scholarly journals Direct Visualization of Horizontal Gene Transfer by Transformation in Live Pneumococcal Cells Using Microfluidics

Genes ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. 675
Author(s):  
Isabelle Mortier-Barrière ◽  
Patrice Polard ◽  
Nathalie Campo
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Expression Profiles ◽  
Physiological State ◽  
Microfluidic System ◽  
Live Cells ◽  
Microfluidic Channels ◽  
Dna Uptake ◽  
Fluorescent Reporters ◽  
Natural Genetic Transformation

Natural genetic transformation is a programmed mechanism of horizontal gene transfer in bacteria. It requires the development of competence, a specialized physiological state during which proteins involved in DNA uptake and chromosomal integration are produced. In Streptococcus pneumoniae, competence is transient. It is controlled by a secreted peptide pheromone, the competence-stimulating peptide (CSP) that triggers the sequential transcription of two sets of genes termed early and late competence genes, respectively. Here, we used a microfluidic system with fluorescence microscopy to monitor pneumococcal competence development and transformation, in live cells at the single cell level. We present the conditions to grow this microaerophilic bacterium under continuous flow, with a similar doubling time as in batch liquid culture. We show that perfusion of CSP in the microfluidic chamber results in the same reduction of the growth rate of individual cells as observed in competent pneumococcal cultures. We also describe newly designed fluorescent reporters to distinguish the expression of competence genes with temporally distinct expression profiles. Finally, we exploit the microfluidic technology to inject both CSP and transforming DNA in the microfluidic channels and perform near real time-tracking of transformation in live cells. We show that this approach is well suited to investigating the onset of pneumococcal competence together with the appearance and the fate of transformants in individual cells.

scholarly journals Natural Genetic Transformation in Monoculture Acinetobacter sp. Strain BD413 Biofilms

2003 ◽  
Vol 69 (3) ◽  
pp. 1721-1727 ◽  
Author(s):  
Larissa Hendrickx ◽  
Martina Hausner ◽  
Stefan Wuertz
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Genetic Transformation ◽  
Exposure Time ◽  
Growth Phase ◽  
Natural Transformation ◽  
Optimal Growth ◽  
Continuous Exposure ◽  
Dna Concentration ◽  
Natural Genetic Transformation

ABSTRACT Horizontal gene transfer by natural genetic transformation in Acinetobacter sp. strain BD413 was investigated by using gfp carried by the autonomously replicating plasmid pGAR1 in a model monoculture biofilm. Biofilm age, DNA concentration, and biofilm mode of growth were evaluated to determine their effects on natural genetic transformation. The highest transfer frequencies were obtained in young and actively growing biofilms when high DNA concentrations were used and when the biofilm developed during continuous exposure to fresh medium without the presence of a significant amount of cells in the suspended fraction. Biofilms were highly amenable to natural transformation. They did not need to advance to an optimal growth phase which ensured the presence of optimally competent biofilm cells. An exposure time of only 15 min was adequate for transformation, and the addition of minute amounts of DNA (2.4 fg of pGAR1 per h) was enough to obtain detectable transfer frequencies. The transformability of biofilms lacking competent cells due to growth in the presence of cells in the bulk phase could be reestablished by starving the noncompetent biofilm prior to DNA exposure. Overall, the evidence suggests that biofilms offer no barrier against effective natural genetic transformation of Acinetobacter sp. strain BD413.

Genetic Competence and Transformation in Oral Streptococci

2001 ◽  
Vol 12 (3) ◽  
pp. 217-243 ◽  
Author(s):  
D.G. Cvitkovitch
Keyword(s):  
Genetic Transformation ◽  
Genetic Information ◽  
Physiological State ◽  
Oral Streptococci ◽  
Dna Uptake ◽  
Genetic Competence ◽  
Gram Positive Bacteria ◽  
Oral Biofilms ◽  
Foreign Dna ◽  
Natural Genetic Transformation

The oral streptococci are normally non-pathogenic residents of the human microflora. There is substantial evidence that these bacteria can, however, act as "genetic reservoirs" and transfer genetic information to transient bacteria as they make their way through the mouth, the principal entry point for a wide variety of bacteria. Examples that are of particular concern include the transfer of antibiotic resistance from oral streptococci to Streptococcus pneumoniae. The mechanisms that are used by oral streptococci to exchange genetic information are not well-understood, although several species are known to enter a physiological state of genetic competence. This state permits them to become capable of natural genetic transformation, facilitating the acquisition of foreign DNA from the external environment. The oral streptococci share many similarities with two closely related Gram-positive bacteria. S. pneumoniae and Bacillus subtilis. In these bacteria, the mechanisms of quorum-sensing, the development of competence, and DNA uptake and integration are well-charaterized. Using this knowledge and the data available in genome databases allowed us to identify putative genes involved in these processes in the oral organism Streptococcus mutans. Models of competence development and genetic transformation in the oral streptococci and strategies to confirm these models are discussed. Future studies of competence in oral biofilms, the natural environment of oral streptococci, will be discussed.

scholarly journals Horizontal gene transfer facilitates the spread of extracellular antibiotic resistance genes in soil

2021 ◽  
Author(s):  
Heather A. Kittredge ◽  
Kevin M. Dougherty ◽  
Sarah E. Evans
Keyword(s):  
Antibiotic Resistance ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Resistance Genes ◽  
Natural Transformation ◽  
Antibiotic Resistance Genes ◽  
Live Cells ◽  
Resistant Bacteria ◽  
Antibiotic Resistant Bacteria ◽  
Antibiotic Resistant

AbstractAntibiotic resistance genes (ARGs) are ubiquitous in the environment and pose a serious risk to human and veterinary health. While many studies focus on the spread of live antibiotic resistant bacteria throughout the environment, it is unclear whether extracellular ARGs from dead cells can transfer to live bacteria to facilitate the evolution of antibiotic resistance in nature. Here, we inoculate antibiotic-free soil with extracellular ARGs (eARGs) from dead Pseudeononas stutzeri cells and track the evolution of antibiotic resistance via natural transformation – a mechanism of horizontal gene transfer involving the genomic integration of eARGs. We find that transformation facilitates the rapid evolution of antibiotic resistance even when eARGs occur at low concentrations (0.25 μg g-1 soil). However, when eARGs are abundant, transformation increases substantially. The evolution of antibiotic resistance was high under soil moistures typical in terrestrial systems (5%-30% gravimetric water content) and was only inhibited at very high soil moistures (>30%). While eARGs transformed into live cells at a low frequency, exposure to a low dose of antibiotic allowed a small number of transformants to reach high abundances in laboratory populations, suggesting even rare transformation events pose a risk to human health. Overall, this work demonstrates that dead bacteria and their eARGs are an overlooked path to antibiotic resistance, and that disinfection alone is insufficient to stop the spread of antibiotic resistance. More generally, the spread of eARGs in antibiotic-free soil suggests that transformation allows genetic variants to establish at low frequencies in the absence of antibiotic selection.ImportanceOver the last decade, antibiotics in the environment have gained increasing attention because they can select for drug-resistant phenotypes that would have otherwise gone extinct. To counter this effect, bacterial populations exposed to antibiotics often undergo disinfection. However, the release of extracellular antibiotic resistance genes (eARGs) into the environment following disinfection can promote the transfer of eARGs through natural transformation. This phenomenon is well-documented in wastewater and drinking water, but yet to be investigated in soil. Our results directly demonstrate that eARGs from dead bacteria are an important, but often overlooked source of antibiotic resistance in soil. We conclude that disinfection alone is insufficient to prevent the spread of ARGs. Special caution should be taken in releasing antibiotics into the environment, even if there are no live antibiotic resistant bacteria in the community, as transformation allows DNA to maintain its biological activity past microbial death.

scholarly journals A Conserved Metal Binding Motif in the Bacillus subtilis Competence Protein ComFA Enhances Transformation

2017 ◽  
Vol 199 (15) ◽  
Author(s):  
Scott S. Chilton ◽  
Tanya G. Falbel ◽  
Susan Hromada ◽  
Briana M. Burton
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Metal Binding ◽  
Dna Uptake ◽  
Content Type ◽  
Efficient Transformation ◽  
Dead Box ◽  
The Dead

ABSTRACT Genetic competence is a process in which cells are able to take up DNA from their environment, resulting in horizontal gene transfer, a major mechanism for generating diversity in bacteria. Many bacteria carry homologs of the central DNA uptake machinery that has been well characterized in Bacillus subtilis. It has been postulated that the B. subtilis competence helicase ComFA belongs to the DEAD box family of helicases/translocases. Here, we made a series of mutants to analyze conserved amino acid motifs in several regions of B. subtilis ComFA. First, we confirmed that ComFA activity requires amino acid residues conserved among the DEAD box helicases, and second, we show that a zinc finger-like motif consisting of four cysteines is required for efficient transformation. Each cysteine in the motif is important, and mutation of at least two of the cysteines dramatically reduces transformation efficiency. Further, combining multiple cysteine mutations with the helicase mutations shows an additive phenotype. Our results suggest that the helicase and metal binding functions are two distinct activities important for ComFA function during transformation. IMPORTANCE ComFA is a highly conserved protein that has a role in DNA uptake during natural competence, a mechanism for horizontal gene transfer observed in many bacteria. Investigation of the details of the DNA uptake mechanism is important for understanding the ways in which bacteria gain new traits from their environment, such as drug resistance. To dissect the role of ComFA in the DNA uptake machinery, we introduced point mutations into several motifs in the protein sequence. We demonstrate that several amino acid motifs conserved among ComFA proteins are important for efficient transformation. This report is the first to demonstrate the functional requirement of an amino-terminal cysteine motif in ComFA.

scholarly journals Fine-tuning cellular levels of DprA ensures transformant fitness in the human pathogenStreptococcus pneumoniae

2018 ◽  
Author(s):  
Calum Johnston ◽  
Isabelle Mortier-Barriere ◽  
Vanessa Khemici ◽  
Patrice Polard
Keyword(s):  
Streptococcus Pneumoniae ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Physiological State ◽  
Fine Tuning ◽  
Primary Role ◽  
Human Pathogen ◽  
Adaptive Potential ◽  
Genetic Traits ◽  
Full Expression

AbstractNatural genetic transformation is a widespread mechanism of bacterial horizontal gene transfer. Transformation involves the internalization of exogenous DNA as single strands, followed by chromosomal integration via homologous recombination, promoting acquisition of new genetic traits. Transformation occurs during a distinct physiological state called competence, during which all proteins required to transform are produced. In the human pathogenStreptococcus pneumoniae, competence is controlled by a two-component system ComDE, which is induced by an exported peptide pheromone. DprA is universal among transformable species, strongly and specifically induced during pneumococcal competence, and crucial for pneumococcal transformation. Pneumococcal DprA plays three crucial roles in transformation and competence. Firstly, DprA protects internalized single-stranded (ss) DNA from degradation. Secondly, DprA loads the homologous recombinase RecA onto transforming ssDNA to promote transformation. Finally, DprA interacts with the response regulator ComE to shut-off pneumococcal competence. Pneumococcal shut-off has been linked to physiology, with long growth delays in competentdprA-cells. Here, we explored the effect of altering the cellular levels of DprA on these three roles. High cellular levels of DprA were not required for the primary role of DprA as a transformation-dedicated recombinase loader or for protection of transforming ssDNA. In contrast, full expression ofdprAwas required for optimal competence shut-off. Full expression ofdprAwas also crucial for transformant fitness. High cellular levels of DprA in competent cells thus ensure the fitness of pneumococcal transformants by promoting competence shut-off. This promotes survival and propagation of transformants, thus maximizing the adaptive potential of this human pathogen.ImportanceTransformation is a widespread mechanism of horizontal gene transfer that allows bacteria to acquire new genetic traits by internalizing foreign DNA and integrating it into their chromosomes. Transformation occurs during a transient physiological state called competence. DprA is conserved in transformable species and crucial for the protection and integration of transforming DNA. In the human pathogenStreptococcus pneumoniae, DprA is highly abundant and is also crucial for competence shut-off. Here, we show that high DprA expression is not required for transformation. In contrast, full expression ofdprAwas required for competence shut-off and transformant fitness. These findings thus link high cellular levels of DprA to survival and propagation of pneumococcal transformants, maximizing the adaptive potential of this human pathogen.

Complexity of genetic sequences modified by horizontal gene transfer and degraded-DNA uptake

2015 ◽  
Author(s):  
George Tremberger ◽  
S. Dehipawala ◽  
A. Nguyen ◽  
E. Cheung ◽  
R. Sullivan ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Degraded Dna ◽  
Dna Uptake ◽  
Genetic Sequences

Mechanisms of DNA Uptake by Naturally Competent Bacteria

2019 ◽  
Vol 53 (1) ◽  
pp. 217-237 ◽  
Author(s):  
David Dubnau ◽  
Melanie Blokesch
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Outer Membrane ◽  
Growth Arrest ◽  
Dna Binding Protein ◽  
Gram Negative Bacteria ◽  
Dna Uptake ◽  
Gram Negative ◽  
Gram Positive Bacteria ◽  
Proton Symport

Transformation is a widespread mechanism of horizontal gene transfer in bacteria. DNA uptake to the periplasmic compartment requires a DNA-uptake pilus and the DNA-binding protein ComEA. In the gram-negative bacteria, DNA is first pulled toward the outer membrane by retraction of the pilus and then taken up by binding to periplasmic ComEA, acting as a Brownian ratchet to prevent backward diffusion. A similar mechanism probably operates in the gram-positive bacteria as well, but these systems have been less well characterized. Transport, defined as movement of a single strand of transforming DNA to the cytosol, requires the channel protein ComEC. Although less is understood about this process, it may be driven by proton symport. In this review we also describe various phenomena that are coordinated with the expression of competence for transformation, such as fratricide, the kin-discriminatory killing of neighboring cells, and competence-mediated growth arrest.

scholarly journals Mechanisms of Transforming DNA Uptake to the Periplasm of Bacillus subtilis

mBio ◽  
2021 ◽  
Author(s):  
Jeanette Hahn ◽  
Micaela DeSantis ◽  
David Dubnau
Keyword(s):  
Antibiotic Resistance ◽  
Bacillus Subtilis ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Molecular Mechanism ◽  
Virulence Genes ◽  
Bacterial Species ◽  
Dna Uptake

Transformation is a widely distributed mechanism of bacterial horizontal gene transfer that plays a role in the spread of antibiotic resistance and virulence genes and more generally in evolution. Although transformation was discovered nearly a century ago and most, if not all the proteins required have been identified in several bacterial species, much remains poorly understood about the molecular mechanism of DNA uptake.

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