scholarly journals Establishment Genes Present on pLS20 Family of Conjugative Plasmids Are Regulated in Two Different Ways

2021 ◽  
Vol 9 (12) ◽  
pp. 2465
Author(s):  
Jorge Val-Calvo ◽  
Andrés Miguel-Arribas ◽  
Fernando Freire ◽  
David Abia ◽  
Ling Juan Wu ◽  
...  
Keyword(s):  
Recipient Cell ◽  
Donor Cell ◽  
Bacillus Species ◽  
Regulatory Sequences ◽  
Defence Mechanisms ◽  
New Host ◽  
Conjugative Plasmids ◽  
Bacterial Chromosomes ◽  
Dna Strand ◽  
Conjugation Process

During conjugation, a conjugative DNA element is transferred from a donor to a recipient cell via a connecting channel. Conjugation has clinical relevance because it is the major route for spreading antibiotic resistance and virulence genes. The conjugation process can be divided into different steps. The initial steps carried out in the donor cell culminate in the transfer of a single DNA strand (ssDNA) of the conjugative element into the recipient cell. However, stable settlement of the conjugative element in the new host requires at least two additional events: conversion of the transferred ssDNA into double-stranded DNA and inhibition of the hosts’ defence mechanisms to prevent degradation of the transferred DNA. The genes involved in this late step are historically referred to as establishment genes. The defence mechanisms of the host must be inactivated rapidly and—importantly—transiently, because prolonged inactivation would make the cell vulnerable to the attack of other foreign DNA, such as those of phages. Therefore, expression of the establishment genes in the recipient cell has to be rapid but transient. Here, we studied regulation of the establishment genes present on the four clades of the pLS20 family of conjugative plasmids harboured by different Bacillus species. Evidence is presented that two fundamentally different mechanisms regulate the establishment genes present on these plasmids. Identification of the regulatory sequences were critical in revealing the establishment regulons. Remarkably, whereas the conjugation genes involved in the early steps of the conjugation process are conserved and are located in a single large operon, the establishment genes are highly variable and organised in multiple operons. We propose that the mosaical distribution of establishment genes in multiple operons is directly related to the variability of defence genes encoded by the host bacterial chromosomes.

scholarly journals Multiple Layered Control of the Conjugation Process of the Bacillus subtilis Plasmid pLS20

2021 ◽  
Vol 8 ◽  
Author(s):  
Wilfried J. J. Meijer ◽  
D. Roeland Boer ◽  
Saúl Ares ◽  
Carlos Alfonso ◽  
Fernando Rojo ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Recipient Cell ◽  
Donor Cell ◽  
Bacterial Conjugation ◽  
Dna Translocation ◽  
The Past ◽  
Number Of Genes ◽  
Conjugation Process ◽  
Exclusive Processes ◽  
Rapid Activation

Bacterial conjugation is the main horizontal gene transfer route responsible for the spread of antibiotic resistance, virulence and toxin genes. During conjugation, DNA is transferred from a donor to a recipient cell via a sophisticated channel connecting the two cells. Conjugation not only affects many different aspects of the plasmid and the host, ranging from the properties of the membrane and the cell surface of the donor, to other developmental processes such as competence, it probably also poses a burden on the donor cell due to the expression of the large number of genes involved in the conjugation process. Therefore, expression of the conjugation genes must be strictly controlled. Over the past decade, the regulation of the conjugation genes present on the conjugative Bacillus subtilis plasmid pLS20 has been studied using a variety of methods including genetic, biochemical, biophysical and structural approaches. This review focuses on the interplay between RcopLS20, RappLS20 and Phr*pLS20, the proteins that control the activity of the main conjugation promoter Pc located upstream of the conjugation operon. Proper expression of the conjugation genes requires the following two fundamental elements. First, conjugation is repressed by default and an intercellular quorum-signaling system is used to sense conditions favorable for conjugation. Second, different layers of regulation act together to repress the Pc promoter in a strict manner but allowing rapid activation. During conjugation, ssDNA is exported from the cell by a membrane-embedded DNA translocation machine. Another membrane-embedded DNA translocation machine imports ssDNA in competent cells. Evidences are reviewed indicating that conjugation and competence are probably mutually exclusive processes. Some of the questions that remain unanswered are discussed.

scholarly journals pLS20 is the archetype of a new family of conjugative plasmids harboured by Bacillus species

2021 ◽  
Vol 3 (4) ◽  
Author(s):  
Jorge Val-Calvo ◽  
Andrés Miguel-Arribas ◽  
David Abia ◽  
Ling Juan Wu ◽  
Wilfried J J Meijer
Keyword(s):  
Antibiotic Resistance Genes ◽  
Distribution Patterns ◽  
Conjugative Plasmid ◽  
Bacillus Species ◽  
Gram Positive ◽  
Conjugative Plasmids ◽  
New Family ◽  
Bacterium Bacillus Subtilis ◽  
Bacterium Bacillus ◽  
Conjugation Process

Abstract Conjugation plays important roles in genome plasticity, adaptation and evolution but is also the major horizontal gene-transfer route responsible for spreading toxin, virulence and antibiotic resistance genes. A better understanding of the conjugation process is required for developing drugs and strategies to impede the conjugation-mediated spread of these genes. So far, only a limited number of conjugative elements have been studied. For most of them, it is not known whether they represent a group of conjugative elements, nor about their distribution patterns. Here we show that pLS20 from the Gram-positive bacterium Bacillus subtilis is the prototype conjugative plasmid of a family of at least 35 members that can be divided into four clades, and which are harboured by different Bacillus species found in different global locations and environmental niches. Analyses of their phylogenetic relationship and their conjugation operons have expanded our understanding of a family of conjugative plasmids of Gram-positive origin.

Abstract P404: Membrane Composition Of Cardiac-derived Extracellular Vesicles Changes With Vesicle Origin And Determines Uptake

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Sruti Bheri ◽  
Jessica R Hoffman ◽  
Hyun-Ji Park ◽  
Michael E Davis
Keyword(s):  
Extracellular Vesicles ◽  
Lipid Membrane ◽  
Recipient Cell ◽  
Donor Cell ◽  
Membrane Lipid ◽  
Least Squares Regression ◽  
Membrane Composition ◽  
Cell Type ◽  
Uptake Mechanism ◽  
Donor Cell Type

Introduction: Myocardial infarction (MI) is a leading cause of mortality worldwide. The potency of cell-based therapies for MI is increasingly attributed to the release of extracellular vesicles (EVs) which consist of a lipid/protein membrane and encapsulate RNA cargo. Specifically, EVs from ckit+ progenitor cells (CPCs) and mesenchymal stromal cells (MSCs) are shown to be pro-reparative, with clinical trials ongoing. Despite copious research into EV cargo, the role of donor cell type on EV membrane composition and its effects on EV uptake mechanism by recipient cells remain unclear. This is crucial for designing EV-based therapeutics as uptake mechanism dictates the functionality of the cargo. Thus, we hypothesized that (1) EV membrane composition varies by donor cell type and (2) this variation covaries with the mechanism of uptake. Methods: EVs were isolated using differential ultracentrifugation from four cardiac cell types: CPCs, MSCs, cardiac endothelial cells (CECs) and rat cardiac fibroblasts (RCFs) grown in normoxia (18% O 2 ) or hypoxia (1% O 2 ) to mimic ischemic conditions. EVs were characterized for size and concentration. EV lipid membrane profile was assessed through LC/MS/MS. Donor cell’s role on EV uptake mechanism was determined by inhibiting known uptake pathways (clathrin, dynamin, macropinocytosis and caveolae/lipid raft) with small molecules and quantifying CEC/RCF endocytosis of EVs with flow cytometry. Finally, partial least squares regression was used to determine the most important lipids involved in EV uptake mechanism. Results: EVs were successfully isolated and characterized. The EV membrane lipid profiles clustered by donor cell type. Uptake mechanism of EVs varied based on both donor and recipient cell type with dynamin mediated endocytosis being the most common. Further, the uptake mechanism was independent of normoxic/hypoxic conditioning. Finally, supervised learning methods revealed specific lipid classes (sphingolipids and glycerophospholipids) covaried with EV uptake mechanism. Conclusion: This work highlights the importance of the understudied EV membrane and its role in delivering therapeutic cargo. Active donor cell selection for efficient EV uptake will allow for more potent EV-based MI therapies.

scholarly journals Functional Analysis of TraA, the Sex Pheromone Receptor Encoded by pPD1, in a Promoter Region Essential for the Mating Response in Enterococcus faecalis

2002 ◽  
Vol 184 (22) ◽  
pp. 6343-6350 ◽  
Author(s):  
Takaaki Horii ◽  
Hiromichi Nagasawa ◽  
Jiro Nakayama
Keyword(s):  
Sex Pheromone ◽  
Enterococcus Faecalis ◽  
Intergenic Region ◽  
Recipient Cell ◽  
Donor Cell ◽  
Northern Analysis ◽  
Mobility Shift ◽  
Transcriptional Initiation ◽  
Mating Response ◽  
Gel Mobility Shift

ABSTRACT Conjugative transfer of a bacteriocin plasmid, pPD1, of Enterococcus faecalis is induced in response to a peptide sex pheromone, cPD1, secreted from plasmid-free recipient cells. cPD1 is taken up by a pPD1 donor cell and binds to an intracellular receptor, TraA. Once a recipient cell acquires pPD1, it starts to produce an inhibitor of cPD1, termed iPD1, which functions as a TraA antagonist and blocks self-induction in donor cells. In this study, we discuss how TraA transduces the signal of cPD1 to the mating response. Gel mobility shift assays indicated that TraA is bound to a traA-ipd intergenic region, which is essential for cPD1 response. DNase I footprinting analysis suggested the presence of one strong (tab1) and two weak (tab2 and tab3) TraA-binding sites in the intergenic region. Primer extension analysis implied that the transcriptional initiation sites of traA and ipd were located in the intergenic region. Northern analysis showed that cPD1 upregulated and downregulated transcription of ipd and traA, respectively. The circular permutation assay showed that TraA bent a DNA fragment corresponding to the tab1 region, and its angle was changed in the presence of cPD1 or iPD1. From these data, we propose a model that TraA changes the conformation of the tab1 region in response to cPD1 and upregulates the transcription of ipd, which may lead to expression of genes required for the mating response.

scholarly journals Chinese hamster ovary cell lysosomes rapidly exchange contents.

1987 ◽  
Vol 105 (6) ◽  
pp. 2703-2712 ◽  
Author(s):  
A L Ferris ◽  
J C Brown ◽  
R D Park ◽  
B Storrie
Keyword(s):  
Cell Fusion ◽  
Lucifer Yellow ◽  
Recipient Cell ◽  
Chinese Hamster ◽  
Donor Cell ◽  
Fluid Phase ◽  
Ovary Cell ◽  
Priming Process

We have used cell fusion to address the question of whether macromolecules are rapidly exchanged between lysosomes. Donor cell lysosomes were labeled by the long-term internalization of the fluid-phase pinocytic markers, invertase (sucrase), Lucifer Yellow, FITC-conjugated dextran, or Texas red-conjugated dextran. Recipient cells contained lysosomes swollen by long-term internalization of dilute sucrose or marked by an overnight FITC-dextran uptake. Cells were incubated for 1 or 2 h in marker-free media before cell fusion to clear any marker from an endosomal compartment. Recipient cells were infected with vesicular stomatitis virus as a fusogen. Donor and recipient cells were co-cultured for 1 or 2 h and then fused by a brief exposure to pH 5. In all cases, extensive exchange of content between donor and recipient cell lysosomes was observed at 37 degrees C. Incubation of cell syncytia at 17 degrees C blocked lysosome/lysosome exchange, although a "priming" process(es) appeared to occur at 17 degrees C. The kinetics of lysosome/lysosome exchange in fusions between cells containing invertase-positive lysosomes and sucrose-positive lysosomes indicated that lysosome/lysosome exchange was as rapid, if not more rapid, than endosome/lysosome exchange. These experiments suggest that in vivo the lysosome is a rapidly intermixing organellar compartment.

scholarly journals Isolation and characterization of Bacillus endophytic strains producers for non ribosomal lipopeptides NRLPs from tomato

2018 ◽  
Vol 9 (1) ◽  
pp. 128
Author(s):  
Walaa Hussein ◽  
Ramadan WA ◽  
Sameh Fahim
Keyword(s):  
High Performance ◽  
Plant Defence ◽  
Plant Diseases ◽  
Bacillus Species ◽  
Vegetable Crops ◽  
Defence Mechanisms ◽  
Isolation And Characterization ◽  
Genes Encoding ◽  
Plant Defence Mechanisms

Tomato (Solanum lycopersicum) are consid­ered one of the most important vegetable crops and infected by numbers of different diseases. Studying the use of biological alternatives, instead of chemical substances against plant diseases became necessary for the treatment by beneficial microorganisms endophytes, which can excrete natural products benefits to plant in reducing disease severity, promoting growth and inducing plant defence mechanisms. In this work, three endophytes strains were isolated from tomato stems and their 16srDNA have been found to belong to Bacillus species. The first strain was named BMG100, the second BMG101 and the third BMG102. Two Bacillus strains BMG100 and BMG101 have been found to harbour synthetases genes from three lipopeptides families; surfactin, plipastatin, and iturin (mycosubtilin) which can be detected by degenerated primers designed to detect the presence of synthetases genes encoding lipopeptides. The lipopeptides production was proved by their quantification using High Performance Liquid Chromatography (HPLC), whereas BMG100 produced 105, 178 and 293 mg/L of plipastatin, mycosubtilin and surfactin, respectively, BMG101 produced 385 mg/L of surfactin and 236 mg/L of mycosubtilin, while BMG102 showed no lipopeptides production. Keywords: Tomato; Endophytic bacteria; Lipopeptides; Bacillus species

scholarly journals Origin of leukemic relapse after bone marrow transplantation: comparison of cytogenetic and molecular analyses

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 2033-2040 ◽  
Author(s):  
J Stein ◽  
PA Zimmerman ◽  
M Kochera ◽  
S Strandjord ◽  
W Golden ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Y Chromosome ◽  
Bone Marrow Cells ◽  
Recipient Cell ◽  
Cell Lineage ◽  
Donor Cell ◽  
Leukemic Cell ◽  
Molecular Techniques ◽  
Molecular Analyses ◽  
Leukemic Relapse

Abstract Leukemic relapse following bone marrow transplant (BMT) is generally due to the recurrence in recipient cells, but may rarely occur as a result of donor cell transformation. Donor cell relapse is generally identified using cytogenetic markers such as the sex chromosomes. Recently, molecular techniques have been used to identify the origin of bone marrow cells by their DNA restriction fragment length polymorphisms. We describe the case of a male pediatric patient who had a leukemic relapse 30 months following BMT from his sister. Both cytogenetic and molecular techniques were used to identify the origin of the leukemic relapse. Cytogenetic analyses indicated the absence of the Y chromosome and the presence of a donor cell type 9qh polymorphism, suggesting a donor cell relapse. Molecular analyses also indicated the absence of the Y chromosome but demonstrated the recurrence of recipient DNA markers from three other chromosomes, suggesting a recipient cell relapse. While the leukemic cell lineage cannot be definitively assigned in this case, our results suggest that caution must be exercised when assigning leukemic cell lineage following post-BMT relapse.

Cloning from stem cells: different lineages, different species, same story

2009 ◽  
Vol 21 (1) ◽  
pp. 83 ◽  
Author(s):  
Björn Oback
Keyword(s):  
Stem Cells ◽  
Recipient Cell ◽  
Donor Cell ◽  
Limiting Factor ◽  
Cloning Efficiency ◽  
Surrogate Mothers ◽  
Divergent Differentiation ◽  
Mouse Cells ◽  
Differentiation Status ◽  
Donor Cell Type

Following nuclear transfer (NT), the most stringent measure of extensive donor cell reprogramming is development into viable offspring. This is referred to as cloning efficiency and quantified as the proportion of cloned embryos transferred into surrogate mothers that survive into adulthood. Cloning efficiency depends on the ability of the enucleated recipient cell to carry out the reprogramming reactions (‘reprogramming ability’) and the ability of the nuclear donor cell to be reprogrammed (‘reprogrammability’). It has been postulated that reprogrammability of the somatic donor cell epigenome is inversely proportional to its differentiation status. In order to test this hypothesis, reprogrammability was compared between undifferentiated stem cells and their differentiated isogenic progeny. In the mouse, cells of divergent differentiation status from the neuronal, haematopoietic and skin epithelial lineage were tested. In cattle and deer, skeletal muscle and antler cells, respectively, were used as donors. No conclusive correlation between differentiation status and cloning efficiency was found, indicating that somatic donor cell type may not be the limiting factor for cloning success. This may reflect technical limitations of the NT-induced reprogramming assay. Alternatively, differentiation status and reprogrammability may be unrelated, making all cells equally difficult to reprogramme once they have left the ground state of pluripotency.

scholarly journals Genetic Analysis of the Role of the Transfer Gene,traN, of the F and R100-1 Plasmids in Mating Pair Stabilization during Conjugation

1998 ◽  
Vol 180 (16) ◽  
pp. 4036-4043 ◽  
Author(s):  
William A. Klimke ◽  
Laura S. Frost
Keyword(s):  
Recipient Cell ◽  
Donor Cell ◽  
Mating Pair ◽  
Wild Type ◽  
Receptor Specificity ◽  
Transfer Gene ◽  
Chloramphenicol Resistance ◽  
Chloramphenicol Resistance Cassette ◽  
Surface Exclusion

ABSTRACT Mating pair stabilization occurs during conjugative DNA transfer whereby the donor and recipient cells form a tight junction which requires pili as well as TraN and TraG in the donor cell. The role of the outer membrane protein, TraN, during conjugative transfer was examined by introduction of a chloramphenicol resistance cassette into the traN gene on an F plasmid derivative, pOX38, to produce pOX38N1::CAT. pOX38N1::CAT was greatly reduced in its ability to transfer DNA, indicating that TraN plays a greater role in conjugation than previously thought. F and R100-1 traN were capable of complementing pOX38N1::CAT transfer equally well when wild-type recipients were used. FtraN, but not R100-1 traN, supported a much lower level of transfer when there was an ompA mutation or lipopolysaccharide (LPS) deficiency in the recipient cell, suggesting receptor specificity. The R100-1traN gene was sequenced, and the gene product was found to exhibit 82.3% overall similarity with F TraN. The differences were mainly located within a central region of the proteins (amino acids 162 to 333 of F and 162 to 348 of R100-1). Deletion analysis of FtraN suggested that this central portion might be responsible for the receptor specificity displayed by TraN. TraN was not responsible for TraT-dependent surface exclusion. Thus, TraN, and not the F pilus, appears to interact with OmpA and LPS moieties during conjugation, resulting in mating pair stabilization, the first step in efficient mobilization of DNA.

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