scholarly journals Unbiased homeologous recombination during pneumococcal transformation allows for multiple chromosomal integration events

2020 ◽  
Author(s):  
Jun Kurushima ◽  
Nathalie Campo ◽  
Renske van Raaphorst ◽  
Guillaume Cerckel ◽  
Patrice Polard ◽  
...  
Keyword(s):  
Integration Site ◽  
Dna Uptake ◽  
Exogenous Dna ◽  
Recombination Efficiency ◽  
Safe Strategy ◽  
Rapid Spread ◽  
Original Genome ◽  
Homeologous Recombination ◽  
Opportunistic Human Pathogen ◽  
Fail Safe

AbstractThe rapid spread of antimicrobial resistance and vaccine escape in the opportunistic human pathogen Streptococcus pneumoniae can be largely attributed to competence-induced transformation. To better understand the dynamics of competence-induced transformation, we studied this process at the single-cell level. We show that within isogenic populations, all cells become naturally competent and bind exogenous DNA. In addition, we find that transformation is highly efficient and that the chromosomal location of the integration site or whether the transformed gene is encoded on the leading or lagging strand has limited influence on recombination efficiency. Indeed, we have observed multiple recombination events in single recipients in real-time. However, because of saturation of the DNA uptake and integration machinery and because a single stranded donor DNA replaces the original allele, we find that transformation efficiency has an upper threshold of approximately 50% of the population. Counterintuitively, in the presence of multiple transforming DNAs, the fraction of untransformed cells increases to more than 50%. The fixed mechanism of transformation results in a fail-safe strategy for the population as half of the population generally keeps an intact copy of the original genome. Together, this work advances our understanding of pneumococcal genome plasticity.

scholarly journals Unbiased homeologous recombination during pneumococcal transformation allows for multiple chromosomal integration events

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Jun Kurushima ◽  
Nathalie Campo ◽  
Renske van Raaphorst ◽  
Guillaume Cerckel ◽  
Patrice Polard ◽  
...  
Keyword(s):  
Transformation Efficiency ◽  
Integration Site ◽  
Cell Level ◽  
Exogenous Dna ◽  
Recombination Efficiency ◽  
Safe Strategy ◽  
Original Genome ◽  
Homeologous Recombination ◽  
Fail Safe ◽  
Lagging Strand

The spread of antimicrobial resistance and vaccine escape in the human pathogen Streptococcus pneumoniae can be largely attributed to competence-induced transformation. Here, we studied this process at the single-cell level. We show that within isogenic populations, all cells become naturally competent and bind exogenous DNA. We find that transformation is highly efficient and that the chromosomal location of the integration site or whether the transformed gene is encoded on the leading or lagging strand has limited influence on recombination efficiency. Indeed, we have observed multiple recombination events in single recipients in real-time. However, because of saturation and because a single-stranded donor DNA replaces the original allele, transformation efficiency has an upper threshold of approximately 50% of the population. The fixed mechanism of transformation results in a fail-safe strategy for the population as half of the population generally keeps an intact copy of the original genome.

scholarly journals Comparison of different methods for exogenous DNA uptake by bovine spermatozoa

2015 ◽  
Vol 52 (1) ◽  
pp. 78 ◽  
Author(s):  
Renata Simões ◽  
Weber Beringui Feitosa ◽  
Marcella Pecora Milazzotto ◽  
Alessandra Corallo Nicacio ◽  
Flavia Regina Oliveira de Barros ◽  
...  
Keyword(s):  
Dna Uptake ◽  
Exogenous Dna ◽  
De Se ◽  
Bovine Spermatozoa

<p>Apesar da manipulação genética de animais domésticos ser de grande interesse para a produção animal e para a indústria farmacêutica, a sua eficiência ainda é insatisfatória. A injeção pronuclear, a técnica mais utilizada para tal proposito, principalmente em camundongos, ainda apresenta limitações para esta espécie. Algumas alternativas têm sido desenvolvidas como o uso de espermatozoides como vetores para transferência genica, na qual a célula espermática tem habilidade espontânea de se ligar a molécula de DNA e internaliza-la. Dado o potencial da transferência genica mediada por espermatozoide para animais domésticos transgênicos, o objetivo do presente trabalho foi a avaliação de quatro métodos de incorporação de DNA para a transferência genica mediada por espermatozoides na espécie bovina: incubação com DNA, alteração da membrana plasmática induzida por cálcio ionóforo seguida por incubação com o DNA exógeno, eletroporação e lipofecção. Espermatozoides não expostos ao DNA exógeno foram usados como grupo controle. Os índices de clivagem, blastocisto e eclosão foram avaliados, respectivamente, as 72 horas após a inseminação dos oócitos, bem como, aos 9 e 12 dias de cultivo embrionário. Os embriões positivos para o DNA exógeno foram avaliados por PCR. Nenhum efeito de tratamento foi observado nos índices de clivagem, blastocisto e eclosão. Além disso, a porcentagem de blastocistos positivos para o DNA exógeno não diferiu entre os grupos experimentais. Apesar do baixo número de embriões positivos para DNA exógeno, os resultados obtidos mostram que todos os tratamentos apresentaram eficiências similares. A conclusão obtida foi que, apesar de os índices de desenvolvimento embrionário terem sido similares e constante em todos os grupos experimentais, outros fatores como a sequência, o tamanho e a concentração do DNA exógeno devem ser avaliados para melhorar a transferência genica mediada por espermatozoides.</p>

scholarly journals Refining the Pneumococcal Competence Regulon by RNA Sequencing

2019 ◽  
Vol 201 (13) ◽  
Author(s):  
Jelle Slager ◽  
Rieza Aprianto ◽  
Jan-Willem Veening
Keyword(s):  
Antibiotic Resistance ◽  
Streptococcus Pneumoniae ◽  
Rna Sequencing ◽  
Genome Annotation ◽  
Stress Factors ◽  
Microarray Technology ◽  
Human Pathogen ◽  
Exogenous Dna ◽  
Content Type ◽  
Opportunistic Human Pathogen

ABSTRACTCompetence for genetic transformation allows the opportunistic human pathogenStreptococcus pneumoniaeto take up exogenous DNA for incorporation into its own genome. This ability may account for the extraordinary genomic plasticity of this bacterium, leading to antigenic variation, vaccine escape, and the spread of antibiotic resistance. The competence system has been thoroughly studied, and its regulation is well understood. Additionally, over the last decade, several stress factors have been shown to trigger the competent state, leading to the activation of several stress response regulons. The arrival of next-generation sequencing techniques allowed us to update the competence regulon, the latest report on which still depended on DNA microarray technology. Enabled by the availability of an up-to-date genome annotation, including transcript boundaries, we assayed time-dependent expression of all annotated features in response to competence induction, were able to identify the affected promoters, and produced a more complete overview of the various regulons activated during the competence state. We show that 4% of all annotated genes are under direct control of competence regulators ComE and ComX, while the expression of a total of up to 17% of all genes is affected, either directly or indirectly. Among the affected genes are various small RNAs with an as-yet-unknown function. Besides the ComE and ComX regulons, we were also able to refine the CiaR, VraR (LiaR), and BlpR regulons, underlining the strength of combining transcriptome sequencing (RNA-seq) with a well-annotated genome.IMPORTANCEStreptococcus pneumoniaeis an opportunistic human pathogen responsible for over a million deaths every year. Although both vaccination programs and antibiotic therapies have been effective in prevention and treatment of pneumococcal infections, respectively, the sustainability of these solutions is uncertain. The pneumococcal genome is highly flexible, leading to vaccine escape and antibiotic resistance. This flexibility is predominantly facilitated by competence, a state allowing the cell to take up and integrate exogenous DNA. Thus, it is essential to obtain a detailed overview of gene expression during competence. This is stressed by the fact that administration of several classes of antibiotics can lead to competence. Previous studies on the competence regulon were performed with microarray technology and were limited to an incomplete set of known genes. Using RNA sequencing combined with an up-to-date genome annotation, we provide an updated overview of competence-regulated genes.

404 DNA TAKE-UP BY SPERM AND IN VITRO EMBRYO DEVELOPMENT BY SPERM-MEDIATED GENE TRANSFER IN THE PIG

2007 ◽  
Vol 19 (1) ◽  
pp. 317
Author(s):  
T. S. Kim ◽  
Y. Cao ◽  
H. T. Cheong ◽  
B. K. Yang ◽  
C. K. Park
Keyword(s):  
Gene Transfer ◽  
Transfection Efficiency ◽  
The Other ◽  
Control Group ◽  
Dna Uptake ◽  
Alkaline Lysis ◽  
Exogenous Dna ◽  
Other Hand ◽  
Dna Complex

Sperm mediated gene transfer (SMGT) is based on the ability of spermatozoa to bind and internalize exogenous DNA and transfer it into the oocytes at fertilization. The purpose of this study was to assess introducing exogenous DNA into boar spermatozoa by DNA solution or DNA/liposome complex under different conditions (period of incubation, exogenous DNA, liposome, and concentration of spermatozoa). Genomic DNA of sperm loaded with DNA by treatment was isolated by alkaline lysis. Quantitation of exogenous DNA amplified by PCR was analyzed by agarose electrophoresis densitometry. The quality of treated spermatozoa under the best conditions or no treatment (control) was evaluated during incubation (0, 2, 4, and 6 h) for viability (SYBR-14/PI), motility (Makler counting chamber), morphology (rose bengal staining), and acrosomal status (Coomassie staining). Sperm loaded with DNA also were used for in vitro fertilization. Immature oocytes incubated in TCM-199 medium for 44 h were fertilized in mTBM medium for 6 h and cultured in PZM-3. Cleavage and development of embryos were assessed on Days 2 and 7 of culture, respectively. Transfection rates at the blastocyst stage were assessed by PCR analysis. Data were evaluated by Duncan&apos;s multiple-range test using the GLM procedure. In the preliminary experiment, DNA uptake of spermatozoa by DNA solution and liposome/DNA complex was completed within 90-120 min. Transfection efficiency of spermatozoa was significantly (P &lt; 0.05) higher in the 105 spermatozoa group than in the other groups (104, 106, and 107 spermatozoa). The transfection efficiency was gradually increased by increasing the concentration of exogenous DNA. On the other hand, viability of transfected spermatozoa by all treatments (control, DNA solution, and DNA/liposome) at 0 h (72.3 � 0.2, 70.8 � 1.8, and 68.0 � 2.2%, respectively) of storage was significantly (P &lt; 0.05) lower than for fresh spermatozoa (83.3 � 1.7%). Survival and motility of all treatments after 4 h of storage were significantly (P &lt; 0.05) lower than at 0 and 2 h. Both abnormality and acrosome reaction of spermatozoa were gradually increased with prolonged storage periods. On the other hand, the cleavage rate of embryos by DNA/liposome complex (56.3 � 2.3%) was significantly (P &lt; 0.05) lower compared to both DNA solution (64.0 � 1.1%) and control (67.8 � 2.3%). The developmental rates of blastocysts were significantly (P &lt; 0.05) lower in the liposome/DNA complex and DNA solution groups (9.1 � 1.3 and 11.3 � 0.8%) than in the control group (22.2 � 0.6%). The transfection rates of blastocysts were higher in the liposome/DNA group (54.3 � 12.0%) than in the DNA solution group (38.7 � 6.6%). These results show that the SMGT method under the control conditions efficiently transfers exogenous DNA into the porcine oocytes. This work was supported by the Research on the Production of Bio-organs (No. 2005 03020302) Ministry of Agriculture and Forestry, Republic of Korea

Liposome-mediated transfection of fetal lung epithelial cells: DNA degradation and enhanced superoxide toxicity

1998 ◽  
Vol 275 (3) ◽  
pp. L452-L460 ◽  
Author(s):  
A. Keith Tanswell ◽  
Olivier Staub ◽  
Richard Iles ◽  
Rosetta Belcastro ◽  
Judy Cabacungan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Reporter Gene ◽  
Fetal Lung ◽  
Reporter Gene Expression ◽  
Lung Epithelial Cells ◽  
Dna Uptake ◽  
Exogenous Dna ◽  
Lipid Ratio ◽  
Lung Epithelial

Cationic liposomes, 1:1 (mol/mol) 1,2-dioleoyldimethylammonium chloride-1,2-dioleoyl- sn-glycero-3-phosphoethanolamine, were used to transfect primary cultures of distal rat fetal lung epithelial cells with pCMV4-based plasmids. A DNA-to-lipid ratio of 1:10 to 1:15 (wt/wt) optimized DNA uptake over a 24-h exposure. At a fixed DNA-to-lipid ratio of 1:15, chloramphenicol acetyltransferase (CAT) reporter gene expression declined at lipid concentrations > 2.5 nmol/cm2 cell surface area, whereas DNA uptake remained concentration dependent. CAT expression peaked 48 h after removal of the liposome-DNA complex, declining thereafter. Reporter gene expression was increased, and supercoiled cDNA degradation was reduced by the addition of 0.2 mM nicotinamide and 10 μM chloroquine. Rat fetal lung epithelial cells transfected with two different expression cassettes had an increased susceptibility to superoxide-mediated cytotoxicity. This could be attributed to a nonspecific delivery of exogenous DNA or some other copurified factor. The DNA-dependent increase in superoxide-mediated cytotoxicity, but not basal levels of cytotoxicity, was inhibited by the addition of 0.2 mM nicotinamide and 10 μM chloroquine.

scholarly journals The Bacillus Subtilis K-State Promotes Stationary-Phase Mutagenesis via Oxidative Damage

Genes ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 190
Author(s):  
Holly A. Martin ◽  
Amanda A. Kidman ◽  
Jillian Socea ◽  
Carmen Vallin ◽  
Mario Pedraza-Reyes ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Oxidative Damage ◽  
Stationary Phase ◽  
Bacterial Cells ◽  
Dna Uptake ◽  
Cellular Division ◽  
Exogenous Dna ◽  
K Cells ◽  
Induced Mutagenesis ◽  
Underlying Mechanisms

Bacterial cells develop mutations in the absence of cellular division through a process known as stationary-phase or stress-induced mutagenesis. This phenomenon has been studied in a few bacterial models, including Escherichia coli and Bacillus subtilis; however, the underlying mechanisms between these systems differ. For instance, RecA is not required for stationary-phase mutagenesis in B. subtilis like it is in E. coli. In B. subtilis, RecA is essential to the process of genetic transformation in the subpopulation of cells that become naturally competent in conditions of stress. Interestingly, the transcriptional regulator ComK, which controls the development of competence, does influence the accumulation of mutations in stationary phase in B. subtilis. Since recombination is not involved in this process even though ComK is, we investigated if the development of a subpopulation (K-cells) could be involved in stationary-phase mutagenesis. Using genetic knockout strains and a point-mutation reversion system, we investigated the effects of ComK, ComEA (a protein involved in DNA transport during transformation), and oxidative damage on stationary-phase mutagenesis. We found that stationary-phase revertants were more likely to have undergone the development of competence than the background of non-revertant cells, mutations accumulated independently of DNA uptake, and the presence of exogenous oxidants potentiated mutagenesis in K-cells. Therefore, the development of the K-state creates conditions favorable to an increase in the genetic diversity of the population not only through exogenous DNA uptake but also through stationary-phase mutagenesis.

Exogenous DNA uptake by South American catfish (Rhamdia quelen) spermatozoa after seminal plasma removal

2011 ◽  
Vol 126 (1-2) ◽  
pp. 136-141 ◽  
Author(s):  
Vinicius F. Campos ◽  
Marta G. Amaral ◽  
Fabiana K. Seixas ◽  
Juvêncio L.F. Pouey ◽  
Lisiane P.R. Selau ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
South American ◽  
Dna Uptake ◽  
Exogenous Dna ◽  
Rhamdia Quelen

scholarly journals Recombination in Streptococcus pneumoniae Lineages Increase with Carriage Duration and Size of the Polysaccharide Capsule

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
Chrispin Chaguza ◽  
Cheryl P. Andam ◽  
Simon R. Harris ◽  
Jennifer E. Cornick ◽  
Marie Yang ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Homologous Recombination ◽  
Relative Rate ◽  
Specific Response ◽  
Dna Uptake ◽  
Exogenous Dna ◽  
Evolutionary Response ◽  
Polysaccharide Capsule ◽  
Capsule Size ◽  
Carriage Prevalence

ABSTRACT Streptococcus pneumoniae causes a high burden of invasive pneumococcal disease (IPD) globally, especially in children from resource-poor settings. Like many bacteria, the pneumococcus can import DNA from other strains or even species by transformation and homologous recombination, which has allowed the pneumococcus to evade clinical interventions such as antibiotics and pneumococcal conjugate vaccines (PCVs). Pneumococci are enclosed in a complex polysaccharide capsule that determines the serotype; the capsule varies in size and is associated with properties including carriage prevalence and virulence. We determined and quantified the association between capsule and recombination events using genomic data from a diverse collection of serotypes sampled in Malawi. We determined both the amount of variation introduced by recombination relative to mutation (the relative rate) and how many individual recombination events occur per isolate (the frequency). Using univariate analyses, we found an association between both recombination measures and multiple factors associated with the capsule, including duration and prevalence of carriage. Because many capsular factors are correlated, we used multivariate analysis to correct for collinearity. Capsule size and carriage duration remained positively associated with recombination, although with a reduced P  value, and this effect may be mediated through some unassayed additional property associated with larger capsules. This work describes an important impact of serotype on recombination that has been previously overlooked. While the details of how this effect is achieved remain to be determined, it may have important consequences for the serotype-specific response to vaccines and other interventions. IMPORTANCE The capsule determines >90 different pneumococcal serotypes, which vary in capsule size, virulence, duration, and prevalence of carriage. Current serotype-specific vaccines elicit anticapsule antibodies. Pneumococcus can take up exogenous DNA by transformation and insert it into its chromosome by homologous recombination. This mechanism has disseminated drug resistance and generated vaccine escape variants. It is hence crucial to pneumococcal evolutionary response to interventions, but there has been no systematic study quantifying whether serotypes vary in recombination and whether this is associated with serotype-specific properties such as capsule size or carriage duration. Larger capsules could physically inhibit DNA uptake, or given the longer carriage duration for larger capsules, this may promote recombination. We find that recombination varies among capsules and is associated with capsule size, carriage duration, and carriage prevalence and negatively associated with invasiveness. The consequence of this work is that serotypes with different capsules may respond differently to selective pressures like vaccines.

scholarly journals Silencing of natural transformation by an RNA chaperone and a multitarget small RNA

2016 ◽  
Vol 113 (31) ◽  
pp. 8813-8818 ◽  
Author(s):  
Laetitia Attaiech ◽  
Aïda Boughammoura ◽  
Céline Brochier-Armanet ◽  
Omran Allatif ◽  
Flora Peillard-Fiorente ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Natural Transformation ◽  
Bacterial Species ◽  
Physiological State ◽  
Uptake System ◽  
Dna Uptake ◽  
Exogenous Dna ◽  
Major Mechanism ◽  
Transcriptional Gene Regulation ◽  
High Level

A highly conserved DNA uptake system allows many bacteria to actively import and integrate exogenous DNA. This process, called natural transformation, represents a major mechanism of horizontal gene transfer (HGT) involved in the acquisition of virulence and antibiotic resistance determinants. Despite evidence of HGT and the high level of conservation of the genes coding the DNA uptake system, most bacterial species appear non-transformable under laboratory conditions. In naturally transformable species, the DNA uptake system is only expressed when bacteria enter a physiological state called competence, which develops under specific conditions. Here, we investigated the mechanism that controls expression of the DNA uptake system in the human pathogenLegionella pneumophila. We found that a repressor of this system displays a conserved ProQ/FinO domain and interacts with a newly characterizedtrans-acting sRNA, RocR. Together, they target mRNAs of the genes coding the DNA uptake system to control natural transformation. This RNA-based silencing represents a previously unknown regulatory means to control this major mechanism of HGT. Importantly, these findings also show that chromosome-encoded ProQ/FinO domain-containing proteins can assisttrans-acting sRNAs and that this class of RNA chaperones could play key roles in post-transcriptional gene regulation throughout bacterial species.

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