scholarly journals Interactions between the surface exclusion systems of some F-like plasmids

1974 ◽  
Vol 24 (1) ◽  
pp. 81-89 ◽  
Author(s):  
Neil Willetts ◽  
John Maule
Keyword(s):  
Recipient Cell ◽  
Receptor Site ◽  
Donor Cell ◽  
E Coli ◽  
Transfer Genes ◽  
Donor Cells ◽  
The One ◽  
Sites Of Action ◽  
Surface Exclusion ◽  
Transfer Inhibition

SUMMARYFour surface exclusion systems have been identified amongst a group of F-like plasmids inE. coli: SfxI(F), SfxII(ColV2 and R538-1fin−), SfxIII(ColVBtrpand R1-19) and SfxIV(R100-1 and R136fin−). None of these systems was expressed in stationary phase cells or, except for ColVBtrp, duringfin+transfer inhibition, showing that the surface exclusion gene(s) is usually co-controlled with the transfer genes.Recipient cells carrying two plasmids specifying different surface exclusion systems did not always express both of these: the overall pattern suggested that the four systems and/or their sites of action are related. There was no surface exclusion between donor cells carrying two plasmids determining different surface exclusion systems and recipient cells carrying a plasmid determining either one of these. Our hypothesis to explain this and other results is that surface exclusion prevents interaction between the tip of the pilus on the donor cell and a receptor site on the recipient cell surface. Pili (probably mixed) with two types of tips would be present on cells carrying two different plasmids, the one unresponsive to the single surface exclusion system of the recipient cells allowing transfer of both plasmids.

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.

scholarly journals ArdC protein overpasses the recipient hsdRMS restriction system broadening conjugation host range

2019 ◽  
Author(s):  
Lorena González-Montes ◽  
Irene del Campo ◽  
Fernando de la Cruz ◽  
Gabriel Moncalian
Keyword(s):  
Host Range ◽  
Recipient Cell ◽  
Broad Host Range ◽  
Rna Seq ◽  
Ssdna Binding ◽  
E Coli ◽  
Broad Host Range Plasmid ◽  
Donor Cells ◽  
Metalloprotease Activity ◽  
Restriction Modification

AbstractPlasmids, when transferred by conjugation, must overpass restriction-modification systems of the recipient cell. We demonstrate that protein ArdC, encoded by broad host range plasmid R388, was required for conjugation from Escherichia coli to Pseudomonas putida, but not from E. coli to E. coli. Surprisingly, expression of ardC was required in the recipient cells, but not in the donor cells. Besides, ardC was not required for conjugation if the hsdRMS system was deleted in P. putida recipient cells. Thus, ArdC has antirestriction activity against HsdRMS system, and consequently broadens R388 plasmid host range. The crystal structure of ArdC was solved both in the absence and in the presence of Mn2+. ArdC is composed of a non-specific ssDNA binding N-terminal domain and a C-terminal metalloprotease domain, although the metalloprotease activity is not needed for antirestriction function. We also observed by RNA-seq that ArdC-dependent conjugation triggers an SOS response in the P. putida recipient cells. Our findings give new insights, and open new questions, into the antirestriction strategies developed by plasmids to counteract bacterial restriction strategies.

scholarly journals Comparative analysis of MOBQ4 plasmids demonstrates that MOBQ is a cis-acting-enriched relaxase protein family

2019 ◽  
Author(s):  
M. Pilar Garcillán-Barcia ◽  
Raquel Cuartas-Lanza ◽  
Ana Cuevas ◽  
Fernando de la Cruz
Keyword(s):  
Bacterial Genome ◽  
Donor Cell ◽  
Cis Acting ◽  
Biologically Relevant ◽  
E Coli ◽  
Conjugative Plasmids ◽  
Transfer Genes ◽  
Plasmid Mobilization ◽  
Helper Plasmids ◽  
Origin Of Transfer

ABSTRACTA group of small mobilizable plasmids is increasingly being reported in epidemiology surveys of enterobacteria. Some of them encode colicins, while others are cryptic. All of them encode a relaxase belonging to a previously non-described group of the MOBQ class, MOBQ4. While highly similar in their mobilization module, two families with unrelated replicons can be distinguished, MOBQ41 and MOBQ42. Members of both groups were compatible between them and stably maintained in E. coli. MOBQ4 plasmids were mobilized by conjugation. They contain two transfer genes, mobA coding for the MOBQ4 relaxase and mobC, which was non-essential but enhanced the plasmid mobilization frequency. The origin of transfer was located between these two divergently transcribed mob genes. MPFI conjugative plasmids were the most efficient helpers for MOBQ4 conjugative transmission. No interference in mobilization was observed when both MOBQ41 and MOBQ42 were present in the same donor cell. Remarkably, MOBQ4 relaxases exhibited a cis-acting preference for their oriTs, a feature already observed in other MOBQ plasmids. These findings indicate that MOBQ4 plasmids can efficiently spread among enterobacteria aided by coresident IncI1, IncK and IncL/M plasmids, while ensuring their self-dissemination over highly-related elements.IMPORTANCEPlasmids are key vehicles of horizontal gene transfer and contribute greatly to bacterial genome plasticity. A group of plasmids, called mobilizable, is able to disseminate aided by helper conjugative plasmids. Here, we studied a group of phylogenetically-related mobilizable plasmids, MOBQ4, commonly found in clinically-relevant enterobacteria, uncovering the helper plasmids responsible for their dissemination. We found that the two plasmid species encompassed in the MOBQ4 group can coexist and transfer orthogonally, despite origin-of-transfer cross-recognition by their relaxases. Specific discrimination among their highly similar oriT sequences is guaranteed by the preferential cis activity of the MOBQ4 relaxases. Such strategy would be biologically relevant in a scenario of co-residence of non-divergent elements to favor self-dissemination.

scholarly journals Ultrafiltration Process in Disinfection and Advanced Treatment of Tertiary Treated Wastewater

Membranes ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 221
Author(s):  
Rafał Tytus Bray ◽  
Katarzyna Jankowska ◽  
Eliza Kulbat ◽  
Aneta Łuczkiewicz ◽  
Aleksandra Sokołowska
Keyword(s):  
Wastewater Treatment ◽  
Wastewater Treatment Plants ◽  
Microscopic Analysis ◽  
Fecal Coliforms ◽  
Treated Wastewater ◽  
Chemical Parameters ◽  
Virus Particles ◽  
E Coli ◽  
The One ◽  
Physical And Chemical

The paper presents the results of research on the use of ultrafiltration, using membranes of 200 and 400 kDa separation, for disinfection of municipal treated wastewater. The research was conducted on a fractional technical scale using real municipal treated wastewater from two large wastewater treatment plants treating most of the wastewater over the one-million polycentric Gdańsk agglomeration (1.2 million inhabitants). UF 200 kDa and UF 400 kDa processes enabled further improvement of the physical and chemical parameters of treated wastewater. Total phosphorus (to below 0.2 mg/L–UF 200 kDa, 0.13 mg/L–UF 400 kDa) and turbid substances (to below 0.2 mg/L, both membranes) were removed in the highest degree. COD was reduced efficiently (to below 25.6 mgO2/L–UF 200 kDa, 26.8 mgO2/L–UF 400 kDa), while total nitrogen was removed to a small extent (to 7.12 mg/L–UF 200 kDa and 5.7 mg/L–UF 400 kDa. Based on the reduction of indicator bacteria; fecal coliforms including E. coli (FC) and fecal enterococci (FE) it was found that the ultrafiltration is an effective method of disinfection. Not much indicator bacterial were observed in the permeate after processes (UF 200 kDa; FC—5 CFU/L; FE—1 CFU/L and UF 400 kDa; FC—70 CFU/L; FE—10 CFU/L. However, microscopic analysis of prokaryotic cells and virus particles showed their presence after the application of both membrane types; TCN 3.0 × 102 cells/mL–UF 200 kDa, 5.0 × 103 cells/mL–UF 400 kDa, VP 1.0 × 105/mL. The presence of potentially pathogenic, highly infectious virus particles means that ultrafiltration cannot be considered a sufficient disinfection method for treated wastewater diverted for reuse or discharged from high load wastewater treatment plants to recreational areas. For full microbiological safety it would be advisable to apply an additional disinfection method (e.g., ozonation).

scholarly journals Dynamic integration of enteric neural stem cells in ex vivo organotypic colon cultures

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Georgina Navoly ◽  
Conor J. McCann
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Organotypic Culture ◽  
Ex Vivo ◽  
Donor Cell ◽  
Colonic Tissue ◽  
Cell Therapies ◽  
Enteric Ganglia ◽  
Donor Cells

AbstractEnteric neural stem cells (ENSC) have been identified as a possible treatment for enteric neuropathies. After in vivo transplantation, ENSC and their derivatives have been shown to engraft within colonic tissue, migrate and populate endogenous ganglia, and functionally integrate with the enteric nervous system. However, the mechanisms underlying the integration of donor ENSC, in recipient tissues, remain unclear. Therefore, we aimed to examine ENSC integration using an adapted ex vivo organotypic culture system. Donor ENSC were obtained from Wnt1cre/+;R26RYFP/YFP mice allowing specific labelling, selection and fate-mapping of cells. YFP+ neurospheres were transplanted to C57BL6/J (6–8-week-old) colonic tissue and maintained in organotypic culture for up to 21 days. We analysed and quantified donor cell integration within recipient tissues at 7, 14 and 21 days, along with assessing the structural and molecular consequences of ENSC integration. We found that organotypically cultured tissues were well preserved up to 21-days in ex vivo culture, which allowed for assessment of donor cell integration after transplantation. Donor ENSC-derived cells integrated across the colonic wall in a dynamic fashion, across a three-week period. Following transplantation, donor cells displayed two integrative patterns; longitudinal migration and medial invasion which allowed donor cells to populate colonic tissue. Moreover, significant remodelling of the intestinal ECM and musculature occurred upon transplantation, to facilitate donor cell integration within endogenous enteric ganglia. These results provide critical evidence on the timescale and mechanisms, which regulate donor ENSC integration, within recipient gut tissue, which are important considerations in the future clinical translation of stem cell therapies for enteric disease.

scholarly journals Resistome Profiles, Plasmid Typing, and Whole-Genome Phylogenetic Tree Analyses of BlaNDM-9 and Mcr-1 Co-Harboring Escherichia coli ST617 from a Patient without a History of Farm Exposure in Korea

Pathogens ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 212 ◽  
Author(s):  
Le Phuong Nguyen ◽  
Naina Adren Pinto ◽  
Thao Nguyen Vu ◽  
Hung Mai ◽  
An HT Pham ◽  
...  
Keyword(s):  
Rectal Swab ◽  
Asymptomatic Carrier ◽  
University Hospital ◽  
Surveillance Culture ◽  
Whole Genome ◽  
E Coli ◽  
Carbapenem Resistant ◽  
Plasmid Replicon ◽  
Donor Cells ◽  
History Of

Recently, a blaNDM-9 and mcr-1 co-harboring E. coli ST 617 isolate was identified from an asymptomatic carrier in Korea. An 81-year-old female was admitted to a university hospital for aortic cardiac valve repair surgery. Following surgery, she was admitted to the intensive care unit (ICU) for three days, and carbapenem-resistant E. coli YMC/2017/02/MS631 was isolated from a surveillance culture (rectal swab). Antimicrobial susceptibility testing (AST) for colistin was not performed at that time. Upon retrospective study, further AST revealed resistance to all tested antibiotics, including meropenem, imipenem, ceftazidime-avibactam, amikacin, gentamicin, ciprofloxacin, trimethoprim-sulfamethoxazole, and colistin, with the exception of tigecycline. Whole genome sequencing analyses showed that this strain belonged to the ST617 serotype O89/162: H10 and harbored three β-lactamase genes (blaTEM-1B, blaCTX-M-55, blaNDM-9), mcr-1, and 14 other resistance genes. Seven plasmid replicon types (IncB, IncFII, IncI2, IncN, IncY, IncR, IncX1) were identified. Horizontal transfer of blaNDM-9 and mcr-1 from donor cells to the recipient E. coli J53 has been observed. blaNDM-9 and mcr-1 were carried by IncB and IncI2 plasmids, respectively. To speculate on the incidence of this strain, routine rectal swab screening to identify asymptomatic carriers might be warranted, in addition to the screening of ICU patients.

scholarly journals Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation

Reproduction ◽  
2007 ◽  
Vol 133 (1) ◽  
pp. 219-230 ◽  
Author(s):  
Feikun Yang ◽  
Ru Hao ◽  
Barbara Kessler ◽  
Gottfried Brem ◽  
Eckhard Wolf ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Nuclear Transfer ◽  
Donor Cell ◽  
Epigenetic Mark ◽  
Cell Type ◽  
Developmental Potential ◽  
Acetylation Status ◽  
Donor Cells ◽  
Donor Cell Type

The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase II oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres fromin vivofertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P<0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF,P<0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that inin vivofertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres fromin vivoderived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and may be a useful epigenetic mark to predict efficiency of SCNT in rabbits.

Production of somatic and germline chimeras in the chicken by transfer of early blastodermal cells

Development ◽  
1990 ◽  
Vol 108 (1) ◽  
pp. 185-189 ◽  
Author(s):  
J.N. Petitte ◽  
M.E. Clark ◽  
G. Liu ◽  
A.M. Verrinder Gibbins ◽  
R.J. Etches
Keyword(s):  
Inbred Line ◽  
Cell Lineage ◽  
Donor Cell ◽  
Black Pigment ◽  
Dominant Allele ◽  
White Leghorn ◽  
Sperm Dna ◽  
Donor Cells ◽  
White Leghorns ◽  
Blastodermal Cells

Cells were isolated from stage X embryos of a line of Barred Plymouth Rock chickens (that have black pigment in their feathers due to the recessive allele at the I locus) and injected into the subgerminal cavity of embryos from an inbred line of Dwarf White Leghorns (that have white feathers due to the dominant allele at the I locus). Of 53 Dwarf White Leghorn embryos that were injected with Barred Plymouth Rock blastodermal cells, 6 (11.3%) were phenotypically chimeric with respect to feather colour and one (a male) survived to hatching. The distribution of black feathers in the recipients was variable and not limited to a particular region although, in all but one case, the donor cell lineage was evident in the head. The male somatic chimera was mated to several Barred Plymouth Rock hens to determine the extent to which donor cells had been incorporated into his testes. Of 719 chicks hatched from these matings, 2 were phenotypically Barred Plymouth Rocks demonstrating that cells capable of incorporation into the germline had been transferred. Fingerprints of the blood and sperm DNA from the germline chimera indicated that both of these tissues were different from those of the inbred line of Dwarf White Leghorns. Bands that were present in fingerprints of blood DNA from the chimera and not present in those of the Dwarf White Leghorns were observed in those of the Barred Plymouth Rocks.(ABSTRACT TRUNCATED AT 250 WORDS)

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 Manipulating the Mitochondrial Genome To Enhance Cattle Embryo Development

2017 ◽  
Vol 7 (7) ◽  
pp. 2065-2080 ◽  
Author(s):  
Kanokwan Srirattana ◽  
Justin C St. John
Keyword(s):  
Embryo Development ◽  
Nuclear Transfer ◽  
Bos Taurus ◽  
Trichostatin A ◽  
Donor Cell ◽  
Blastocyst Stage ◽  
Genetic Integrity ◽  
Rna Seq ◽  
Mtdna Copy Number ◽  
Donor Cells

Abstract The mixing of mitochondrial DNA (mtDNA) from the donor cell and the recipient oocyte in embryos and offspring derived from somatic cell nuclear transfer (SCNT) compromises genetic integrity and affects embryo development. We set out to generate SCNT embryos that inherited their mtDNA from the recipient oocyte only, as is the case following natural conception. While SCNT blastocysts produced from Holstein (Bos taurus) fibroblasts were depleted of their mtDNA, and oocytes derived from Angus (Bos taurus) cattle possessed oocyte mtDNA only, the coexistence of donor cell and oocyte mtDNA resulted in blastocysts derived from nondepleted cells. Moreover, the use of the reprogramming agent, Trichostatin A (TSA), further improved the development of embryos derived from depleted cells. RNA-seq analysis highlighted 35 differentially expressed genes from the comparison between blastocysts generated from nondepleted cells and blastocysts from depleted cells, both in the presence of TSA. The only differences between these two sets of embryos were the presence of donor cell mtDNA, and a significantly higher mtDNA copy number for embryos derived from nondepleted cells. Furthermore, the use of TSA on embryos derived from depleted cells positively modulated the expression of CLDN8, TMEM38A, and FREM1, which affect embryonic development. In conclusion, SCNT embryos produced by mtDNA depleted donor cells have the same potential to develop to the blastocyst stage without the presumed damaging effect resulting from the mixture of donor and recipient mtDNA.

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