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‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’) is a crop pathogen that is a member of the 16SrXII-A ribosomal subgroup. It is also known as stolbur phytoplasma and causes yield losses in several important crops, especially in Solanaceous crops. Different strains of the pathogen are regularly reported all over the world, particularly in the Mediterranean region. In this study, the determination of genetic diversity for the pathogen infecting tomatoes and potatoes was carried out by using multilocus sequence typing analysis for the Tuf, SecY, and Vmp1 genes to gain insight into the epidemiology of ‘Ca. P. solani’ in Turkey. Genetic diversity of the phytoplasmas was investigated by sequence-based phylogenetic analyses and in silico RFLP analysis of related genes. It was determined that all ‘Ca. P. solani’-related strains infecting tomatoes and potatoes were tuf-b, which is linked to field bindweed (Convolvulus arvensis L.). Tomato or potato-infecting ‘Ca. P. solani’-related strains showed similarities with each other; however, the isolates collected from different plants showed genetic differences in terms of the SecY gene. This study indicates that the highest genetic variability of collected samples was found in the Vmp1 gene. RsaI-RFLP analysis of TYPH10F/R amplicons showed that potato-infecting ‘Ca. P. solani’-related strains were found to be similar to some existing V types. However, the V-type of tomato-infecting isolates is not similar to any previously reported V-type. The results indicate that there could be an important genetic diversity of ‘Ca. P. solani’-related phytoplasmas in Turkey. This could indicate various ways in which the pathogen has adapted to the two host plants as a consequence of the various Vmp1 gene rearrangements seen in these two plant hosts. Obtained results also indicate that the epidemiology of ‘Ca. P. solani’-related phytoplasmas in the tomato and potato agroecosystem may be better understood with the use of molecular data on the complex of vmp-types.

As the causal agent of the grapevine yellows disease Bois noir, ‘Candidatus Phytoplasma solani' has a major economic impact on grapevines. To improve the control of Bois noir, it is critical to understand the very complex epidemiological cycles that involve the multiple “Ca. P. solani” host plants and insect vectors, of which Hyalesthes obsoletus is the most important. In the present study, multiple genotyping of the tuf, secY, stamp, and vmp1 genes was performed. This involved archived grapevine samples that were collected during an official survey of grapevine yellows throughout the wine-growing regions of Slovenia (from 2003 to 2016), plus samples from Austrian grapevines, stinging nettle, field bindweed, and insect samples (collected from 2012 to 2019). The data show that the tuf-b2 type of the tuf gene has been present in eastern Slovenia since at least 2003. The hypotheses that the occurrence of the haplotypes varies due to the geographical position of Slovenia on the Italian–Slovenian Karst divide and that the haplotypes are similar between Slovenian and Austrian Styria were confirmed. The data also show haplotype changes for host plants and H. obsoletus associated with ‘Ca. P. solani,' which might be linked to new epidemiological cycles of this phytoplasma that involve not just new plant sources and new insect vectors, but also climate and land-use changes.

Background Protein transporters translocate hydrophilic segments of polypeptide across hydrophobic cell membranes. Two protein transporters are ubiquitous and date back to the last universal common ancestor: SecY and YidC. SecY consists of two pseudosymmetric halves, which together form a membrane-spanning protein-conducting channel. YidC is an asymmetric molecule with a protein-conducting hydrophilic groove that partially spans the membrane. Although both transporters mediate insertion of membrane proteins with short translocated domains, only SecY transports secretory proteins and membrane proteins with long translocated domains. The evolutionary origins of these ancient and essential transporters are not known. Results The features conserved by the two halves of SecY indicate that their common ancestor was an antiparallel homodimeric channel. Structural searches with SecY’s halves detect exceptional similarity with YidC homologs. The SecY halves and YidC share a fold comprising a three-helix bundle interrupted by a helical hairpin. In YidC, this hairpin is cytoplasmic and facilitates substrate delivery, whereas in SecY, it is transmembrane and forms the substrate-binding lateral gate helices. In both transporters, the three-helix bundle forms a protein-conducting hydrophilic groove delimited by a conserved hydrophobic residue. Based on these similarities, we propose that SecY originated as a YidC homolog which formed a channel by juxtaposing two hydrophilic grooves in an antiparallel homodimer. We find that archaeal YidC and its eukaryotic descendants use this same dimerisation interface to heterodimerise with a conserved partner. YidC’s sufficiency for the function of simple cells is suggested by the results of reductive evolution in mitochondria and plastids, which tend to retain SecY only if they require translocation of large hydrophilic domains. Conclusions SecY and YidC share previously unrecognised similarities in sequence, structure, mechanism, and function. Our delineation of a detailed correspondence between these two essential and ancient transporters enables a deeper mechanistic understanding of how each functions. Furthermore, key differences between them help explain how SecY performs its distinctive function in the recognition and translocation of secretory proteins. The unified theory presented here explains the evolution of these features, and thus reconstructs a key step in the origin of cells.

Bacterial cells utilize monitoring substrates, which undergo force-sensitive translation elongation arrest, to feedback-regulate a Sec-related gene. Vibrio alginolyticus VemP controls the expression of SecD/F that stimulates a late step of translocation by undergoing export-regulated elongation arrest. Here, we attempted at delineating the pathway of the VemP nascent-chain interaction with Sec-related factors, and identified the signal recognition particle (SRP) and PpiD (a membrane-anchored periplasmic chaperone) in addition to other translocon components and a ribosomal protein as interacting partners. Our results showed that SRP is required for the membrane-targeting of VemP, whereas PpiD acts cooperatively with SecD/F in the translocation and arrest-cancelation of VemP. We also identified the conserved Arg-85 residue of VemP as a crucial element that confers PpiD-dependence to VemP and plays an essential role in the regulated arrest-cancelation. We propose a scheme of the arrest-cancelation processes of VemP, which likely monitors late steps in the protein translocation pathway.

The oldest prokaryotic photoautotrophic organisms, cyanobacteria, produce many different metabolites. Among them is the water-soluble neurotoxic non-protein amino acid beta-N-methylamino-L-alanine (BMAA), whose biological functions in cyanobacterial metabolism are of fundamental scientific and practical interest. An early BMAA inhibitory effect on nitrogen fixation and heterocyst differentiation was shown in strains of diazotrophic cyanobacteria Nostoc sp. PCC 7120, Nostoc punctiforme PCC 73102 (ATCC 29133), and Nostoc sp. strain 8963 under conditions of nitrogen starvation. Herein, we present a comprehensive proteomic study of Nostoc (also called Anabaena) sp. PCC 7120 in the heterocyst formation stage affecting by BMAA treatment under nitrogen starvation conditions. BMAA disturbs proteins involved in nitrogen and carbon metabolic pathways, which are tightly co-regulated in cyanobacteria cells. The presented evidence shows that exogenous BMAA affects a key nitrogen regulatory protein, PII (GlnB), and some of its protein partners, as well as glutamyl-tRNA synthetase gltX and other proteins that are involved in protein synthesis, heterocyst differentiation, and nitrogen metabolism. By taking into account the important regulatory role of PII, it becomes clear that BMAA has a severe negative impact on the carbon and nitrogen metabolism of starving Nostoc sp. PCC 7120 cells. BMAA disturbs carbon fixation and the carbon dioxide concentrating mechanism, photosynthesis, and amino acid metabolism. Stress response proteins and DNA repair enzymes are upregulated in the presence of BMAA, clearly indicating severe intracellular stress. This is the first proteomic study of the effects of BMAA on diazotrophic starving cyanobacteria cells, allowing a deeper insight into the regulation of the intracellular metabolism of cyanobacteria by this non-protein amino acid.

The oldest prokaryotic photoautotrophic organisms, cyanobacteria, produce many different metabolites. Among them is the water-soluble neurotoxic non-protein amino acid beta-N-methylamino-L-alanine (BMAA), whose biological functions in cyanobacterial metabolism are of fundamental scientific and practical interest. An early BMAA inhibitory effect on nitrogen fixation and heterocyst differentiation was shown in strains of diazotrophic cyanobacteria Nostoc sp. PCC 7120, Nostoc punctiforme PCC 73102 (ATCC 29133), and Nostoc sp. strain 8963 under conditions of nitrogen starvation. Herein, we present a comprehensive proteomic study of Nostoc (also called Anabaena) sp. PCC 7120 in the heterocyst formation stage affecting by BMAA treatment under nitrogen starvation conditions. BMAA disturbs proteins involved in nitrogen and carbon metabolic pathways, which are tightly co-regulated in cyanobacteria cells. The presented evidence shows that exogenous BMAA affects a key nitrogen regulatory protein, PII (GlnB), and some of its protein partners, as well as glutamyl-tRNA synthetase gltX and other proteins that are involved in protein synthesis, heterocyst differentiation, and nitrogen metabolism. By taking into account the important regulatory role of PII, it becomes clear that BMAA has a severe negative impact on the carbon and nitrogen metabolism of starving Nostoc sp. PCC 7120 cells. BMAA disturbs carbon fixation and the carbon dioxide concentrating mechanism, photosynthesis, and amino acid metabolism. Stress response proteins and DNA repair enzymes are upregulated in the presence of BMAA, clearly indicating severe intracellular stress. This is the first proteomic study of the effects of BMAA on diazotrophic starving cyanobacteria cells, allowing a deeper insight into the regulation of the intracellular metabolism of cyanobacteria by this non-protein amino acid.

Bois noir (BN), associated with ‘Candidatus Phytoplasma solani’ (CaPsol), is the most widespread disease of the grapevine yellows complex worldwide. In this work, BN epidemiology was investigated in a case study vineyard where an unusual CaPsol strain, previously detected only in other host plants, was found to be prevalent in grapevine. Experimental activities included: symptom observation; sampling of symptomatic vines, Auchenorrhyncha specimens, and weeds; molecular detection and typing of CaPsol strains; statistical analyses for determining possible relationships between CaPsol relative concentration, strain type, and symptom severity. Among insects, Reptalus quinquecostatus was the most abundant and was found to be highly infected by CaPsol, while Hyalesthes obsoletus, the main CaPsol vector, was not caught. Moreover, R. quinquecostatus harbored CaPsol strains carrying uniquely the stamp sequence variant St10, also identified as prevalent in vines and in the majority of weeds, and all the secY variants identified in the vineyard. Statistical analyses revealed that CaPsol strains carrying the St10 variant are not associated with severe symptoms, suggesting their possible moderate virulence. Based on such evidence, a new BN epidemiological pattern related to these CaPsol strains and involving grapevine, R. quinquecostatus, and/or weeds is proposed. Furthermore, the possible presence of other players (vectors and weeds) involved in CaPsol transmission to grapevines was highlighted.

The bacterial channel SecYEG efficiently translocates both hydrophobic and hydrophilic proteins across the plasma membrane. Translocating polypeptide chains may dislodge the plug, a half helix that blocks the permeation of small molecules, from its position in the middle of the aqueous translocation channel. Instead of the plug, six isoleucines in the middle of the membrane supposedly seal the channel, by forming a gasket around the translocating polypeptide. However, this hypothesis does not explain how the tightness of the gasket may depend on membrane potential. Here, we demonstrate voltage-dependent closings of the purified and reconstituted channel in the presence of ligands, suggesting that voltage sensitivity may be conferred by motor protein SecA, ribosomes, signal peptides, and/or translocating peptides. Yet, the presence of a voltage sensor intrinsic to SecYEG was indicated by voltage driven closure of pores that were forced-open either by crosslinking the plug to SecE or by plug deletion. We tested the involvement of SecY&rsquo, s half-helix 2b (TM2b) in voltage sensing, since clearly identifiable gating charges are missing. The mutation L80D accelerated voltage driven closings by reversing TM2b&rsquo, s dipolar orientation. In contrast, the L80K mutation decelerated voltage induced closings by increasing TM2b&rsquo, s dipole moment. The observations suggest that TM2b is part of a larger voltage sensor. By partly aligning the combined dipole of this sensor with the orientation of the membrane-spanning electric field, voltage may drive channel closure.

Bois noir (BN), associated with &lsquo, Candidatus Phytoplasma solani&rsquo, (CaPsol), is the most widespread disease of the grapevine yellows complex worldwide. In this work, BN epidemiology was investigated in a case study vineyard where an unusual CaPsol strain, previously detected only in other host plants, was found to be prevalent in grapevine. Experimental activities included: symptom observation, sampling of symptomatic vines, Auchenorrhyncha specimens, and weeds, molecular detection and typing of CaPsol strains, statistical analyses for determining possible relationships between CaPsol relative concentration, strain type, and symptom severity. Among insects, Reptalus quinquecostatus was the most abundant and was found to be highly infected by CaPsol, while Hyalesthes obsoletus, the main CaPsol vector, was not caught. Moreover, R. quinquecostatus harbored CaPsol strains carrying uniquely the stamp sequence variant St10, also identified as prevalent in vines and in the majority of weeds, and all the secY variants identified in the vineyard. Statistical analyses revealed that CaPsol strains carrying the St10 variant are not associated with severe symptoms, suggesting their possible moderate virulence. Based on such evidence, a new BN epidemiological pattern related to these CaPsol strains and involving grapevine, R. quinquecostatus, and/or weeds is proposed. Furthermore, the possible presence of other players (vectors and weeds) involved in CaPsol transmission to grapevines was highlighted.

International audience; Bois noir (BN) is an important grapevine yellows endemic to the Euro-Mediterranean basin caused by ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’), a non culturable plant pathogenic Mollicute. Bois noir symptoms could be associated with ‘Ca. P. solani’ in two Azerbaijanian vineyards where disease incidence and severity were recorded for five local Vitis vinifera cultivars. In order to gain insight into the epidemiology of Bois noir in Azerbaijan, ‘Ca. P. solani’ isolates infecting plants were characterized by multi-locus sequence analysis and their secY and stamp gene sequences compared to that of the strains detected in other plants and in local Cixiidae planthoppers. Genotypes were determined for two non-ribosomal house-keeping genes, namely tuf and secY, as well as two variable markers namely Stamp and mleP1 genes, that respectively encode the antigenic membrane protein AMP and a 2-Hydroxycarboxylate transporter. The Azerbaijanian BN phytoplasma isolates corresponded to three tufB and secY genotypes. A finer differentiation of Azerbaijanian ‘Ca. P. solani’ isolates was obtained with mleP1 as five different mleP1 genetic variants were found. Finally, Stamp gene allowed differentiating four new genotypes in grapevine among the 10 new Stamp genotypes detected in various plants in Azerbaijan. The preliminary survey for infected insects conducted in northern Azerbaijan, led to the identification of Hyalesthes obsoletus and Reptalus noahi as potential vectors for two ‘Ca. P. solani’ new genotypes phylogenetically distant from the known genetic clusters. Altogether these results indicate an important genetic diversity of BN phytoplasmas in Azerbaijan that certainly result from spread through local insect vectors.

This review focusses on the energetics of protein translocation via the Sec translocation machinery. First we complement structural data about SecYEG’s conformational rearrangements by insight obtained from functional assays. These include measurements of SecYEG permeability that allow assessment of channel gating by ligand binding and membrane voltage. Second we will discuss the power stroke and Brownian ratcheting models of substrate translocation and the role that the two models assign to the putative driving forces: (i) ATP (SecA) and GTP (ribosome) hydrolysis, (ii) interaction with accessory proteins, (iii) membrane partitioning and folding, (iv) proton motive force (PMF), and (v) entropic contributions. Our analysis underlines how important energized membranes are for unravelling the translocation mechanism in future experiments.

The Sec translocon provides a polypeptide-conducting channel, which is insulated from the hydrophobic lipidic environment of the membrane, for translocation of hydrophilic passenger polypeptides. Its lateral gate allows a downstream hydrophobic segment (stop-transfer sequence) to exit the channel laterally for integration into the lipid phase. We note that this channel model only partly accounts for the translocon function. The other essential role of translocon is to facilitate de novo insertion of the N-terminal topogenic segment of a substrate polypeptide into the membrane. Recent structural studies suggest that de novo insertion does not use the polypeptide-conducting channel; instead, it takes place directly at the lateral gate, which is prone to opening. We propose that the de novo insertion process, in concept, is similar to that of insertases (such as YidC in bacteria and EMC3 in eukaryotes), in which an intramembrane surface of the machinery provides the halfway point of insertion.

Bacterial protein transport via the conserved SecYEG translocon is generally classified as either cotranslational, i.e., when transport is coupled to translation, or posttranslational, when translation and transport are separated. We show here that the ATPase SecA, which is considered to bind its substrates posttranslationally, already scans the ribosomal tunnel for potential substrates. In the presence of a nascent chain, SecA retracts from the tunnel but maintains contact with the ribosomal surface. This is remarkably similar to the ribosome-binding mode of the signal recognition particle, which mediates cotranslational transport. Our data reveal a striking plasticity of protein transport pathways, which likely enable bacteria to efficiently recognize and transport a large number of highly different substrates within their short generation time., Bacteria execute a variety of protein transport systems for maintaining the proper composition of their different cellular compartments. The SecYEG translocon serves as primary transport channel and is engaged in transporting two different substrate types. Inner membrane proteins are cotranslationally inserted into the membrane after their targeting by the signal recognition particle (SRP). In contrast, secretory proteins are posttranslationally translocated by the ATPase SecA. Recent data indicate that SecA can also bind to ribosomes close to the tunnel exit. We have mapped the interaction of SecA with translating and nontranslating ribosomes and demonstrate that the N terminus and the helical linker domain of SecA bind to an acidic patch on the surface of the ribosomal protein uL23. Intriguingly, both also insert deeply into the ribosomal tunnel to contact the intratunnel loop of uL23, which serves as a nascent chain sensor. This binding pattern is remarkably similar to that of SRP and indicates an identical interaction mode of the two targeting factors with ribosomes. In the presence of a nascent chain, SecA retracts from the tunnel but maintains contact with the surface of uL23. Our data further demonstrate that ribosome and membrane binding of SecA are mutually exclusive, as both events depend on the N terminus of SecA. Our study highlights the enormous plasticity of bacterial protein transport systems and reveals that the discrimination between SRP and SecA substrates is already initiated at the ribosome.

'Candidatus Phytoplasma solani' je bakterija brez celične stene, ki je obligatni parazit rastlin. Je povzročiteljica bolezni na številnih rastlinah na vinski trti na primer povzroča navadno trsno rumenico. S sekvenciranjem produktov ugnezdene verižne reakcije s polimerazo (nPCR) genov tuf, secY in stamp smo določili molekulsko raznolikost sevov 'Ca. P. solani' izoliranih iz vinske trte. Z analizo njihovega časovnega in geografskega pojavljanja v Sloveniji ter primerjavo rezultatov z rezultati podobnih analiz v Evropi, še posebej v okoliških državah, smo določili možno pot širjenja navadne trsne rumenice med Slovenijo in okoliškimi državami. 'Candidatus Phytoplasma solani' is a bacteria without a cell wall, which is an obligatory parasite of plants. It causes diseases in many plants, for example: Bois Noir on grapevine. By sequencing nested polymerase chain reaction products of tuf, secY and stamp genes, we determined the molecular diversity of 'Ca. P. solani' strains isolated from grapevines. By analyzing their occurrence in Slovenia on a geographical and time scale and then comparing the results with similar analysis in Europe, especially in surrounding countries, we determined possible pathways by which spreading of Bois Noir grapevine disease, between Slovenia and its neighbouring countries, can occur.

Proteins routed to the secretory pathway start their journey by being transported across biological membranes, such as the endoplasmic reticulum. The essential nature of this protein translocation process has led to the evolution of several factors that specifically target the translocon and block translocation. In this review, various translocation pathways are discussed together with known inhibitors of translocation. Properties of signal peptide-specific systems are highlighted for the development of new therapeutic and antimicrobial applications, as compounds can target signal peptides from either host cells or pathogens and thereby selectively prevent translocation of those specific proteins. Broad inhibition of translocation is also an interesting target for the development of new anticancer drugs because cancer cells heavily depend on efficient protein translocation into the endoplasmic reticulum to support their fast growth. ispartof: Cellular and Molecular Life Sciences vol:75 issue:9 pages:1541-1558 ispartof: location:Switzerland status: published

This review focusses on the energetics of protein translocation via the Sec translocation machinery. First we complement structural data about SecYEG’s conformational rearrangements by insight obtained from functional assays. These include measurements of SecYEG permeability that allow assessment of channel gating by ligand binding and membrane voltage. Second we will discuss the power stroke and Brownian ratcheting models of substrate translocation and the role that the two models assign to the putative driving forces: (i) ATP (SecA) and GTP (ribosome) hydrolysis, (ii) interaction with accessory proteins, (iii) membrane partitioning and folding, (iv) proton motive force (PMF), and (v) entropic contributions. Our analysis underlines how important energized membranes are for unravelling the translocation mechanism in future experiments. Version of record

Bois noir (BN) associated with ‘Candidatus Phytoplasma solani’ (Stolbur) is regularly found in Austrian vine growing regions. Investigations between 2003 and 2008 indicated sporadic presence of the confirmed disease vector Hyalesthes obsoletus and frequent infections of bindweed and grapevine. Infections of nettles were rare. In contrast present investigations revealed a mass occurrence of H. obsoletus almost exclusively on stinging nettle. The high population densities of H. obsoletus on Urtica dioica were accompanied by frequent occurrence of ‘Ca. P. solani’ in nettles and planthoppers. Sequence analysis of the molecular markers secY, stamp, tuf and vmp1 of stolbur revealed a single genotype named CPsM4_At1 in stinging nettles and more than 64 and 90 % abundance in grapevine and H. obsoletus, respectively. Interestingly, this genotype showed tuf b type restriction pattern previously attributed to bindweed associated ‘Ca. P. solani’ strains, but a different sequence assigned as tuf b2 compared to reference tuf b strains. All other marker genes of CPsM4_At1 clustered with tuf a and nettle derived genotypes verifying distinct nettle phytoplasma genotypes. Transmission experiments with H. obsoletus and Anaceratagallia ribauti resulted in successful transmission of five different strains including the major genotype to Catharanthus roseus and in transmission of the major genotype to U. dioica. Altogether, five nettle and nine bindweed associated genotypes were described. Bindweed types were verified in 34 % of grapevine samples, in few positive Reptalus panzeri, rarely in bindweeds and occasionally in Catharanthus roseus infected by H. obsoletus or A. ribauti. ‘Candidatus Phytoplasma convolvuli‘(bindweed yellows) was ascertained in nettle and bindweed samples.

Protein secretion and membrane insertion occur through the ubiquitous Sec machinery. In this system, insertion involves the targeting of translating ribosomes via the signal recognition particle and its cognate receptor to the SecY (bacteria and archaea)/Sec61 (eukaryotes) translocon. A common mechanism then guides nascent transmembrane helices (TMHs) through the Sec complex, mediated by associated membrane insertion factors. In bacteria, the membrane protein ‘insertase’ YidC ushers TMHs through a lateral gate of SecY to the bilayer. YidC is also thought to incorporate proteins into the membrane independently of SecYEG. Here, we show the bacterial holo-translocon (HTL) — a supercomplex of SecYEG–SecDF–YajC–YidC — is a bona fide resident of the Escherichia coli inner membrane. Moreover, when compared with SecYEG and YidC alone, the HTL is more effective at the insertion and assembly of a wide range of membrane protein substrates, including those hitherto thought to require only YidC.

International audience; Bovine leptospirosis causes substantial reproductive failure in cattle, mainly due to infections with serovar (sv) Hardjo infection. Notwithstanding, other serovars from the serogroup (sg) Sejroe could also have important roles in bovine leptospirosis. The objective was to investigate genetic diversity of serogroup Sejroe isolates obtained from asymptomatic cattle in the state of Rio de Janeiro, Brazil. Urine and vaginal fluid (VF) were collected from clinically healthy cattle immediately after slaughter. Five isolates were recovered and characterized (serogrouping) as belonging to sg Sejroe. Sequencing of rrs and secY genes further identified them as Leptospira santarosai. Analysis of secY sequences indicated a high level of sequence homology to sv Guaricura strains. Based on culture and sequence data, we inferred that other members of sg Sejroe may be important in bovine leptospiral infection, particularly genotypes of L. santarosai serovar Guaricura.

Uncultivable and insect-transmitted bacteria from the genus ‘Candidatus Phytoplasma’ are associated with diseases affecting various plant species worldwide, including many economically important crops. Classification and detection of phytoplasmas are based mainly on the 16S rRNA gene analysis. However, for the elucidation of a complex pathosystem or a distinction between strains, genotyping of additional housekeeping and specific variable genes is applied. Multi-locus sequence typing (MLST) is widely used in population, evolutionary and epidemiological studies of diverse bacterial pathogens. Yet unassigned to a species taxon within the ‘Ca. Phytoplasma’ genus, Flavescence dorée (FD) phytoplasma is one of the main agents associated with grapevine yellows diseases in Europe. It is transmitted by insect vector Scaphoideus titanus Ball. and possesses a high epidemic potential. First report of FD phytoplasma in Croatia was given in 2009 for restricted continental areas of the country. The main objective of this study was to demonstrate tracking and tracing of FD phytoplasmas emergence and spread by MLST approach. In the 4-year period, over 500 samples of grapevine, S. titanus and wild plants from different regions of Croatia were analyzed. Triplex real- time PCR was performed for detection together with PCR/RFLP of 16S rDNA. Subsequently, secY, map and uvrB-degV genes were amplified and sequenced and the phylogenetic analysis was performed. Continuous emergence of new locations affected by FD over the years and a clear trend of the disease spread with Istrian peninsula heavily affected since 2014 was revealed. FD-related phytoplasmas were found for the first time in alder and invasive tree species Ailanthus altissima Mill. Phylogenetic analyses of map gene revealed the existence of 2 distinct FD clusters: map-FD2 and map-FD3, with the latter being prevalent. Different genotypes within both FD clusters could be distinguished based on additional SNPs. Furthermore, MLST revealed a strong relatedness between strains detected in grapevines and insects, showing their affiliation to the same pathosystem. Finding of distinct FD clusters and different distribution of genotypes based on MLST suggests separate routes of the disease introduction in Croatia and spread of the disease.

Insect-transmitted plant pathogens from the genus ‘Candidatus Phytoplasma’ are associated with numerous diseases affecting plant species worldwide. Since their axenic cultivation in a pure culture is still challenging, phytoplasma detection, diagnosis and classification are still based primarily on the 16S rRNA gene analysis. However, when a distinction between closely related strains or the elucidation of a complex pathosystem is needed, information obtained solely on the basis of highly conserved 16S rDNA is not adequate. Thus, the genotyping of additional housekeeping genes together with specific variable genes is performed. Multi-locus sequence typing (MLST) is commonly used tool in population, evolutionary and epidemiological studies of various bacterial pathogens and is recognized as a reproducible approach. In Euro-Mediterranean region, economically important grapevine yellows (GY) diseases are mainly associated with phytoplasmas belonging to the ribosomal groups 16SrV-C and -D (flavescence dorée ; FD) and 16SrXII-A (stolbur ; bois noir ; BN, ‘Ca. Phytoplasma solani’) causing similar symptoms but differing in epidemiology. The main objective of this work was to demonstrate tracking and tracing of both BN and FD phytoplasmas emergence and spread by MLST approach. In the period 2012-2016, over 500 samples of grapevine, insect vectors and wild plants were taken from different viticultural regions of Croatia. Total nucleic acids were extracted and triplex real-time PCR assay was performed, together with PCR/RFLP. Subsequently, distinctive sets of gene fragments differing in conservation and evolution were amplified and sequenced and the phylogenetic analysis was performed. Real-time PCR revealed the continuous emergence of new locations and areas affected by FD over the years and a clear trend of the disease spread with Istria as a new hotspot heavily affected since 2014. For FD phytoplasma, MLST demonstrated the existence of 2 map, 4 uvrB-degV and 5 secY genotypes with a strong relatedness between strains, showing their affiliation to the same pathosystem. MLST analysis of BN isolates revealed the presence of at least 17 different comprehensive genotypes encompassing 3 tuf, 5 secY, 7 vmp1 and 10 stamp genotypes and the prevalent ones were identified. Finding of distinct BN and FD clusters and different distribution of genotypes based on MLST suggests separate routes of the disease introduction in Croatia and spread of the disease.

Most bacterial secretory proteins pass across the cytoplasmic membrane via the translocase, which consists of a protein-conducting channel SecYEG and an ATP-dependent motor protein SecA. The ancillary SecDF membrane protein complex promotes the final stages of translocation. Recent years have seen a major advance in our understanding of the structural and biochemical basis of protein translocation, and this has led to a detailed model of the translocation mechanism.

About 25% to 30% of the bacterial proteins function in the cell envelope or outside of the cell. These proteins are synthesized in the cytosol, and the vast majority is recognized as a ribosome-bound nascent chain by the signal recognition particle (SRP) or by the secretion-dedicated chaperone SecB. Subsequently, they are targeted to the Sec translocase in the cytoplasmic membrane, a multimeric membrane protein complex composed of a highly conserved protein-conducting channel, SecYEG, and a peripherally bound ribosome or ATP-dependent motor protein SecA. The Sec translocase mediates the translocation of proteins across the membrane and the insertion of membrane proteins into the cytoplasmic membrane. Translocation requires the energy sources of ATP and the proton motive force (PMF) while the membrane protein insertion is coupled to polypeptide chain elongation at the ribosome. This review summarizes the present knowledge of the mechanism and structure of the Sec translocase, with a special emphasis on unresolved questions and topics of current research.

Protein translocation in Escherichia coli is mediated by the translocase that, in its minimal form, comprises a protein-conducting pore (SecYEG) and a motor protein (SecA). The SecYEG complex forms a narrow channel in the membrane that allows passage of secretory proteins (preproteins) in an unfolded state only. It has been suggested that the SecA requirement for translocation depends on the folding stability of the mature preprotein domain. Here we studied the effects of the signal sequence and SecB on the folding and translocation of folding stabilizing and destabilizing mutants of the mature maltose binding protein (MBP). Although the mutations affect the folding of the precursor form of MBP, these are drastically overruled by the combined unfolding stabilization of the signal sequence and SecB. Consequently, the translocation kinetics, the energetics and the SecA and SecB dependence of the folding mutants are indistinguishable from those of wild-type preMBP. These data indicate that unfolding of the mature domain of preMBP is likely not a rate-determining step in translocation when the protein is targeted to the translocase via SecB. (c) 2008 Elsevier Ltd. All rights reserved.

About 25% to 30% of the bacterial proteins function in the cell envelope or outside of the cell. These proteins are synthesized in the cytosol, and the vast majority is recognized as a ribosome-bound nascent chain by the signal recognition particle (SRP) or by the secretion-dedicated chaperone SecB. Subsequently, they arc targeted to the See translocase in the cytoplasmic membrane, a multimeric membrane protein complex composed of a highly conserved protein-conducting channel, SecYEG, and a peripherally bound ribosome or ATP-dependent motor protein SecA. The See translocase mediates the translocation of proteins across the membrane and the insertion of membrane proteins into the cytoplasmic membrane. Translocation requires the energy sources of ATP and the proton motive force (PMF) while the membrane protein insertion is coupled to polypeptide chain elongation at the ribosome. This review summarizes the present knowledge of the mechanism and structure of the Sec translocase, with a special emphasis on unresolved questions and topics of current research.

The Escherichia coli SecYEG complex forms a transmembrane channel for both protein export and membrane protein insertion. Secretory proteins and large periplasmic domains of membrane proteins require for translocation in addition the SecA ATPase. The conserved arginine 357 of SecY is essential for a yet unidentified step in the SecA catalytic cycle. To further dissect its role, we have analysed the requirement for 8357 in membrane protein insertion. Although 8357 substitutions abolish post-translational translocation, they allow the translocation of periplasmic domains targeted co-translationally by an N-terminal transmembrane segment. We propose that 8357 is essential for the initiation of SecA-dependent translocation only. (C) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Mechanical properties such as physical constraint and pushing of chromosomes are thought to be important for chromosome segregation in Escherichia coli and it could be mediated by a hypothetical molecular “tether.” However, the actual tether that mediates these features is not known. We previously described that SecA (Secretory A) and Secretory Y (SecY), components of the membrane protein translocation machinery, and AcpP (Acyl carrier protein P) were involved in chromosome segregation and homeostasis of DNA topology. In the present work, we performed three-dimensional deconvolution of microscopic images and time-lapse experiments of these proteins together with MukB and DNA topoisomerases, and found that these proteins embraced the structures of tortuous nucleoids with condensed regions. Notably, SecA, SecY, and AcpP dynamically localized in cells, which was interdependent on each other requiring the ATPase activity of SecA. Our findings imply that the membrane protein translocation machinery plays a role in the maintenance of proper chromosome partitioning, possibly through “tethering” of MukB [a functional homolog of structural maintenance of chromosomes (SMC) proteins], DNA gyrase, DNA topoisomerase IV, and SeqA (Sequestration A).

The motor protein SecA drives the translocation of (pre-)proteins across the SecYEG channel in the bacterial cytoplasmic membrane by nucleotide-dependent cycles of conformational changes often referred to as membrane insertion/de-insertion. Despite structural data on SecA and an archaeal homolog of SecYEG, the identity of the sites of interaction between SecA and SecYEG are unknown. Here, we show that SecA can be cross-linked to several residues in cytoplasmic loop 5 (C5) of SecY, and that SecA directly interacts with a part of transmembrane segment 4 (TMS4) of SecY that is buried in the membrane region of SecYEG. Mutagenesis of either the conserved Arg357 in C5 or Glu176 in TMS4 interferes with the catalytic activity of SecA but not with binding of SecA to SecYEG. Our data explain how conformational changes in SecA could be directly coupled to the previously proposed opening mechanism of the SecYEG channel. (c) 2006 Elsevier Ltd. All rights reserved.

Conditional lethal YidC mutants have been isolated to decipher the role of YidC in the assembly of Sec-dependent and Sec-independent membrane proteins. We now show that the membrane insertion of the Sec-independent M13 procoat-lep protein is inhibited in a short time in a temperature-sensitive mutant when shifted to the nonpermissive temperature. This provides an additional line of evidence that YidC plays a direct role in the insertion of the Sec-independent M13 procoat protein. However, in the temperature-sensitive mutant, the insertion of the Sec-independent Pf3 phage coat protein and the Sec-dependent leader peptidase were not strongly inhibited at the restricted temperatures. Conversely, using a cold-sensitive YidC strain, we find that the membrane insertion of the Sec-independent Pf3 coat protein is blocked, and the Sec-dependent leader peptidase is inhibited at the nonpermissive temperature, whereas the insertion of the M13 procoat protein is nearly normal. These data show that the YidC function for procoat and its function for Pf3 coat and possibly leader peptidase are genetically separable and suggest that the YidC structural requirements are different for the Sec-independent M13 procoat and Pf3 coat phage proteins that insert by different mechanisms.

Conditional lethal YidC mutants have been isolated to decipher the role of YidC in the assembly of Sec-dependent and Sec-independent membrane proteins. We now show that the membrane insertion of the Sec-independent M13 procoat-lep protein is inhibited in a short time in a temperature-sensitive mutant when shifted to the nonpermissive temperature. This provides an additional line of evidence that YidC plays a direct role in the insertion of the Sec-independent M13 procoat protein. However, in the temperature-sensitive mutant, the insertion of the Sec-independent Pf3 phage coat protein and the Sec-dependent leader peptidase were not strongly inhibited at the restricted temperatures. Conversely, using a cold-sensitive YidC strain, we find that the membrane insertion of the Sec-independent Pf3 coat protein is blocked, and the Sec-dependent leader peptidase is inhibited at the nonpermissive temperature, whereas the insertion of the M13 procoat protein is nearly normal. These data show that the YidC function for procoat and its function for Pf3 coat and possibly leader peptidase are genetically separable and suggest that the YidC structural requirements are different for the Sec-independent M13 procoat and Pf3 coat phage proteins that insert by different mechanisms.

The Escherichia coli inner membrane protein (IMP) YidC is involved in the membrane integration of IMPs both in concert with and independently from the Sec translocase. YidC seems to be dispensable for the assembly of Sec-dependent IMPs, and so far it has been shown to be essential only for the proper Sec-independent integration of some phage coat proteins. Here, we studied the physiological consequences of YidC depletion in an effort to understand the essential function of YidC. The loss of YidC rapidly and specifically induced the Psp stress response, which is accompanied by a reduction of the proton-motive force. This reduction is due to defects in the functional assembly of cytochrome o oxidase and the F 1 F o ATPase complex, which is reminiscent of the effects of mutations in the yidC homologue OXA1 in the yeast mitochondrial inner membrane. The integration of CyoA (subunit II of the cytochrome o oxidase) and F o c (membrane subunit of the F 1 F o ATPase) appeared exceptionally sensitive to depletion of YidC, suggesting that these IMPs are natural substrates of a membrane integration and assembly pathway in which YidC plays an exclusive or at least a pivotal role.

Fitoplazme (rod 'Candidatus Phytoplasma’) su bakterije bez stanične stijenke iz razreda Mollicutes koje imaju domaćine u dva carstva jer ih nalazimo u floemu biljaka kao i stanicama kukaca. Nije ih moguće uzgojiti u čistoj kulturi in vitro jer se radi o unutarstaničnim organizmima kod kojih je došlo do redukcije genoma i gubitka mnogih metabolički važnih gena. Uzročnici su ekonomski značajnih bolesti biljaka u čitavom svijetu. Fitoplazmatska vrsta 'Ca. Phytoplasma solani' jedna je od fitoplazmi s najširim krugom prirodnih domaćinskih vrsta, a karakteristična je za europski kontinent. Cilj ovog diplomskog rada bio je karakterizirati izolate bakterije 'Ca. Phytoplasma solani' iz madagaskarskog zimzelena (Catharanthus roseus (L.) G. Don) prikupljenih na području užeg centra grada Zagreba. U multigensku tipizaciju izolata bili su uključeni sljedeći fitoplazmatski geni: 16S rRNA, secY, tufB, vmp1 i stamp. Fragmenti gena za 16S rRNA, tufB i vmp1 umnoženi su lančanom reakcijom polimerazom korištenjem specifičnih početnica te nakon toga analizirani metodom RFLP (polimorfizam duljine restrikcijskih fragmenata). Specifično umnoženi fragmenti gena secY i stamp sekvencirani su te analizirani bioinformatičkim metodama. Filogenetske analize dobivenih nukleotidnih sljedova gena secY i stamp kao i rezultati analize RFLP ostalih gena, potvrdile su pripadnost izolata fitoplazmatskoj vrsti 'Ca. Phytoplasma solani' te njihovu varijabilnost unutar vrste. Analize gena secY i tufB pokazale su očuvanost ovih gena dok je kod gena stamp i vmp1, specifičnih za vrstu, uočena veća raznolikost genotipova. Phytoplasmas (genus "Candidatus Phytoplasma") are bacteria from the class Mollicutes without cell wall found in hosts from two kingdoms, in plant phloem and insect hemolymph. They are intracellular organisms that have undergone reduction of genome with a loss of many metabolically important genes. Therefore they cannot be cultured in vitro in cell-free media. Phytoplasma infect plants all over the world causing significant economic damage. Species 'Ca. Phytoplasma solani' is phytoplasma with widest natural host species range which is characteristic for the European continent. The aim of this thesis was to characterize 'Ca. Phytoplasma solani' isolates from periwinkle (Catharanthus roseus (L.) G. Don) collected at the Zagreb downtown area. In multigene typization of isolates following genes were analyzed: 16S rRNA, secY, tufB, vmp1 and stamp. Fragments of 16S rRNA, tufB and vmp1 genes were amplified by polymerase chain reaction using phytoplasma specific primers and further analyzed by RFLP (restriction fragment length polymorphism) analyses. Amplified fragments of genes secY and stamp were sequenced and analyzed by using bioinformatics tools. Phylogenetic analysis of secY and stamp nucleotide sequences together with RFLP analysis of other genes confirmed the affiliation of all isolates to the 'Ca. Phytoplasma solani' species and their intraspecies variability. Analyses of housekeeping genes secY and tufB showed that these sequences were highly conserved, while the analyses of stamp i vmp1 species specific genes, revealed more genotype variability.

Aim Molecular characterization of a pathogenic complex infecting winter oilseed rape (Brassica napus ssp. oleifera (DC.) Metzg.) plants showing typical rape phyllody symptoms along with some atypical changes. Methods and Results Phytoplasma (‘Candidatus Phytoplasma’) presence was confirmed by PCR-RFLP and 16S rRNA gene sequencing. Phylogenetic analyses of phytoplasma amp, tufB, secY, groEL and ribosomal protein genes confirmed its affiliation to the ‘Ca. P. asteris’ species. However, in the amp gene encoding a specific protein crucial for insect transmission specificity, significant SNPs were found. Biological and serological tests revealed the co-infection with Turnip mosaic virus (TuMV). The phylogenetic analysis of full TuMV genome sequence, the first reported from the Balkans, classified it into the world-B phylogenetic lineage. Conclusions A pathogenic complex consisting of ‘Ca. P. asteris’ and TuMV found to co-infect oilseed rape plants for the first time was molecularly characterized. Significance and Impact of the Study Rape phyllody is a serious problem in rapeseed production. The molecular information from this first multi-gene analysis of ‘Ca. P. asteris’ strain associated with rape phyllody as well as the first report of the complete sequence of TuMV isolate from the Balkans is a starting point for understanding the disease complexity and management.

Proliferacija jabuke (apple proliferation; AP) je bolest uzrokovana bakterijom ‘Candidatus Phytoplasma mali' koja je prisutna i rasprostranjena u većini europskih zemalja. Iako zaraza ne dovodi do potpunog propadanja biljke-domaćina, zbog značajnog smanjenja veličine i kvalitete plodova ekonomski gubici su značajni. Fitoplazme (rod ‘Candidatus Phytoplasma') su unutarstanični, pleomorfni biljni patogeni iz razreda Mollicutes. Smještene su u žilnom staničju zaražene biljke, a umnažaju se i žive samo unutar sitastih cijevi floema biljke i stanica kukaca. Bolest se prenosi zaraženim sadnim materijalom i kukcima – vektorima iz reda Hemiptera. Kako je još uvijek gotovo nemoguće uzgojiti čistu kulturu fitoplazmi na umjetnom mediju, za karakterizaciju se koristi metoda multigenske tipizacije. Uzročnik bolesti, ‘Candidatus Phytoplasma mali' tek je nedavno u Hrvatskoj po prvi puta identificiran molekularnim metodama. U ovom diplomskom radu glavni cilj bio je karakterizirati novootkrivene izolate bakterije 'Ca. Phytoplasma mali' iz zaraženog tkiva jabuke i kukca-vektora Cacopsylla picta analizom gena secY i imp. Fragmenti ovih gena umnoženi su metodom PCR korištenjem specifičnih početnica te su sekvencirani. Prisutnost zaraze fitoplazmom 'Ca. Phytoplasma mali' dokazana je umnažanjem i restrikcijskom analizom gena za 16S rRNA. Filogenetskom analizom dobivenih nukleotidnih sljedova dobiveni su podaci o varijabilnosti ovih gena kao i njihovoj srodnosti s izolatima iz čitavog svijeta. Rezultati analize varijabilnosti dodatnih molekularnih markera uz 16S rRNA važni su za razjašnjavanje molekularne epidemiologije, posebno stoga što se ova fitoplazma prenosi kukcima – vektorima iz roda Cacopsylla koje su prisutne u Hrvatskoj. Apple poliferation (AP) is a disease caused by 'Candidatus Phytoplasma mali' that is present and widespread in most European countries. Although infection does not lead to the degradation of plant hosts, due to a significant reduction in the size and quality of the fruits of economic losses are significant. Phytoplasma (genus 'Candidatus Phytoplasma') are intracellular, pleomorphic plant pathogens from the class Mollicutes. They are located in the core of tissues of infected plants are propagated and live only in the phloem plant and insect cells. Disease is transmitted by infected planting material and insects - vectors of the order Hemiptera. As it is still almost impossible to obtain pure culture of phytoplasma in an artificial medium, multigene characterization in often used for their characterization. 'Ca. Phytoplasma mali' has only recently been identified in Croatia for the first time by molecular methods. This thesis main objective is to characterize the newly discovered 'Ca. Phytoplasma mali' isolates from infected tissue apples and insect-vector Cacopsylla picta analysis of gene secY and imp. The presence of phytoplasma infection 'Ca. Phytoplasma mali' was demonstrated by amplification and restriction analysis of 16S rRNA.Fragments of these genes were amplified by PCR using specific primers and sequenced. Phylogenetic analysis of the nucleotide sequences obtained provided information on the variability of these genes as well as their relatedness to already described isolates. Results of the analysis of variability of additional molecular markers to the 16S rRNA are important for elucidating the molecular epidemiology, especially because this phytoplasma is transmitted by insects of the genus Cacopsylla that are present in Croatia.

The thylakoid Delta pH pathway is a protein transport system with unprecedented characteristics. To investigate its mechanism, the topology of precursor insertion was determined. A fusion protein comprising a large polypeptide domain fused to the amino terminus of pOE17 (a Delta pH pathway precursor) was efficiently processed by thylakoid membranes. The amino terminus, including the targeting peptide, remained on the cis side of the membrane. Mature OE17 was transported to the lumen. These experiments demonstrate that Delta pH directed precursors enter the thylakoid membrane in a loop, implying that the Delta pH pathway has evolved from an export-type protein translocation system.

International audience; Tuf and secY genotyping techniques have been developed to distinguish phytoplasma strains. Tuf polymerase chain reaction sequence analyses are available for phytoplasma taxonomic groups 16SrI, 16SrV, 16SrXII-A, and XII-B. In addition to their use to confirm the taxonomic status of phytoplasma strains, they allow the spread of phytoplasma strains in host plants and insect vectors to be traced. SecY is more variable than tuf and is therefore more discriminatory than tuf, but secY and tuf phylogenies show congruence.

The genetic diversity of three temperate fruit tree phytoplasmas ‘Candidatus Phytoplasma prunorum’, ‘Ca. P. mali’ and ‘Ca. P. pyri’ has been established by multilocus sequence analysis. Among the four genetic loci used, the genes imp and aceF distinguished 30 and 24 genotypes, respectively, and showed the highest variability. Percentage of substitution for imp ranged from 50 to 68 % according to species. Percentage of substitution varied between 9 and 12 % for aceF, whereas it was between 5 and 6 % for pnp and secY. In the case of ‘Ca P. prunorum’ the three most prevalent aceF genotypes were detected in both plants and insect vectors, confirming that the prevalent isolates are propagated by insects. The four isolates known to be hypo-virulent had the same aceF sequence, indicating a possible monophyletic origin. Haplotype network reconstructed by eBURST revealed that among the 34 haplotypes of ‘Ca. P. prunorum’, the four hypo-virulent isolates also grouped together in the same clade. Genotyping of some Spanish and Azerbaijanese ‘Ca. P. pyri’ isolates showed that they shared some alleles with ‘Ca. P. prunorum’, supporting for the first time to our knowledge, the existence of inter-species recombination between these two species.

This study focused on evaluating the genetic diversity among ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) populations in orchards of north-western Italy, where apple proliferation (AP) disease is widespread and induces severe economic losses. ‘Ca. P. mali’ was detected through restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S rDNA in 101 of 114 samples examined. Collective RFLP patterns, obtained by restriction analyses of four amplified genomic segments (16S/23S rDNA, PR-1, PR-2 and PR-3 non-ribosomal region, ribosomal protein genes rplV-rpsC and secY gene), revealed the presence of 12 distinct genetic lineages among 60 selected representative ‘Ca. P. mali’ isolates, underscoring an unexpected high degree of genetic heterogeneity among AP phytoplasma populations in north-western Italy. Prevalence of distinct genetic lineages in diverse geographic regions opens new interesting avenues for studying the epidemiology of AP disease. Furthermore, lineage-specific molecular markers identified in this work could be useful for investigating the biological life cycle of ‘Ca. P. mali’.

Elucidation of high-resolution structures of integral membrane proteins is drastically lagging behind that of cytoplasmic proteins. In vitro synthesis and insertion of membrane proteins into synthetic membranes could circumvent bottlenecks associated with the overexpression of membrane proteins, producing sufficient membrane-inserted, correctly folded protein for structural studies. Using the mechanosensitive channel of large conductance, MscL, as a model protein we show that in vitro synthesized MscL inserts into YidC-containing proteoliposomes and oligomerizes to form a homopentamer. Using planar membrane bilayers, we show that MscL forms functional ion channels capable of ion transport. These data demonstrate that membrane insertion of MscL is YidC mediated, whereas subsequent oligomerization into a functional homopentamer is a spontaneous event. (C) 2010 Published by Elsevier B. V. on behalf of the Federation of European Biochemical Societies.

The SecY/E protein of Escherichia coli was coreconstituted with the proton pump bacteriorhodopsin and cytochrome c oxidase yielding proteoliposomes capable of sustaining a protonmotive force (DELTA-p) of defined polarity and composition. Proteoliposomes support the ATP- and SecA-dependent translocation of proOmpA which is stimulated by a DELTA-p, inside acid and positive. DELTA-p of opposite polarity, inside alkaline and negative, suppresses translocation while SecA-mediated ATP hydrolysis continues unabated. DELTA-psi and DELTA-pH are equally effective in promoting or inhibiting translocation. Membrane-spanning translocation intermediates move backwards in the presence of a reversed DELTA-p. These results support a model [Schiebel,E., Driessen, A.J.M., Hartl,F.U. and Wickner,W. (1991) Cell, 64, 927-939] in which the DELTA-p defines the direction of translocation after ATP hydrolysis has released proOmpA from its association with SecA. The polarity effect of the DELTA-p challenges models involving DELTA-p-dependent membrane destabilization and provides further evidence for a role of the DELTA-p as driving force in precursor protein translocation.

The SecY/E protein of Escherichia coli was coreconstituted with the proton pump bacteriorhodopsin and cytochrome c oxidase yielding proteoliposomes capable of sustaining a protonmotive force (DELTA-p) of defined polarity and composition. Proteoliposomes support the ATP- and SecA-dependent translocation of proOmpA which is stimulated by a DELTA-p, inside acid and positive. DELTA-p of opposite polarity, inside alkaline and negative, suppresses translocation while SecA-mediated ATP hydrolysis continues unabated. DELTA-psi and DELTA-pH are equally effective in promoting or inhibiting translocation. Membrane-spanning translocation intermediates move backwards in the presence of a reversed DELTA-p. These results support a model [Schiebel,E., Driessen, A.J.M., Hartl,F.U. and Wickner,W. (1991) Cell, 64, 927-939] in which the DELTA-p defines the direction of translocation after ATP hydrolysis has released proOmpA from its association with SecA. The polarity effect of the DELTA-p challenges models involving DELTA-p-dependent membrane destabilization and provides further evidence for a role of the DELTA-p as driving force in precursor protein translocation.

The SecY/E protein of Escherichia coli was coreconstituted with the proton pump bacteriorhodopsin and cytochrome c oxidase yielding proteoliposomes capable of sustaining a protonmotive force (delta p) of defined polarity and composition. Proteoliposomes support the ATP- and SecA-dependent translocation of proOmpA which is stimulated by a delta p, inside acid and positive. delta p of opposite polarity, inside alkaline and negative, suppresses translocation while SecA-mediated ATP hydrolysis continues unabated. delta psi and delta pH are equally effective in promoting or inhibiting translocation. Membrane-spanning translocation intermediates move backwards in the presence of a reversed delta p. These results support a model [Schiebel, E., Driessen, A.J.M., Hartl, F.-U. and Wickner, W. (1991) Cell, 64, 927-939] in which the delta p defines the direction of translocation after ATP hydrolysis has released proOmpA from its association with SecA. The polarity effect of the delta p challenges models involving delta p-dependent membrane destabilization and provides further evidence for a role of the delta p as driving force in precursor protein translocation.

OmpT, an outer membrane-localized protease of Escherichia coli, cleaves a number of exogenous and endogenous proteins during their purification. SecY, an endogenous membrane protein, is a target of this artificial proteolysis in vitro. Here we report that SecY cleavage occurs even in cell extracts from ompT-disrupted cells, if they carry an F plasmid derivative. A gene, ompP, on the F plasmid was shown to be responsible for this proteolysis. These results indicate that the absence of an F-like plasmid should be checked when choosing a host strain for E. coli-based protein production.

The presence and distribution of grapevine phytoplasmas was investigated from 2003 to 2005 in some of the most important viticultural areas of Serbia, considering in particular the susceptibility and sensitiveness of both local and imported grapevine cultivars. Both flavescence dorée (FD) and bois noir (BN) phytoplasmas were detected using molecular techniques. The presence of FD phytoplasma at the moment seems limited, while BN phytoplasma appears to be present in the majority of grape growing regions in Serbia. Field surveys demonstrate that grapevine yellows (GY) epidemics in the vineyards inspected in Serbia spread very fast, indeed the incidence of symptomatic plants increased considerably year by year. In particular, the average rate of FD diffusion increased from 45.5 to 93.0 % in the Sićevačko region, while the spread of BN resulted lower. The local cultivar 'Plovdina' appeared to be extremely sensitive to FD phytoplasma showing a percentage of infected plants ranging from 91 to 100 %. PCR-RFLP and phylogenetic analyses based on ribosomal protein (rp) and secY gene sequences performed on Serbian FD grapevine strains demonstrated their close relationship with the Italian FD-C strain present in north-east Italy. Based on both phylogenetic markers, Serbian FD strains represent a new distinct lineage and together with the FD-C strain form a major phylogenetic group within the elm yellows group., VITIS - Journal of Grapevine Research, Vol. 47 No. 2 (2008): Vitis

The SecY protein and other membrane proteins of Escherichia coli were solubilized by mixed micelles of n-octyl beta-D-glucopyranoside, phospholipids, and glycerol. Proteoliposomes formed from this extract by detergent dialysis supported energy-dependent translocation and processing of pro-OmpA. Translocation required ATP, SecY, and SecA and was stimulated by a proton-motive force. These results provide an important assay for the isolation and identification of membrane components involved in protein translocation.

In bacteria, two major pathways exist to secrete proteins across the cytoplasmic membrane. The general Secretion route, termed Sec-pathway, catalyzes the transmembrame translocation of proteins in their unfolded conformation, whereupon they fold into their native structure at the trans-side of the membrane. The Twin-arginine translocation pathway, termed Tat-pathway, catalyses the translocation of secretory proteins in their folded state. Although the targeting signals that direct secretory proteins to these pathways show a high degree of similarity, the translocation mechanisms and translocases involved are vastly different. (C) 2007 Elsevier B.V. All rights reserved.