Search

Bovine leptospirosis is a worldwide disease that causes reproductive issues, including early embryonic death, stillbirths and infertility, which result in significant economic losses. Although bovine leptospirosis is well-documented in cows, the role of bulls in harbouring and potentially transmitting genital leptospirosis has been largely neglected. We examined genital and blood samples from 16 slaughtered bulls using microscopic agglutination testing (MAT), polymerase chain reaction (PCR) and sequencing of amplicons. Our results showed that 81.2% of bulls were seroreactive, and 75% were genitally infected. The amplicons displayed an identity greater than 99% with Leptospira interrogans strains from the Sejroe serogroup, specifically serovar Hardjoprajitno. This study demonstrates that bulls can harbour in their genital tract the same leptospires associated with reproductive syndromes in cows from the same geographic region, highlighting the importance of bulls in maintaining and transmitting Sejroe serogroup strains associated with reproductive disease., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Pathogenic bacteria Leptospira spp. are commonly associated with bovine leptospirosis, characterized chiefly by chronic and subclinical reproductive disorders. Strains from the Sejroe serogroup play a significant role in these chronic genital infections known as Bovine Genital Leptospirosis (BGL), which notably impact cattle health. This study aims to deepen our understanding of BGL by investigating the genetic diversity, geographical distribution, and specific anatomical sites of infection of the causative agents. Initially, uterine fragments and cervicovaginal mucus were collected from 47 cows and subjected to PCR targeting the lipL32 gene. Positive samples in lipL32-PCR (9 samples) underwent genotyping based on the secY gene. Subsequently, sequences were aligned with GenBank entries (108 sequences) and analyzed in silico. All nine sequences from this study were identified as L. interrogans with an identity >99 % to serogroup Sejroe reference strains (Norma and L53). In the broader analysis, the most prevalent species observed was L. borgpetersenii, followed by L. interrogans and L. santarosai. The haplotype network of L. interrogans revealed that haplogroups B and C exclusively included L. interrogans strains of genital origin, while haplogroup A encompassed strains from renal sources as well. These findings underscore the significance of the L. borgpetersenii genotype Hardjobovis and L. interrogans genotype Hardjoprajitno as the predominant circulating strains and highlight the existence of distinct haplogroups of pathogenic leptospires originating from genital sources. We advocate for the use of secY as an effective genetic marker for Leptospira spp. and stress the necessity for additional research prioritizing the genital tract. The outcomes of this study contribute to the development of improved control measures for chronic cattle diseases and provide valuable guidance for future investigations., Competing Interests: Declaration of Competing Interest None of the authors has any other financial or personal relationships that could inappropriately influence or bias the content of the paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Canine leptospirosis is a bacterial disease caused by spirochetes of the genus Leptospira . Infections can vary from asymptomatic and chronic infections to clinical acute diseases. The disease is endemic in tropical areas, such as Latin American countries, but a broad understanding of the dynamics of circulation of strains, based on molecular data, has not yet been performed. Based on in silico analyses, the present study aims to analyze the genetic diversity and circulation patterns of haplotypes from pathogenic leptospires infecting dogs in Latin America. DNA sequences were obtained from GenBank platform, curated, and aligned. Genetic distances were calculated, and a maximum likelihood tree and haplotype network were constructed. According to the inclusion criteria adopted, a total of 148 sequences were identified. Most of the records were from Brazil, including sequences from L. interrogans serogroup Icterohaemorrhagiae. Phylogenetic analysis showed a genetically closely related cluster, consisting of a larger haplogroup that includes the reference strain Fiocruz L1-130, known to be the major circulating strain in humans. Moreover, no genetic variations were observed according to clinical history and/or geographical localization. We described the molecular epidemiology of leptospires circulating among dogs in Latin America and demonstrated a very genetically homogeneous group, elucidating its ubiquitous circulation pattern and drawing attention to the important role of dogs in the One Health transmission dynamics of leptospirosis.

Leptospirosis is a bacterial disease caused by the infection of pathogenic strains of the genus Leptospira, endemic in tropical and subtropical regions. Although well documented in terrestrial animals and humans, little information is available on its distribution and impact on marine animals. Despite clinical manifestations that may occur, the occurrence of carriers was suggested in some species. Nevertheless, there are few studies regarding the infection by Leptospira sp. in marine mammals. In this context, and considering the One Health approach, the present aimed to investigate pinnipeds' role as Leptospira sp. carriers. Kidneys of 47 pinnipeds of two species, Arctocephalus australis (n = 40) and Arctocephalus tropicalis (n = 7) were collected. DNA was extracted and the diagnosis was performed through LipL32-PCR and genetic characterization based on secY gene sequencing. Phylogenetic analysis and haplotype networks were constructed. Pathogenic Leptospira sp. DNA was detected in 31.9% (15/47) of the tested pinnipeds. It was possible to amplify and sequence eight strains (6 for A. australis, 2 for A. tropicalis), all identified as L. interrogans, with high similarity with sequences from Icterohaemorrhagiae serogroup. Phylogenetic analysis revealed sequences from the present study grouped in species-specific unique clusters, but very close to others from humans, wild animals, and domestic animals. We demonstrate that pinnipeds could act as carriers, and play an important role in leptospirosis dynamics., (Copyright © 2022. Published by Elsevier Ltd.)

A large number of nascent polypeptides have to get across a membrane in targeting to the proper subcellular locations. The SecYEG protein complex, a homolog of the Sec61 complex in eukaryotic cells, has been viewed as the common translocon at the inner membrane for targeting proteins to three extracytoplasmic locations in Gram-negative bacteria, despite the lack of direct verification in living cells. Here, via unnatural amino acid-mediated protein-protein interaction analyses in living cells, in combination with genetic studies, we unveiled a hitherto unreported SecA N protein that seems to be directly involved in translocationg nascent outer membrane proteins across the plasma membrane; it consists of the N-terminal 375 residues of the SecA protein and exists as a membrane-integrated homooligomer. Our new findings place multiple previous observations related to bacterial protein targeting in proper biochemical and evolutionary contexts., Competing Interests: Declaration of Competing Interest We declare that we have no conflict of interest to this work., (Copyright © 2022. Published by Elsevier B.V.)

Bovine Genital Leptospirosis (BGL) is an important syndrome that leads to reproductive failures. The present study aimed to perform a molecular analysis of Leptospira spp. identified from genital and urine samples from in vivo naturally infected cows with poor reproductive performance. A total of 48 cows destined for culling due to low reproductive efficiency were selected and submitted to sampling. Uterine fragments, cervicovaginal mucus (CVM), and urine were collected from all of the cows and processed for culturing and PCR. One isolate was recovered from the uterus of one cow. Other 25 animals were PCR-positive, totaling 26 positive cows. Of them, 18 animals were positive in lipL32-PCR to genital samples, while only seven animals were positive in urine. From those, sequencing of secY gene was performed. Of the 21 good sequences obtained, 16 were L. interrogans, two were L. noguchii, two were L. santarosai and one was L. borgpetersenii. In order to evaluate the genetic similarity of sequences found herein and other sequences from bovines worldwide, a phylogenetic analysis and haplotype networks were performed. Cows with reproductive failures had a significant association (p < 0.05) with positive PCR of genital samples when compared to PCR of urine. None of the animals were positive for genital samples and urine simultaneously. A high diversity of leptospiral strains were found, even in animals of the same epidemiological region. Haplotype networks of L. interrogans showed clusters of sequences from the uterus and CVM with high similarity to other genital sequences originating from previous studies. L. borgpetersenii haplotype networks presented two major clusters with high similarity, even from worldwide sequences, while L. santarosai showed clusters with high genetic distances, even with all the sequences being from Brazil. This study reinforces the theory that BGL and renal infection are distinct diseases, as well as, genital samples are crucial for the diagnosis of cows with reproductive failures caused by leptospires. In addition, haplotype networks confirmed a high genetic similarity between sequences from the present study and Sejroe strains, reinforcing Sejroe strains as the main BGL agents., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

' 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 , Sec Y, and Vmp 1 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 Sec Y gene. This study indicates that the highest genetic variability of collected samples was found in the Vmp 1 gene. Rsa I-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 Vmp 1 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.

Leptospirosis is known to determine reproductive disorders on livestock, and Leptospira interrogans and Leptospira borgpetersenii are the most frequently reported species. Leptospira noguchii is an emerging pathogen, but its association with reproductive disease is unclear. We have detected L. noguchii as the agent of an outbreak with reproductive disorders in a Brazilian dairy goat flock. In the kidding season, five out of 10 Saanen had abortions in the final month of pregnancy and two newborn kids had acute clinical signs. After necropsy of three foetuses and one newborn kid, fragments of liver, lung and kidney were submitted to lipL32-PCR. It yielded positive results in at least one fragment from each animal. After, a nested secY-PCR, followed by sequencing, could identify L. noguchii, with 99-100% of identity with sequences obtained from cattle in the same region. For the first time, L. noguchii was detected in goats and, most importantly, the association of this leptospiral species with reproductive failures in ruminants has been demonstrated., (© 2021 Wiley-VCH GmbH.)

Leptospirosis in ruminants causes reproductive failures leading to important economic losses. This study assessed the occurrence and genetically identified Leptospira spp. in the follicular fluid (FF) of naturally infected live cows. A total of 251 asymptomatic cows from different commercial dairy herds were subjected to ovum-pick up technique for follicular fluid sampling. PCR was performed for Leptospira spp. detection and phylogenetic analysis was later implemented for sequencing. From 251 samples analyzed, 67 (26.7 %) were lipL32-PCR positive, confirming the presence of leptospiral DNA on FF. Furthermore, it was possible to amplify and sequence nine strains after secY nested-PCR. All of them were identified as L. interrogans, with 100 % of identity with strains belonging to Sejroe serogroup. Our findings reveal a high occurrence of infection of Leptospira in the ovarium of asymptomatic cows, highlighting the importance of considering the silent leptospirosis syndrome when screening animals for assisted reproductive biotechniques., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mehle, Kavčič, Mermal, Vidmar, Pompe Novak, Riedle-Bauer, Brader, Kladnik and Dermastia.)

Bovine Genital Leptospirosis (BGL) is an important reproductive disease. The main agents are Sejroe strains, particularly the Hardjo genotypes from Leptospira interrogans and L. borgpetersenii. Although other Sejroe strain, L. santarosai genotype Guaricura, has been frequently isolated from asymptomatic and slaughtered cattle, even from vaginal fluid samples, the role of this strain as real agent of BGL remains uncertain. This study aimed to reinforce L. santarosai strain Guaricura as an important BGL agent, through genetic characterization of a uterine isolate from a live subfertile cow. Urine, cervicovaginal mucus (CVM) and uterine fragment (UF) were collected. In a set up field laboratory, urine, CVM and UF were immediately seeded in T80/40LH medium with antimicrobial cocktail STAFF. Cultures were subcultured in T80/40LH without cocktails, stored at 29ºC and weekly examined. DNA from urine, CVM and UF samples were submitted to PCR targeting lipL32 and secY genes. One leptospiral isolate was recovered from uterine sample; it was serogrouped as Sejroe (titre 25,600) and secY sequencing revealed high genetic similarity with L. santarosai strains from Guaricura serovar. The isolation of this strain from uterus of a live subfertile cow represents substantial evidence that L. santarosai strain Guaricura indeed plays an important role as a BGL agent., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Swine genital leptospirosis is an infectious disease that leads to economic losses due to abortions, stillbirths, and reproductive failures. Considering the scarcity of studies regarding this condition, the objective of the present study was to identify and analyse leptospires infecting the reproductive tract of female pigs slaughtered in the Amazon region. Cervical-vaginal mucus (CVM) from 150 non-pregnant females were collected and submitted to molecular analysis. Initially, polymerase chain reaction (PCR) based on the lipL32 gene was performed. A total of 26.7% (40/150) samples were positive, indicating the presence of Leptospira sp. DNA. Subsequently, positive lipL32-PCR samples were evaluated using secY nested-PCR and sequencing procedures. Eleven amplicons could be sequenced and were identified as Leptospira interrogans (100% identity). Results from phylogenetic analyses led to identification of a putative strain of L. interrogans serogroup Australis, which is indicative of this being a serogroup. In the present study, there was detection of female pigs with leptospires in CVM indicating the possibility of venereal transmission. The large number of genital positive cases could indicate that genital leptospirosis syndrome could also be relevant onto swine production., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., (© 2021. The Author(s).)

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., Competing Interests: RM, YA, HM No competing interests declared, (© 2020, Miyazaki et al.)

Bovine genital leptospirosis is a chronic disease that causes reproductive disorders such as abortions, stillbirths, and estrus repetition, as well as economic losses. Despite clinical signs related to reproductive failure, the majority of studies have focused on the detection of Leptospira spp. in the urine, while few have considered the reproductive tract. Consequently, the aim of the present study was to investigate the uterus as an important extra-renal site of leptospiral infection in cows. A total of 42 non-pregnant cows were studied at a slaughterhouse. Blood samples and uterine fragments were collected for serology and molecular analysis, respectively. Concerning serologic results, 20.5 % presented as reactive, all of them against the Sejroe serogroup. Regarding lipL32 PCR, 26.2 % (11/42) of samples were positive for pathogenic Leptospira sp. Sequencing the secY gene short region enabled nine strains to be characterized, all of which were L. interrogans, with high identity (98.8 %-99.8 %) with serovar Hardjo. The use of molecular tools substantially improved the sensitivity of Leptospira sp. detection at species level and demonstrated that the uterus is an important site of bovine leptospiral infection. The findings of the present study reinforce our understanding that leptospiral uterine infection are associated to members of the Sejroe serogroup., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest.

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'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's dipolar orientation. In contrast, the L80K mutation decelerated voltage induced closings by increasing TM2b'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., Competing Interests: The authors declare no conflict of interest.

Single-molecule techniques provide insights into the heterogeneity and dynamics of ensembles and enable the extraction of mechanistic information that is complementary to high-resolution structural techniques. Here, we describe the application of single-molecule Förster resonance energy transfer to study the dynamics of integral membrane protein complexes on timescales spanning sub-milliseconds to minutes (10 -9 -10 2  s).

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., Competing Interests: No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed., (Copyright: © 2019 Ito K et al.)

Pseudomonas plecoglossicida is a Gram-negative aerobic bacterium that causes high mortality and serious economic losses in some commercial marine fish. Expression of secY was found to be significantly upregulated at 18 °C compared to 28 °C by RNA-seq and qRT-PCR. All five tested recombinant vectors (pCM130/tac + shRNA) significantly reduced secY mRNA levels in P. plecoglossicida. The recombinant vector encoding shRNA-1165 exhibited the best gene-silencing efficiency, 82.4% and was used to create an RNAi strain for further studies. Compared with the wildtype strain, infections of Larimichthys crocea with the RNAi strain resulted in a 2-day delay in onset time and a 35% reduction in mortality, as well as the alleviation of spleen symptoms. The spleens of L. crocea infected by the wild type or RNAi strain of P. plecoglossicida were subjected to dual RNA-seq at 2 dpi. Compared with the wildtype strain, infection of P. plecoglossicida with the RNAi strain resulted in significant changes in the transcriptomes of both host and pathogen. KEGG analysis showed that the complement and coagulation cascade and the Toll-like receptor signalling pathway were the most enriched host pathways. In the pathogen, genes of the "Sec secretion system" were significantly downregulated. This downregulation of "Sec secretion system" genes hindered the secretion of bacterial proteins and reduced the virulence of P. plecoglossicida. Thus, it was easier for L. crocea to clear the RNAi strain of P. plecoglossicida, and the immune response was similarly reduced. The results indicated that secY was a virulence gene of P. plecoglossicida and played roles in the host-pathogen interactions of L. crocea and P. plecoglossicida., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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. IMPORTANCE 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., (Copyright © 2019 Knüpffer et al.)

In the current context of the emergence of certain infectious diseases and discussion of the One Health concept for many of these, the study of leptospirosis - both in domestic and wild hosts - cannot be neglected. The study of animal leptospirosis has evolved in recent years. It has been demonstrated that the human-animal-environment interface is more important than previously thought. In the present study, 35 strains of five pathogenic Leptospira species were isolated from different animal species in Brazil and characterized by rrs, secY, and Multilocus Sequence Typing (MLST) sequencing. Phylogenetic inferences were performed and the molecular diversity of the populations (intra- and inter-population levels) was evaluated. Among the five studied species, 18 different sequence types (STs) were found (22 new alleles and 11 new STs). eBURST analysis revealed two clonal complexes (CCs) and seven singletons. A high genetic diversity was demonstrated (H = 0.954 ± 0.017), mainly for the L. santarosai population (H = 0.942 ± 0.034, n = 20). The same strain was identified in different host species, as well as strains with zoonotic potential circulating in the country. Although the difficulty of culturing Leptospira strains is well known, the high variability of the strains found in Brazil highlights the importance of animals in maintaining the biological cycle of the bacterium in nature. Moreover, the selection of autochthonous strains for the development of vaccines becomes a challenge., (Copyright © 2019 Elsevier B.V. All rights reserved.)

SecA belongs to the large class of ATPases that use the energy of ATP hydrolysis to perform mechanical work resulting in protein translocation across membranes, protein degradation, and unfolding. SecA translocates polypeptides through the SecY membrane channel during protein secretion in bacteria, but how it achieves directed peptide movement is unclear. Here, we use single-molecule FRET to derive a model that couples ATP hydrolysis-dependent conformational changes of SecA with protein translocation. Upon ATP binding, the two-helix finger of SecA moves toward the SecY channel, pushing a segment of the polypeptide into the channel. The finger retracts during ATP hydrolysis, while the clamp domain of SecA tightens around the polypeptide, preserving progress of translocation. The clamp opens after phosphate release and allows passive sliding of the polypeptide chain through the SecA-SecY complex until the next ATP binding event. This power-stroke mechanism may be used by other ATPases that move polypeptides., (© 2019 The Authors.)

Differentiation and classification of phytoplasmas have been primarily based on the highly conserved 16S rRNA gene, for which "universal" primers are available. To date, 36 ribosomal (16Sr) groups and more than 150 subgroups have been delineated by RFLP analysis of 16S rRNA gene sequences. However, in recent years, the use of moderately conserved genes as additional genetic markers has enhanced the resolving power in delineating distinct phytoplasma strains among members of some 16Sr subgroups.This chapter describes the methodology of amplification, differentiation, and classification of phytoplasma based on less-conserved non-ribosomal genes, named rp and secY. Actual and virtual RFLP analyses of amplicons obtained by semi-universal or group-specific rp and secY gene-based primers are used for finer differentiation of phytoplasma strains within a given group. The rp and secY gene-based classification not only readily resolves 16Sr subgroups within a given 16Sr group, but also provides finer differentiation of closely related phytoplasma strains within a given 16Sr subgroup.

Inactivation or deletion of genes allows for investigation and understanding of gene function. To facilitate markerless gene deletion in Listeria monocytogenes, we developed a new suicide plasmid (pHoss1). pHoss1 contains the pMAD backbone, the secY antisense cassette from pIMAY driven by an inducible Pxyl/tetO promoter, a heat-sensitive origin of replication, four unique restriction sites (SalI, EcoRI, SmaI, and NcoI), and erythromycin resistance gene. We demonstrated that pHoss1 is very efficient for introducing mutations into different L. monocytogenes strains. In this chapter, we include a brief description of pHoss1 and the method used for gene deletion in L. monocytogenes using pHoss1.

Chloroplasts inherited systems and strategies for protein targeting, translocation, and integration from their cyanobacterial ancestor. Unlike cyanobacteria however, chloroplasts in green algae and plants contain two distinct SEC translocase/integrase systems: the SEC1 system in the thylakoid membrane and the SEC2 system in the inner envelope membrane. This review summarizes the mode of action of SEC translocases, identification of components of the SEC2 system, evolutionary history of SCY and SECA genes, and previous work on the co- and post-translational targeting of lumenal and thylakoid membrane proteins to the SEC1 system. Recent work identifying substrates for the SEC2 system and potential features that may contribute to inner envelope targeting are also discussed.

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.

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.

The prion protein (PrP) is composed of two major domains of similar size. The structured C-terminal domain contains three alpha-helical regions and a short two-stranded beta-sheet, while the N-terminal domain is intrinsically disordered. The analysis of PrP mutants with deletions in the C-terminal globular domain provided the first hint that intrinsically disordered domains are inefficiently transported into the endoplasmic reticulum through the Sec61 translocon. Interestingly, C-terminally truncated PrP mutants have been linked to inherited prion disease in humans and are characterized by inefficient ER import and the formation of neurotoxic PrP conformers. In a recent study we found that the Sec61 translocon in eukaryotic cells as well as the SecY translocon in bacteria is inherently deficient in translocating intrinsically disordered proteins. Moreover, our results suggest that translocon-associated components in eukaryotic cells enable the Sec61 complex to transport secretory proteins with extended unstructured domains such as PrP and shadoo.

Reproductive tract is an important site of infection for chronic leptospirosis and cooperate in pathogenesis of reproductive failure, leading to economic losses. Since serology techniques cannot detect chronic carriers, the molecular analysis of clinical samples is an alternative to detect these animals on livestock. The aim of the present study was to perform a retrospective study in order to detect leptospiral uterine carriers in slaughtered cows. Tissue samples were collected from 50 post-pubertal, nonpregnant cows. These samples were fixed in 10% buffered formalin, paraffin-embedded and stored. PCR targeting lipL32 gene and molecular characterization by secY sequence was performed. Leptospiral DNA was identified in 18% (9/50) examined blocks. Two sequences were characterized as L. interrogans. These findings suggest that the presence of infectious leptospires in uterus is associated with the physiopathogenesis of the reproductive failure., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

About one-quarter to nearly one-third of the proteins synthesized in the cytosol of eukaryotic cells are integrated into the plasma membrane or are secreted. Translocation of secretory proteins into the lumen of the endoplasmic reticulum or the periplasm of bacteria is mediated by a highly conserved heterotrimeric membrane protein complex denoted Sec61 in eukaryotes and SecYEG in bacteria. To evaluate a possible modulation of the translocation efficiency by secondary structures of the nascent peptide chain, we performed a comparative analysis in bacteria, yeast, and mammalian cells. Strikingly, neither the bacterial SecY nor the eukaryotic Sec61 translocon was able to efficiently transport proteins entirely composed of intrinsically disordered domains (IDDs) or β-strands. However, translocation could be restored by α-helical domains in a position- and organism-dependent manner. In bacteria, we found that the α-helical domains have to precede the IDD or β-strands, whereas in mammalian cells, C-terminally located α-helical domains are sufficient to promote translocation. Our study reveals an evolutionarily conserved deficiency of the Sec61/SecY complex to translocate IDDs and β-strands in the absence of α-helical domains. Moreover, our results may suggest that adaptive pathways co-evolved with the expansion of IDDs in the proteome of eukaryotic cells to increase the transport capacity of the Sec61 translocon., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

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., (© 2016 The Author(s).)

We came together in Leeds to commemorate and celebrate the life and achievements of Prof. Stephen Baldwin. For many years we, together with Sheena Radford and Roman Tuma (colleagues also of the University of Leeds), have worked together on the problem of protein translocation through the essential and ubiquitous Sec system. Inspired and helped by Steve we may finally be making progress. My seminar described our latest hypothesis for the molecular mechanism of protein translocation, supported by results collected in Bristol and Leeds on the tractable bacterial secretion process-commonly known as the Sec system; work that will be published elsewhere. Below is a description of the alternative and contested models for protein translocation that we all have been contemplating for many years. This review will consider their pros and cons., (© 2016 The Author(s).)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

SecYEG protein of bacteria or Sec61αβγ of eukaryotes is a universally conserved heterotrimeric protein channel complex that accommodates the partitioning of membrane proteins into the lipid bilayer as well as the secretion of proteins to the trans side of the plasma or endoplasmic reticular membrane, respectively. SecYEG function is facilitated by cytosolic partners, mainly a nascent chain-ribosome complex or the SecA ATPase motor protein. Extensive efforts utilizing both biochemical and biophysical approaches have been made to determine whether SecYEG functions as a monomer or a dimer, but such approaches have often generated conflicting results. Here we have employed site-specific in vivo photo-cross-linking or cysteine cross-linking, along with co-immunoprecipitation or SecA footprinting techniques to readdress this issue. Our findings show that the SecY dimer to monomer ratio is relatively constant regardless of whether translocons are actively engaged with protein substrate or not. Under the former conditions the SecY dimer can be captured associated with a translocon-jammed substrate, indicative of SecY dimer function. Furthermore, SecA ATPase can be cross-linked to two copies of SecY when the complex contains a translocation intermediate. Collectively, our results suggest that SecYEG dimers are functional units of the translocon., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The aim of this study was to identify Leptospira in urine samples of cattle by direct sequencing of the secY gene. The validity of this approach was assessed using ten Leptospira strains obtained from cattle in Brazil and 77 DNA samples previously extracted from cattle urine, that were positive by PCR for the genus-specific lipL32 gene of Leptospira. Direct sequencing identified 24 (31·1%) interpretable secY sequences and these were identical to those obtained from direct DNA sequencing of the urine samples from which they were recovered. Phylogenetic analyses identified four species: L. interrogans, L. borgpetersenii, L. noguchii, and L. santarosai with the most prevalent genotypes being associated with L. borgpetersenii. While direct sequencing cannot, as yet, replace culturing of leptospires, it is a valid additional tool for epidemiological studies. An unexpected finding from this study was the genetic diversity of Leptospira infecting Brazilian cattle.

Most noncytoplasmic bacterial proteins are exported through the SecYEG channel in the cytoplasmic membrane. This channel and its associated proteins, collectively referred to as the Sec pathway, have strong appeal as a possible antibiotic drug target, yet progress toward new drugs targeting this pathway has been slow, perhaps due partly to many researchers' focus on a single component, the SecA ATPase. Here we report on a pathway-based screen in which beta-galactosidase (β-gal) activity is trapped in the cytoplasm of Escherichia coli cells if translocation through SecYEG is impaired. Several hit compounds passed a counterscreen distinguishing between β-gal overexpression and impaired β-gal export. However, the most extensively characterized hit gave limited E. coli growth inhibition (EC(50) ≥ 400 µM), and growth inhibition could not be unambiguously linked to the compound's effect on the Sec pathway. Our study and others underscore the challenges of finding potent druglike hits against this otherwise promising drug target., (© 2015 Society for Laboratory Automation and Screening.)

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).

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&#95;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&#95;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.

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., (© 2014 The Society for Applied Microbiology.)

SecYEG functions as a membrane channel for protein export. SecY constitutes the protein-conducting pore, which is enwrapped by SecE in a V-shaped manner. In its minimal form SecE consists of a single transmembrane segment that is connected to a surface-exposed amphipathic α-helix via a flexible hinge. These two domains are the major sites of interaction between SecE and SecY. Specific cleavage of SecE at the hinge region, which destroys the interaction between the two SecE domains, reduced translocation. When SecE and SecY were disulfide bonded at the two sites of interaction, protein translocation was not affected. This suggests that the SecY and SecE interactions are static, while the hinge region provides flexibility to allow the SecY pore to open., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)