P receptor (S1PR) activation on NMDA receptor subunits. We used acute rat hippocampal slices as a model system, and determined the effects of the active phosphorylated S1P analog, fingolimod (FTY720P) on various NMDA receptors. Treatment with FTY720P significantly increased phosphorylation of GluN2B-containing NMDA receptors at Tyr1472. This effect appears rather specific, as treatment with FTY720P did not modify GluN2B-Tyr1336, GluN2B-Ser1480, GluN2A-Tyr1325 or GluN1-Ser897 phosphorylation. Pre-treatment of hippocampal slices with the compounds W146 and PP1 indicated that FTY720P-induced GluN2B phosphorylation at Tyr1472 epitopes was dependent on activation of S1PR subunit 1 (S1PR1) and Src/Fyn kinase, respectively. Cell surface biotinylation experiments indicated that FTY720P-induced GluN2B phosphorylation at Tyr1472 was also associated with increased levels of GluN1 and GluN2B subunits on membrane surface, whereas no change was observed for GluN2A subunits. We finally demonstrate that FTY720P is inclined to favor Tau and Fyn accumulation on plasma membranes. These results suggest that activation of S1PR1 by FTY720P enhances GluN2B receptor phosphorylation in rat hippocampal slices, resulting in increased levels of GluN1 and GluN2B receptor subunits in neuronal membranes through a mechanism probably involving Fyn and Tau.

Constraining the Lateral Helix of Respiratory Complex I by Cross-linking Does Not Impair Enzyme Activity or Proton Translocation

Complex I (NADH:ubiquinone oxidoreductase) is a multisubunit, membrane-bound enzyme of the respiratory chain. The energy from NADH oxidation in the peripheral region of the enzyme is used to drive proton translocation across the membrane. One of the integral membrane subunits, nuoL in Escherichia coli, has an unusual lateral helix of ∼75 residues that lies parallel to the membrane surface and has been proposed to play a mechanical role as a piston during proton translocation (Efremov, R. G., Baradaran, R., and Sazanov, L. A. (2010) Nature 465, 441-445). To test this hypothesis we have introduced 11 pairs of cysteine residues into Complex I; in each pair one is in the lateral helix, and the other is in a nearby region of subunit N, M, or L. The double mutants were treated with Cu(2+) ions or with bi-functional methanethiosulfonate reagents to catalyze cross-link formation in membrane vesicles. The yields of cross-linked products were typically 50-90%, as judged by immunoblotting, but in no case did the activity of Complex I decrease by >10-20%, as indicated by deamino-NADH oxidase activity or rates of proton translocation. In contrast, several pairs of cysteine residues introduced at other interfaces of N:M and M:L subunits led to significant loss of activity, in particular, in the region of residue Glu-144 of subunit M. The results do not support the hypothesis that the lateral helix of subunit L functions like a piston, but rather, they suggest that conformational changes might be transmitted more directly through the functional residues of the proton translocation apparatus.

Production of new amilorides as potent inhibitors of mitochondrial respiratory complex I.

Amilorides, well-known inhibitors of Na(+)/H(+) antiporters, have also shown to inhibit bacterial and mitochondrial NADH-quinone oxidoreductase (complex I). Since the membrane subunits ND2, ND4, and ND5 of bovine mitochondrial complex I are homologous to Na(+)/H(+) antiporters, amilorides have been thought to bind to any or all of the antiporter-like subunits; however, there is no direct experimental evidence in support of this notion. Photoaffinity labeling is a powerful technique to identify the binding site of amilorides in bovine complex I. Commercially available amilorides such as 5-(N-ethyl-N-isopropyl)amiloride are not suitable as design templates to synthesize photoreactive amilorides because of their low binding affinities to bovine complex I. Thereby, we attempted to modify the structures of commercially available amilorides in order to obtain more potent derivatives. We successfully produced two photoreactive amilorides (PRA1 and PRA2) with a photolabile azido group at opposite ends of the molecule.

Expression systems for soluble metal-dependent formate dehydrogenase

Abstract Molybdenum or tungsten-dependent formate dehydrogenases (FDH) can reduce carbon dioxide (CO 2 ) to formate under ordinary conditions, and therefore, are considered promising catalysts for CO 2 fixation. However, to our knowledge, no study on the modification of metal-dependent FDHs has been published, likely because of a lack of convenient expression systems for the recombinant enzymes. We attempted to establish an methodology for the preparation and modification of soluble oxygen-tolerant metal-dependent FDHs, for the following three strategies: (1) Escherichia coli FDH is converted from a membrane bound protein into a soluble protein by deleting the C-terminal membrane-anchor of small subunit (FdoH) and the whole membrane subunit (FdoI) and expressed homologously in E . coli ; (2) originally soluble FDHs from Desulfovibrio are expressed heterologously in E. coli ; and (3) Desulfovibrio FDHs are genetically engineered by the homologous gene recombination method and expressed homologously in Desulfovibrio . We successfully established the expression systems for (1) and (3), and succeeded in purification of the soluble FDHs with a tag by using affinity columns.

Organization of Subunits in the Membrane Domain of the Bovine F-ATPase Revealed by Covalent Cross-linking

The F-ATPase in bovine mitochondria is a membrane-bound complex of about 30 subunits of 18 different kinds. Currently, ∼85% of its structure is known. The enzyme has a membrane extrinsic catalytic domain, and a membrane intrinsic domain where the turning of the enzyme's rotor is generated from the transmembrane proton-motive force. The domains are linked by central and peripheral stalks. The central stalk and a hydrophobic ring of c-subunits in the membrane domain constitute the enzyme's rotor. The external surface of the catalytic domain and membrane subunit a are linked by the peripheral stalk, holding them static relative to the rotor. The membrane domain contains six additional subunits named ATP8, e, f, g, DAPIT (diabetes-associated protein in insulin-sensitive tissues), and 6.8PL (6.8-kDa proteolipid), each with a single predicted transmembrane α-helix, but their orientation and topography are unknown. Mutations in ATP8 uncouple the enzyme and interfere with its assembly, but its roles and the roles of the other five subunits are largely unknown. We have reacted accessible amino groups in the enzyme with bifunctional cross-linking agents and identified the linked residues. Cross-links involving the supernumerary subunits, where the structures are not known, show that the C terminus of ATP8 extends ∼70 Å from the membrane into the peripheral stalk and that the N termini of the other supernumerary subunits are on the same side of the membrane, probably in the mitochondrial matrix. These experiments contribute significantly toward building up a complete structural picture of the F-ATPase.

P47phox contributes to albuminuria and kidney fibrosis in mice

Reactive oxygen species (ROS) play an important pathogenic role in the development of many diseases, including kidney disease. Major ROS generators in the glomerulus of the kidney are the p47phox-containing NAPDH oxidases NOX1 and NOX2. The cytosolic p47phox subunit is a key regulator of the assembly and function of NOX1 and NOX2 and its expression and phosphorylation are up-regulated in the course of renal injury, and have been shown to exacerbate diabetic nephropathy. However, its role in non-diabetic-mediated glomerular injury is unclear. To address this, we subjected p47phox-null mice to either adriamycin-mediated or partial renal ablation-mediated glomerular injury. Deletion of p47phox protected the mice from albuminuria and glomerulosclerosis in both injury models. Integrin α1-null mice develop more severe glomerulosclerosis than wild type mice in response to glomerular injury mainly due to increased production of ROS. Interestingly, the protective effects of p47phox knockout were more profound in p47phox/integrin α1 double knockout mice. In vitro analysis of primary mesangial cells showed that deletion of p47phox led to reduced basal levels of superoxide and collagen IV production. Thus, p47phox-dependent NADPH oxidases are a major glomerular source of ROS, contribute to kidney injury, and are potential targets for antioxidant therapy in fibrotic disease.

Abstract 655: Increased Nadph-Oxidase Activity Is Associated With Reduced Telomere Length in the Human Vascular Wall: The Influence of Oxidative Stress on Biological Aging

Background: Accelerated telomere shortening is a hallmark of cellular senescence and has been associated with cardiovascular disease (CVD). Oxidative stress has been proposed as a cause of telomere damage. We explored the association between vascular redox state and telomere length in patients with advanced atherosclerosis. Methods: We recruited xx patients undergoing bypass surgery. Saphenous vein (SV) samples were used to measure superoxide (O2-) generation using lucigenin chemiluminescence (+NADPH 100 uM to estimate NADPH-oxidase activity). DNA was extracted from whole-blood and SV samples and used to measure relative telomere length (TL) using qPCR (which determines T/S ratio using albumin as a single-copy gene). DNA was also used to genotype for two functional single nucleotide polymorphisms (SNPs rs4673 and rs1049255) in the CYBA locus, which encodes for the p22phox membrane subunit of NADPH-oxidase and is associated with activity of the enzyme. Results: Shorter vascular TL was associated with increased NADPH-stimulated O2- generation in SV tissue (A). The additive effect of the rs4673C and rs1049255G alleles was linked to increased vascular NADPH-oxidase activity (B). In addition, the same combination of SNP alleles was linked to reduced T/S ratio in whole blood DNA (C), suggesting a causal association between high oxidative stress and accelerated telomere shortening. Conclusions: We demonstrate for the first time in humans that genetically determined high NADPH-oxidase activity could be causally linked to reduced TL in vascular and blood cells. These novel findings highlight the importance of oxidative stress on cellular senescence and provide an important link between aging and cardiovascular disease.

Development of a novel antimicrobial screening system targeting the pyoverdine-mediated iron acquisition system and xenobiotic efflux pumps.

The iron acquisition systems in Pseudomonas aeruginosa are inducible in response to low-iron conditions and important for growth of this organism under iron limitation. OprM is the essential outer membrane subunit of the MexAB-OprM xenobiotic efflux pump. We designed and constructed a new model antimicrobial screening system targeting both the iron-uptake system and xenobiotic efflux pumps. The oprM gene was placed immediately downstream of the ferri-pyoverdine receptor gene, fpvA, in the host lacking chromosomal oprM and the expression of oprM was monitored by an antibiotic susceptibility test under iron depleted and replete conditions. The recombinant cells showed wild-type susceptibility to pump substrate antibiotics, e.g., aztreonam, under iron limitation and became supersusceptible to them under iron repletion, suggesting that expression of oprM is under control of the iron acquisition system. Upon screening of a chemical library comprising 2952 compounds using this strain, a compound—ethyl 2-(1-acetylpiperidine-4-carboxamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate—was found to enhance the efficacy of aztreonam under iron limitation, suggesting that the compound inhibits either the iron acquisition system or the MexAB-OprM efflux pump. This compound was subsequently found to inhibit the growth of wild-type cells in the presence of sublethal amounts of aztreonam, regardless of the presence or absence of dipyridyl, an iron-chelator. The compound was eventually identified to block the function of the MexAB-OprM efflux pump, showing the validity of this new method.

Amilorides bind to the quinone binding pocket of bovine mitochondrial complex I.

Amilorides, well-known inhibitors of Na(+)/H(+) antiporters, were previously shown to inhibit bacterial and mitochondrial NADH-quinone oxidoreductase (complex I) but were markedly less active for complex I. Because membrane subunits ND2, ND4, and ND5 of bovine complex I are homologous to Na(+)/H(+) antiporters, amilorides have been thought to bind to any or all of the antiporter-like subunits; however, there is currently no direct experimental evidence that supports this notion. To identify the binding site of amilorides in bovine complex I, we synthesized two photoreactive amilorides (PRA1 and PRA2), which have a photoreactive azido (-N3) group and terminal alkyne (-C≡CH) group at the opposite ends of the molecules, respectively, and conducted photoaffinity labeling with bovine heart submitochondrial particles. The terminal alkyne group allows various molecular tags to covalently attach to it via Cu(+)-catalyzed click chemistry, thereby allowing purification and/or detection of the labeled peptides. Proteomic analyses revealed that PRA1 and PRA2 label none of the antiporter-like subunits; they specifically label the accessory subunit B14.5a and core subunit 49 kDa (N-terminal region of Thr25-Glu115), respectively. Suppressive effects of ordinary inhibitors (bullatacin, fenpyroximate, and quinazoline), which bind to the putative quinone binding pocket, on labeling were fairly different between the B14.5a and 49 kDa subunits probably because the binding positions of the three inhibitors differ within the pocket. The results of this study clearly demonstrate that amilorides inhibit complex I activity by occupying the quinone binding pocket rather than directly blocking translocation of protons through the antiporter-like subunits (ND2, ND4, and ND5). The accessory subunit B14.5a may be located adjacent to the N-terminal region of the 49 kDa subunits. The structural features of the quinone binding pocket in bovine complex I were discussed on the basis of these results.

Effects of pathogenic mutations in membrane subunits of mitochondrial Complex I on redox activity and proton translocation studied by modeling in Escherichia coli

Effects of Complex I mutations were studied by modeling in NuoH, NuoJ or NuoK subunits of Escherichia coli NDH-1 by simultaneous optical monitoring of deamino-NADH oxidation and proton translocation and fitting to the data a model equation of transmembrane proton transport. A homolog of the ND1-E24 LHON/MELAS mutation caused 95% inhibition of d-NADH oxidation and proton translocation. The NuoJ-Y59F replacement decreased proton translocation. The NuoK-E72Q mutation lowered the enzyme activity, but proton pumping could be rescued by the double mutation NuoK-E72Q/I39D. Moving the NuoK-E72/E36 pair one helix turn towards the periplasm did not affect redox activity but decreased proton pumping.

Group 16SrXII phytoplasma strains, including subgroup 16SrXII-E (‘Candidatus Phytoplasma fragariae’) and a new subgroup, 16SrXII-I, are associated with diseased potatoes (Solanum tuberosum) in the Yunnan and Inner Mongolia regions of China

In China, potato is widely cultivated economic crop. Recently, potato diseases with characteristic symptoms of phytoplasma infections were found in potato fields. In 2006 and 2007, samples exhibiting symptoms including rosette and upright growth, upward rolling, yellowing and purpling of leaves, shortened and thickened internodes and formation of aerial tubers were collected from plants in Yunnan and Inner Mongolia and analyzed for the presence of phytoplasmas. DNA was extracted from tissues of 63 symptomatic and 10 asymptomatic plants. Phytoplasma 16S rRNA was amplified by PCR with primer pair P1/P7, followed by nested PCR with P1A/P7A, P1A/16S-SR or R16F2n/R16R2n. Twenty nine symptomatic plants (46 %), but no asymptomatic plants, tested positive for phytoplasmas. Nested PCR products were cloned and sequenced. Sequence analysis indicated that the phytoplasmas from diseased potatoes shared 98.1–99.8 % similarity with ‘Candidatus Phytoplasma fragariae’ (16SrXII-E) and other strains in 16SrXII subgroups. RFLP and phylogenetic analyses also indicated that phytoplasmas of group 16SrXII were associated with phytoplasma infected potatoes in China; these strains are most closely related to subgroup 16SrXII-E. Our results showed that five strains belonged to 16SrXII-E; 11 strains were designated as a new 16SrXII subgroup, 16SrXII-I; and subgroup affiliations of all others were not determined. The genetic diversity of the strains was corroborated by sequence analysis of ribosomal protein genes, the elongation factor Tu gene (tuf) and the pre-protein translocase membrane subunit gene (secY). The results illustrated the complexity and diversity of phytoplasmas associated with potatoes in China.

Tackling codon usage bias for heterologous expression in Rhodobacter sphaeroides by supplementation of rare tRNAs

The photosynthetic Rhodobacter species are promising alternative expression hosts in bioproduction and biorefinery due to their unique metabolic capacities. With prominent inner membrane areas and efficient endogenous translocation machineries, they are especially attractive for membrane protein expression. However, codon usage bias could be a limitation in the engineering of Rhodobacter species and has seldom been investigated. In this study, we tackled the codon bias of Rhodobacter by functionally expressing 8 rare tRNAs of Rhodobacter sphaeroides with a multi-copy vector. The impact of tRNA supplementation was evaluated through monitoring expression levels of two heterologous proteins with different phylogenetic origins, a membrane subunit of the riboflavin transporter, RibU, from Lactobacillus acidophilus La-14 and a decaheme cytochrome, MtrA, from Shewanella oneidensis. Our results showed that the performances were closely related to medium composition and rare codon percentages of raw DNA sequences. Provision of rare tRNAs has increased RibU production by 7.7-folds and 2.86-fold in minimal medium and rich medium, respectively, while MtrA levels were increased by 1-fold in minimal medium. The present study confirms the presence of codon bias in R. sphaeroides and offers a facile tool for improving heterologous expression of rare-codon containing genes. We anticipate that this tRNA supplementation system can be further extended to other species of Rhodobacter, and thus will facilitate the engineering of purple bacteria for interesting applications in microbial technology.