Natural transformation of the filamentous cyanobacterium Phormidium lacuna

Mapping Intimacies ◽

10.1101/870006 ◽

2019 ◽

Author(s):

Fabian Nies ◽

Marion Mielke ◽

Janko Pochert ◽

Tilman Lamparter

Keyword(s):

High Resistance ◽

Natural Transformation ◽

Selection Marker ◽

Filamentous Cyanobacterium ◽

Biotechnological Applications ◽

Natural Competence ◽

Bioreactor Design ◽

Homologous Integration

AbstractResearch for biotechnological applications of cyanobacteria focuses on synthetic pathways and bioreactor design, while little effort is devoted to introduce new, promising organisms in the field. Applications are most often based on recombinant work, and the establishment of transformation can be a risky, time-consuming procedure. In this work we demonstrate the natural transformation of the filamentous cyanobacterium Phormidium lacuna and insertion of a selection marker into the genome by homologous integration. This is the first example for natural transformation of a member of the order Oscillatoriales. We found that Phormidium lacuna is polyploid, each cell has about 20-100 chromosomes. Transformed filaments were resistant against up to 15 mg/ml of kanamycin, and the high resistance feature allowed for rapid segregation into all chromosomes. Formerly, natural transformation in cyanobacteria has been considered a rare and exclusive feature of a few unicellular species. Our finding suggests that natural competence is more distributed among cyanobacteria than previously thought. This is supported by bioinformatic analyses which show that all protein factors for natural transformation are present in the majority of the analyzed cyanobacteria.

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Serum Albumin and Ca2+Are Natural Competence Inducers in the Human Pathogen Acinetobacter baumannii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00529-16 ◽

2016 ◽

Vol 60 (8) ◽

pp. 4920-4929 ◽

Cited By ~ 23

Author(s):

German Matias Traglia ◽

Brettni Quinn ◽

Sareda T. J. Schramm ◽

Alfonso Soler-Bistue ◽

Maria Soledad Ramirez

Keyword(s):

Acinetobacter Baumannii ◽

Natural Transformation ◽

Hospital Environment ◽

Human Pathogen ◽

Nosocomial Pathogen ◽

Natural Competence ◽

Exogenous Dna ◽

Content Type ◽

Resistance Determinants ◽

Main Protein

ABSTRACTThe increasing frequency of bacteria showing antimicrobial resistance (AMR) raises the menace of entering into a postantibiotic era. Horizontal gene transfer (HGT) is one of the prime reasons for AMR acquisition.Acinetobacter baumanniiis a nosocomial pathogen with outstanding abilities to survive in the hospital environment and to acquire resistance determinants. Its capacity to incorporate exogenous DNA is a major source of AMR genes; however, few studies have addressed this subject. The transformation machinery as well as the factors that induce natural competence inA. baumanniiare unknown. In this study, we demonstrate that naturally competent strain A118 increases its natural transformation frequency upon the addition of Ca2+or albumin. We show thatcomEAandpilQare involved in this process since their expression levels are increased upon the addition of these compounds. An unspecific protein, like casein, does not reproduce this effect, showing that albumin's effect is specific. Our work describes the first specific inducers of natural competence inA. baumannii. Overall, our results suggest that the main protein in blood enhances HGT inA. baumannii, contributing to the increase of AMR in this threatening human pathogen.

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Spread of Recombinant DNA by Roots and Pollen of Transgenic Potato Plants, Identified by Highly Specific Biomonitoring Using Natural Transformation of an Acinetobacter sp

Applied and Environmental Microbiology ◽

10.1128/aem.69.8.4455-4462.2003 ◽

2003 ◽

Vol 69 (8) ◽

pp. 4455-4462 ◽

Cited By ~ 66

Author(s):

Johann de Vries ◽

Martin Heine ◽

Klaus Harms ◽

Wilfried Wackernagel

Keyword(s):

Recombinant Dna ◽

Natural Transformation ◽

High Sensitivity ◽

Transgenic Potato ◽

Kanamycin Resistance ◽

Selection Marker ◽

Potato Plants ◽

Gene Coding ◽

Transgenic Potato Plants ◽

Field Plots

ABSTRACT Transgenic potato plants with the nptII gene coding for neomycin phosphotransferase (kanamycin resistance) as a selection marker were examined for the spread of recombinant DNA into the environment. We used the recombinant fusion of nptII with the tg4 terminator for a novel biomonitoring technique. This depended on natural transformation of Acinetobacter sp. strain BD413 cells having in their genomes a terminally truncated nptII gene (nptII′; kanamycin sensitivity) followed by the tg4 terminator. Integration of the recombinant fusion DNA by homologous recombination in nptII′ and tg4 restored nptII, leading to kanamycin-resistant transformants. DNA of the transgenic potato was detectable with high sensitivity, while no transformants were obtained with the DNA of other transgenic plants harboring nptII in different genetic contexts. The recombinant DNA was frequently found in rhizosphere extracts of transgenic potato plants from field plots. In a series of field plot and greenhouse experiments we identified two sources of this DNA: spread by roots during plant growth and by pollen during flowering. Both sources also contributed to the spread of the transgene into the rhizospheres of nontransgenic plants in the vicinity. The longest persistence of transforming DNA in field soil was observed with soil from a potato field in 1997 sampled in the following year in April and then stored moist at 4°C in the dark for 4 years prior to extract preparation and transformation. In this study natural transformation is used as a reliable laboratory technique to detect recombinant DNA but is not used for monitoring horizontal gene transfer in the environment.

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Natural Transformation of Gallibacterium anatis

Applied and Environmental Microbiology ◽

10.1128/aem.00412-12 ◽

2012 ◽

Vol 78 (14) ◽

pp. 4914-4922 ◽

Cited By ~ 18

Author(s):

Bodil M. Kristensen ◽

Sunita Sinha ◽

John D. Boyce ◽

Anders M. Bojesen ◽

Joshua C. Mell ◽

...

Keyword(s):

Bioinformatics Analysis ◽

Natural Transformation ◽

Genetic Manipulation ◽

Targeted Mutagenesis ◽

Chromosomal Dna ◽

Natural Competence ◽

Content Type ◽

Transformation Procedure ◽

High Transformation ◽

Dna Types

ABSTRACTGallibacterium anatisis a pathogen of poultry. Very little is known about its genetics and pathogenesis. To enable the study of gene function inG. anatis, we have established methods for transformation and targeted mutagenesis. The genusGallibacteriumbelongs to thePasteurellaceae, a group with several naturally transformable members, includingHaemophilus influenzae. Bioinformatics analysis identifiedG. anatishomologs of theH. influenzaecompetence genes, and natural competence was induced inG. anatisby the procedure established forH. influenzae: transfer from rich medium to the starvation medium M-IV. This procedure gave reproducibly high transformation frequencies withG. anatischromosomal DNA and with linearized plasmid DNA carryingG. anatissequences. Both DNA types integrated into theG. anatischromosome by homologous recombination. Targeted mutagenesis gave transformation frequencies of >2 × 10−4transformants CFU−1. Transformation was also efficient with circular plasmid containing noG. anatisDNA; this resulted in the establishment of a self-replicating plasmid. Nine diverseG. anatisstrains were found to be naturally transformable by this procedure, suggesting that natural competence is common and the M-IV transformation procedure widely applicable for this species. TheG. anatisgenome is only slightly enriched for the uptake signal sequences identified in other pasteurellaceaen genomes, butG. anatisdid preferentially take up its own DNA over that ofEscherichia coli. Transformation by electroporation was not effective for chromosomal integration but could be used to introduce self-replicating plasmids. The findings described here provide important tools for the genetic manipulation ofG. anatis.

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Interplay between meropenem and human serum albumin on expression of carbapenem resistance genes and natural competence in Acinetobacter baumannii

10.1101/2021.05.14.444273 ◽

2021 ◽

Author(s):

Camila Pimentel ◽

Casin Le ◽

Marisel Romina Tuttobene ◽

Tomas Subils ◽

Brent Nishimura ◽

...

Keyword(s):

Human Serum Albumin ◽

Serum Albumin ◽

Human Serum ◽

Acinetobacter Baumannii ◽

Resistance Genes ◽

Natural Transformation ◽

Natural Competence ◽

Expression Levels ◽

Carbapenem Resistant ◽

Synergistic Enhancement

Acinetobacter baumannii A118, a mostly susceptible strain, and AB5075, carbapenem-resistant, were cultured in L-broth or L-broth with different supplements: 3.5% human serum albumin (HSA), human serum (HS), meropenem, or meropenem plus 3.5% HSA. Natural transformation levels were enhanced in A. baumannii A118 and AB5075 cultured in medium supplemented with 3.5 % HSA. Addition of meropenem plus 3.5% HSA caused synergistic enhancement of natural transformation inA. baumannii A118. Medium containing 3.5% HSA or meropenem enhanced the expression levels of the competence and type IV pilus associated genes. The combination meropenem plus 3.5% HSA produced a synergistic augmentation in the expression levels of many of these genes. The addition of HS, which has a high content of HSA, was also an inducer of these genes. Cultures in medium supplemented with HS or 3.5% HSA also affected resistance genes, which were expressed at higher or lower levels depending on the modification required to enhance resistance. The inducing or repressing activity of these modulators also occurred in three more carbapenem-resistant strains tested. An exception was the A. baumanniiAMA16 blaNDM-1 gene, which was repressed in the presence of 3.5% HSA. In conclusion, HSA produces an enhancement of natural transformation and a modification in expression levels of competence genes and antibiotic resistance. Furthermore, when HSA is combined with carbapenems, which may produce stronger cellular stress, the A. baumannii responds increasing the levels of expression of genes involved in natural competence. This process may favor the acquisition of foreign DNA and accelerate evolution.

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Characterization of a ComE3 Homologue Essential for DNA Transformation in Helicobacter pylori

Infection and Immunity ◽

10.1128/iai.71.9.5427-5431.2003 ◽

2003 ◽

Vol 71 (9) ◽

pp. 5427-5431 ◽

Cited By ~ 26

Author(s):

Yu-Ching Yeh ◽

Tzu-Lung Lin ◽

Kai-Chih Chang ◽

Jin-Town Wang

Keyword(s):

Helicobacter Pylori ◽

Dna Binding ◽

Natural Transformation ◽

Open Reading Frames ◽

Dna Transformation ◽

Knockout Mutant ◽

Natural Competence ◽

Mutant Complementation ◽

Reading Frames

ABSTRACT To find genes involved in natural competence in Helicobacter pylori, we used a bioinformatics database search and found two transformation-related open reading frames (ORFs): a comE3 homologue (HP1361 ORF) of Bacillus subtilis and a comL homologue (HP1378 ORF) of Neisseria gonorrhoeae. We failed to obtain an HP1378 ORF knockout mutant, while an HP1361 ORF knockout mutant was obtained by transposon shuttle mutagenesis. The DNA transformation abilities of both natural transformation and electroporation were severely impaired (frequency, <10−9) in the HP1361− mutant. Complementation with a pHel2 vector carrying the HP1361 ORF restored the capabilities of natural competence (to a frequency of 4.21 × 10−7) and electroporation (to 3.62 × 10−7). The HP1361− mutant showed impairment in DNA binding and uptake. The results suggest that HP1361 is a comE3 homologue and is required for DNA binding and uptake during DNA transformation.

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Association of Metronidazole Resistance and Natural Competence in Helicobacter pylori

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.5.1564-1567.2002 ◽

2002 ◽

Vol 46 (5) ◽

pp. 1564-1567 ◽

Cited By ~ 12

Author(s):

Yu-Ching Yeh ◽

Kai-Chin Chang ◽

Jyh-Chin Yang ◽

Chi-Tai Fang ◽

Jin-Town Wang

Keyword(s):

Helicobacter Pylori ◽

Natural Transformation ◽

23S Rrna ◽

Dilution Method ◽

Agar Dilution Method ◽

Rrna Gene ◽

Natural Competence ◽

Metronidazole Resistance ◽

23S Rrna Gene ◽

Agar Dilution

ABSTRACT To study whether the capability of horizontal DNA transfer is associated with metronidazole resistance in Helicobacter pylori, a total of 81 clinical isolates were tested for MICs of metronidazole (MTZ). The MIC assays were performed by using the E-test and reconfirmed by the agar dilution method. Natural competence assays were performed by transferring a chloramphenicol acetyltransferase cassette and a 23S rRNA gene from a clarithromycin-resistant strain (with an A-to-G mutation at nucleotide 2143) by using natural transformation. Of the 81 isolates, 65 (80.2%) were naturally competent while 16 were not. Among the 65 naturally competent strains, 39 (60%) were highly resistant to MTZ (MICs, >32 μg/ml) while only 2 of 16 (12.5%) noncompetent strains were highly MTZ resistant (P, <0.001). Therefore, there is an association between natural competence and MTZ resistance.

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Natural competence in Chlorogloeopsis fritschii PCC 6912 and other ramified cyanobacteria

10.1101/2020.06.19.162503 ◽

2020 ◽

Author(s):

Benjamin L. Springstein ◽

Fabian Nies ◽

Tal Dagan

Keyword(s):

Natural Transformation ◽

Photosynthetic Apparatus ◽

Arid Environments ◽

Natural Competence ◽

Type Iv ◽

Chlorogloeopsis Fritschii ◽

Competence Proteins ◽

Global Biogeochemical Cycles ◽

Dna Acquisition

AbstractLateral DNA transfer plays an important role in the evolution of genetic diversity in prokaryotes. DNA acquisition via transformation involves the uptake of DNA from the environment. The ability of recipient cells to actively transport DNA into the cytoplasm – termed natural competence – depends on the presence of type IV pili and competence proteins. Natural competence has been described in cyanobacteria for several organisms including unicellular and filamentous species. However, the presence of natural competence in ramified cyanobacteria, which are considered the peak of cyanobacterial morphological complexity, remains unknown. Here we show that ramified cyanobacteria harbour the genes essential for natural competence and experimentally demonstrate natural competence in the ramified cyanobacterium Chlorogloeopsis fritschii PCC 6912 (hereafter Chlorogloeopsis). Searching for homologs to known natural competence genes in ramified cyanobacteria showed that these genes are conserved in the majority of tested isolates. Experimental validation of natural competence using several alternative protocols demonstrates that Chlorogloeopsis could be naturally transformed with a replicative plasmid. Our results show that natural competence is a common trait in ramified cyanobacteria and that natural transformation is likely to play an important role in cyanobacteria evolution.ImportanceCyanobacteria are crucial players in the global biogeochemical cycles where they contribute to CO2- and N2-fixation. Their main ecological significance is the oxygen-producing photosynthetic apparatus that contributes to contemporary food chains. Ramified cyanobacteria form true-branching and multiseriate cell filament structures that represent a peak of prokaryotic multicellularity. Species in that group inhabit fresh and marine water habitats, thermal springs, arid environments, as well as endolithic and epiphytic habitats. Here we show that ramified cyanobacteria harbor the mechanisms required for DNA acquisition via natural transformation. The prevalence of mechanisms for natural uptake of DNA has implications for the role of DNA acquisition in the evolution of cyanobacteria. Furthermore, presence of mechanisms for natural transformation in ramified cyanobacteria opens up new possibilities for genetic modification of ramified cyanobacteria.

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Engineering natural competence into the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973

Applied and Environmental Microbiology ◽

10.1128/aem.01882-21 ◽

2021 ◽

Author(s):

Kristen E. Wendt ◽

Patricia Walker ◽

Annesha Sengupta ◽

Justin Ungerer ◽

Himadri B. Pakrasi

Keyword(s):

Natural Transformation ◽

Model Organism ◽

Model System ◽

Research Community ◽

Synechococcus Elongatus ◽

Model Systems ◽

Extracellular Dna ◽

Natural Competence ◽

Fast Growing ◽

Cyanobacterial Species

Natural transformation is the process by which bacteria actively take up and integrate extracellular DNA into their genomes. In cyanobacteria, natural transformation has only been experimentally demonstrated in a handful of species. Although, cyanobacteria are important model systems for studying photosynthesis and circadian cycling, natural transformation in cyanobacteria has not been characterized to the degree that the process has been studied in other gram-negative bacteria. Two cyanobacterial species that are 99.8% genetically identical provide a unique opportunity to better understand the nuances of natural transformation in cyanobacteria: Synechococcus elongatus PCC 7942 and Synechococcus elongatus UTEX 2973 (hereafter Synechococcus 7942 and Synechococcus 2973 respectively). Synechococcus 7942 is a naturally transformable model system, while Synechococcus 2973 is a recently discovered species that is not naturally competent. Taking only 1.5 hours to replicate, Synechococcus 2973 is the fastest growing cyanobacterial species known, and thus is a strong candidate for serving as a model organism. However, the organism’s inability to undergo natural transformation has prevented it from becoming a widely used model system. By substituting polymorphic alleles from Synechococcus 7942 for native Synechococcus 2973 alleles, natural transformation was introduced into Synechococcus 2973. Two genetic loci were found to be involved in differential natural competence between the two organisms: transformation pilus component pilN and circadian transcriptional master regulator rpaA . By using targeting genome editing and enrichment outgrowth, a strain that was both naturally transformable and fast-growing was created. This new Synechococcus 2973-T strain will serve as a valuable resource to the cyanobacterial research community. Importance Certain bacterial species have the ability to take up naked extracellular DNA and integrate it into their genomes. This process is known as natural transformation and is widely considered to play a major role in bacterial evolution. Because of the ease of introducing new genes into naturally transformable organisms, this capacity is also highly valued in the laboratory. Cyanobacteria are photosynthetic and can therefore serve as model systems for some important aspects of plant physiology. Here, we describe the creation of a modified cyanobacterial strain ( Synechococcus 2973-T) that is capable of undergoing natural transformation and has a replication time that is on par with the fastest-growing cyanobacterium that has been discovered to date. This new cyanobacterium has the potential to serve as a new model organism for the cyanobacterial research community and will allow experiments to be completed in a fraction of the time that it took to complete previous assays.

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CRISPR-Cas Inhibits Natural Transformation Through Altruistic Group Defense and Self-Sacrifice

10.1101/2021.09.16.460680 ◽

2021 ◽

Author(s):

Robert M. Cooper ◽

Jeff Hasty

Keyword(s):

Cell Death ◽

Natural Transformation ◽

Experimental Studies ◽

Fitness Costs ◽

Acinetobacter Baylyi ◽

Natural Competence ◽

New Genes ◽

Crispr Array ◽

Genomic Analyses ◽

Group Defense

SummaryCRISPR-Cas systems present an evolutionary tradeoff: does defense against phages and other parasitic DNA also prevent cells from acquiring potentially helpful new genes? Genomic analyses of this conundrum have arrived at often contradictory conclusions. Meanwhile, experimental studies have focused mainly on phages, conjugation, or artificial transformation, but less work has examined natural competence, a major driver of evolution and antibiotic resistance. Here, we use Acinetobacter baylyi, which combines high natural competence with a functional CRISPR-Cas system, to experimentally probe the interactions between CRISPR-Cas and natural competence. In these bacteria, the endogenous CRISPR array largely allows natural transformation by targeted DNA. However, CRISPR-Cas then kills the newly autoimmune cells in a form of programmed cell death. CRISPR-Cas often allows self-targeting cells to form colonies, albeit with fitness costs. Thus CRISPR-Cas appears to block natural transformation in a process more akin to altruistic group defense than an individual immune system.

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