scholarly journals Natural Transformation of Gallibacterium anatis

2012 ◽  
Vol 78 (14) ◽  
pp. 4914-4922 ◽  
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.

scholarly journals Use of Natural Transformation To Establish an Easy Knockout Method in Riemerella anatipestifer

2017 ◽  
Vol 83 (9) ◽  
Author(s):  
MaFeng Liu ◽  
Li Zhang ◽  
Li Huang ◽  
Francis Biville ◽  
DeKang Zhu ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Natural Transformation ◽  
Genetic Manipulation ◽  
Gene Knockout ◽  
Targeted Mutagenesis ◽  
Chromosomal Dna ◽  
Content Type ◽  
Riemerella Anatipestifer ◽  
The Family ◽  
First Time

ABSTRACT Riemerella anatipestifer is a member of the family Flavobacteriaceae and a major causative agent of duck serositis. Little is known about its genetics and pathogenesis. Several bacteria are competent for natural transformation; however, whether R. anatipestifer is also competent for natural transformation has not been investigated. Here, we showed that R. anatipestifer strain ATCC 11845 can uptake the chromosomal DNA of R. anatipestifer strain RA-CH-1 in all growth phases. Subsequently, a natural transformation-based knockout method was established for R. anatipestifer ATCC 11845. Targeted mutagenesis gave transformation frequencies of ∼10−5 transformants. Competition assay experiments showed that R. anatipestifer ATCC 11845 preferentially took up its own DNA rather than heterogeneous DNA, such as Escherichia coli DNA. Transformation was less efficient with the shuttle plasmid pLMF03 (transformation frequencies of ∼10−9 transformants). However, the efficiency of transformation was increased approximately 100-fold using pLMF03 derivatives containing R. anatipestifer DNA fragments (transformation frequencies of ∼10−7 transformants). Finally, we found that the R. anatipestifer RA-CH-1 strain was also naturally transformable, suggesting that natural competence is widely applicable for this species. The findings described here provide important tools for the genetic manipulation of R. anatipestifer. IMPORTANCE Riemerella anatipestifer is an important duck pathogen that belongs to the family Flavobacteriaceae. At least 21 different serotypes have been identified. Genetic diversity has been demonstrated among these serotypes. The genetic and pathogenic mechanisms of R. anatipestifer remain largely unknown because no genetic tools are available for this bacterium. At present, natural transformation has been found in some bacteria but not in R. anatipestifer. For the first time, we showed that natural transformation occurred in R. anatipestifer ATCC 11845 and R. anatipestifer RA-CH-1. Then, we established an easy gene knockout method in R. anatipestifer based on natural transformation. This information is important for further studies of the genetic diversity and pathogenesis in R. anatipestifer.

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

2016 ◽  
Vol 60 (8) ◽  
pp. 4920-4929 ◽  
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.

scholarly journals Unleashing Natural Competence in Lactococcus lactis by Induction of the Competence Regulator ComX

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Joyce Mulder ◽  
Michiel Wels ◽  
Oscar P. Kuipers ◽  
Michiel Kleerebezem ◽  
Peter A. Bron
Keyword(s):  
Lactococcus Lactis ◽  
Genetic Manipulation ◽  
Bacterial Species ◽  
Streptococcus Thermophilus ◽  
Starter Cultures ◽  
Natural Competence ◽  
Content Type ◽  
Competence Gene ◽  
Gene Sets ◽  
Novel Traits

ABSTRACT In biotechnological workhorses like Streptococcus thermophilus and Bacillus subtilis, natural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starter Lactococcus lactis, natural competence has not been established to date. However, in silico analysis of the complete genome sequences of 43 L. lactis strains revealed complete late competence gene sets in 2 L. lactis subsp. cremoris strains (KW2 and KW10) and at least 10 L. lactis subsp. lactis strains, including the model strain IL1403 and the plant-derived strain KF147. The remainder of the strains, including all dairy isolates, displayed genomic decay in one or more of the late competence genes. Nisin-controlled expression of the competence regulator comX in L. lactis subsp. lactis KF147 resulted in the induction of expression of the canonical competence regulon and elicited a state of natural competence in this strain. In contrast, comX expression in L. lactis NZ9000, which was predicted to encode an incomplete competence gene set, failed to induce natural competence. Moreover, mutagenesis of the comEA-EC operon in strain KF147 abolished the comX-driven natural competence, underlining the involvement of the competence machinery. Finally, introduction of nisin-inducible comX expression into nisRK-harboring derivatives of strains IL1403 and KW2 allowed the induction of natural competence in these strains also, expanding this phenotype to other L. lactis strains of both subspecies. IMPORTANCE Specific bacterial species are able to enter a state of natural competence in which DNA is taken up from the environment, allowing the introduction of novel traits. Strains of the species Lactococcus lactis are very important starter cultures for the fermentation of milk in the cheese production process, where these bacteria contribute to the flavor and texture of the end product. The activation of natural competence in this industrially relevant organism can accelerate research aiming to understand industrially relevant traits of these bacteria and can facilitate engineering strategies to harness the natural biodiversity of the species in optimized starter strains.

scholarly journals Natural Competence Is a Major Mechanism for Horizontal DNA Transfer in the Oral Pathogen Porphyromonas gingivalis

mBio ◽  
2012 ◽  
Vol 3 (1) ◽  
Author(s):  
Gena D. Tribble ◽  
Todd W. Rigney ◽  
Doan-Hieu V. Dao ◽  
Cindy T. Wong ◽  
Jennifer E. Kerr ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Dna Transfer ◽  
Extracellular Dna ◽  
Minor Role ◽  
Chromosomal Dna ◽  
Natural Competence ◽  
Content Type ◽  
A Minor ◽  
First Time

ABSTRACTPorphyromonas gingivalisis a Gram-negative anaerobe that resides exclusively in the human oral cavity. Long-term colonization byP. gingivalisrequires the bacteria to evade host immune responses while adapting to the changing host physiology and alterations in the composition of the oral microflora. The genetic diversity ofP. gingivalisappears to reflect the variability of its habitat; however, little is known about the molecular mechanisms generating this diversity. Previously, our research group established that chromosomal DNA transfer occurs betweenP. gingivalisstrains. In this study, we examine the role of putative DNA transfer genes in conjugation and transformation and demonstrate that natural competence mediated bycomFis the dominant form of chromosomal DNA transfer, with transfer by a conjugation-like mechanism playing a minor role. Our results reveal that natural competence mechanisms are present in multiple strains ofP. gingivalis, and DNA uptake is not sensitive to DNA source or modification status. Furthermore, extracellular DNA was observed for the first time inP. gingivalisbiofilms and is predicted to be the major DNA source for horizontal transfer and allelic exchange between strains. We propose that exchange of DNA in plaque biofilms by a transformation-like process is of major ecological importance in the survival and persistence ofP. gingivalisin the challenging oral environment.IMPORTANCEP. gingivaliscolonizes the oral cavities of humans worldwide. The long-term persistence of these bacteria can lead to the development of chronic periodontitis and host morbidity associated with tooth loss.P. gingivalisis a genetically diverse species, and this variability is believed to contribute to its successful colonization and survival in diverse human hosts, as well as evasion of host immune defenses and immunization strategies. We establish here that natural competence is the major driving force behindP. gingivalisDNA exchange and that conjugative DNA transfer plays a minor role. Furthermore, we reveal for the first time the presence of extracellular DNA inP. gingivalisbiofilms, which is most likely the source of DNA exchanged between strains within dental plaque. These studies expand our understanding of the mechanisms used by this important member of the human oral flora to transition its relationship with the host from a commensal to a pathogenic relationship.

scholarly journals Theapt/6-Methylpurine Counterselection System and Its Applications in Genetic Studies of the Hyperthermophilic Archaeon Sulfolobus islandicus

2016 ◽  
Vol 82 (10) ◽  
pp. 3070-3081 ◽  
Author(s):  
Changyi Zhang ◽  
Qunxin She ◽  
Hongkai Bi ◽  
Rachel J. Whitaker
Keyword(s):  
Evolutionary Ecology ◽  
Population Genomics ◽  
Genetic Manipulation ◽  
Model Organisms ◽  
Loss Of Function ◽  
Chromosomal Dna ◽  
Genetic Studies ◽  
Content Type ◽  
Sulfolobus Islandicus ◽  
Mutation Assay

ABSTRACTSulfolobus islandicusserves as a model for studying archaeal biology as well as linking novel biology to evolutionary ecology using functional population genomics. In the present study, we developed a new counterselectable genetic marker inS. islandicusto expand the genetic toolbox for this species. We show that resistance to the purine analog 6-methylpurine (6-MP) inS. islandicusM.16.4 is due to the inactivation of a putative adenine phosphoribosyltransferase encoded byM164_0158(apt). The application of theaptgene as a novel counterselectable marker was first illustrated by constructing an unmarked α-amylase deletion mutant. Furthermore, the 6-MP counterselection feature was employed in a forward (loss-of-function) mutation assay to reveal the profile of spontaneous mutations inS. islandicusM.16.4 at theaptlocus. Moreover, the general conservation ofaptgenes in the crenarchaea suggests that the same strategy can be broadly applied to other crenarchaeal model organisms. These results demonstrate that theaptlocus represents a new tool for genetic manipulation and sequence analysis of the hyperthermophilic crenarchaeonS. islandicus.IMPORTANCECurrently, thepyrEF/5-fluoroorotic acid (5-FOA) counterselection system remains the sole counterselection marker in crenarchaeal genetics. Since mostSulfolobusmutants constructed by the research community were derived from genetic hosts lacking thepyrEFgenes, thepyrEF/5-FOA system is no longer available for use in forward mutation assays. Demonstration of theapt/6-MP counterselection system for theSulfolobusmodel renders it possible to again study the mutation profiles in mutants that have already been constructed by the use of strains with apyrEF-deficient background. Furthermore, additional counterselectable markers will allow us to conduct more sophisticated genetic studies, i.e., investigate mechanisms of chromosomal DNA transfer and quantify recombination frequencies amongS. islandicusstrains.

scholarly journals Control of Gene Expression in Leptospira spp. by Transcription Activator-Like Effectors Demonstrates a Potential Role for LigA and LigB in Leptospira interrogans Virulence

2015 ◽  
Vol 81 (22) ◽  
pp. 7888-7892 ◽  
Author(s):  
Christopher J. Pappas ◽  
Mathieu Picardeau
Keyword(s):  
Genetic Manipulation ◽  
Immunoblot Analysis ◽  
Targeted Mutagenesis ◽  
Pcr Analysis ◽  
Leptospira Interrogans ◽  
Transcription Activator ◽  
Promoter Regions ◽  
Control Of Gene Expression ◽  
Content Type

ABSTRACTLeptospirosis is a zoonotic disease that affects ∼1 million people annually, with a mortality rate of >10%. Currently, there is an absence of effective genetic manipulation tools for targeted mutagenesis in pathogenic leptospires. Transcription activator-like effectors (TALEs) are a recently described group of repressors that modify transcriptional activity in prokaryotic and eukaryotic cells by directly binding to a targeted sequence within the host genome. To determine the applicability of TALEs withinLeptospiraspp., two TALE constructs were designed. First, a constitutively expressed TALE gene specific for thelacO-like region upstream ofbgaLwastransinserted in the saprophyteLeptospira biflexa(the TALEβgalstrain). Reverse transcriptase PCR (RT-PCR) analysis and enzymatic assays demonstrated that BgaL was not expressed in the TALEβgalstrain. Second, to study the role of LigA and LigB in pathogenesis, a constitutively expressed TALE gene with specificity for the homologous promoter regions ofligAandligBwascisinserted into the pathogenLeptospira interrogans(TALElig). LigA and LigB expression was studied by using three independent clones: TALElig1, TALElig2, and TALElig3. Immunoblot analysis of osmotically induced TALEligclones demonstrated 2- to 9-fold reductions in the expression levels of LigA and LigB, with the highest reductions being noted for TALElig1and TALElig2, which were avirulentin vivoand nonrecoverable from animal tissues. This study reconfirms galactosidase activity in the saprophyte and suggests a role for LigA and LigB in pathogenesis. Collectively, this study demonstrates that TALEs are effective at reducing the expression of targeted genes within saprophytic and pathogenic strains ofLeptospiraspp., providing an additional genetic manipulation tool for this genus.

scholarly journals Electroporation-Based Genetic Manipulation in Type I Methanotrophs

2016 ◽  
Vol 82 (7) ◽  
pp. 2062-2069 ◽  
Author(s):  
Xin Yan ◽  
Frances Chu ◽  
Aaron W. Puri ◽  
Yanfen Fu ◽  
Mary E. Lidstrom
Keyword(s):  
Process Development ◽  
Transformation Efficiency ◽  
Genetic Manipulation ◽  
Industrial Process ◽  
Type I ◽  
Content Type ◽  
High Transformation Efficiency ◽  
New Antibiotics ◽  
Industrial Strains ◽  
High Transformation

ABSTRACTMethane is becoming a major candidate for a prominent carbon feedstock in the future, and the bioconversion of methane into valuable products has drawn increasing attention. To facilitate the use of methanotrophic organisms as industrial strains and accelerate our ability to metabolically engineer methanotrophs, simple and rapid genetic tools are needed. Electroporation is one such enabling tool, but to date it has not been successful in a group of methanotrophs of interest for the production of chemicals and fuels, the gammaproteobacterial (type I) methanotrophs. In this study, we developed electroporation techniques with a high transformation efficiency for three different type I methanotrophs:Methylomicrobium buryatense5GB1C,Methylomonassp. strain LW13, andMethylobactertundripaludum21/22. We further developed this technique inM. buryatense, a haloalkaliphilic aerobic methanotroph that demonstrates robust growth with a high carbon conversion efficiency and is well suited for industrial use for the bioconversion of methane. On the basis of the high transformation efficiency ofM. buryatense, gene knockouts or integration of a foreign fragment into the chromosome can be easily achieved by direct electroporation of PCR-generated deletion or integration constructs. Moreover, site-specific recombination (FLP-FRT [FLP recombination target] recombination) andsacBcounterselection systems were employed to perform marker-free manipulation, and two new antibiotics, zeocin and hygromycin, were validated to be antibiotic markers in this strain. Together, these tools facilitate the rapid genetic manipulation ofM. buryatenseand other type I methanotrophs, promoting the ability to perform fundamental research and industrial process development with these strains.

scholarly journals Natural Transformation in a Classical-Biotype Vibrio cholerae Strain

2021 ◽  
Vol 87 (10) ◽  
Author(s):  
Cameron J. Lloyd ◽  
Adrian Mejia-Santana ◽  
Triana N. Dalia ◽  
Ankur B. Dalia ◽  
Karl E. Klose
Keyword(s):  
Vibrio Cholerae ◽  
Natural Transformation ◽  
Genetic Manipulation ◽  
Regulatory Gene ◽  
Dna Uptake ◽  
Exogenous Dna ◽  
Content Type ◽  
Classical Strain ◽  
Artificial Induction ◽  
El Tor

ABSTRACT Vibrio cholerae causes the gastrointestinal illness cholera, which spreads throughout the globe in large pandemics. The current pandemic is caused by O1 El Tor biotype strains, whereas previous pandemics were caused by O1 classical biotype strains. El Tor V. cholerae is noted for its ability to acquire exogenous DNA through chitin-induced natural transformation, which has been exploited for genetic manipulation of El Tor strains in the laboratory. In contrast, the prototypical classical strain O395 lacks this ability, which was suspected to be due to a mutation in the regulatory gene hapR. HapR and the regulator TfoX control expression of a third competence regulator, QstR. We found that artificial induction of both TfoX and QstR in the presence of HapR in O395 was required for efficient DNA uptake. However, natural transformation in the classical strain is still orders of magnitude below that of an El Tor strain. O395 expressing HapR could also undergo natural transformation after growth on chitin, which could be increased by artificial induction of TfoX and/or QstR. A plasmid that expresses both TfoX and QstR was created that allowed for consistent DNA uptake in O395 carrying a hapR plasmid. This technique was also used to facilitate cotransformation into O395 of unmarked DNA (ΔlacZ, ΔflaA, ΔflgG) for multiplex genome editing by natural transformation (MuGENT). These results demonstrate that the classical biotype O395 strain is functionally capable of DNA uptake, which allows for the rapid genetic manipulation of its genome. IMPORTANCE Natural transformation (uptake of exogenous DNA) in Vibrio cholerae has contributed to the evolution of these human pathogens. Classical biotype V. cholerae strains were responsible for the first six cholera pandemics but were replaced by El Tor biotype V. cholerae in the current pandemic. This study demonstrates that classical V. cholerae is functionally capable of natural transformation, but inactivation of the transformation regulator HapR and inherent levels of transformation that are lower than those of El Tor V. cholerae suggest that the classical biotype may be less able to utilize natural transformation for horizontal gene transfer.

scholarly journals Acinetobacter baumannii Strain M2 Produces Type IV Pili Which Play a Role in Natural Transformation and Twitching Motility but Not Surface-Associated Motility

mBio ◽  
2013 ◽  
Vol 4 (4) ◽  
Author(s):  
Christian M. Harding ◽  
Erin N. Tracy ◽  
Michael D. Carruthers ◽  
Philip N. Rather ◽  
Luis A. Actis ◽  
...  
Keyword(s):  
Experimental Evidence ◽  
Acinetobacter Baumannii ◽  
Natural Transformation ◽  
Genetic Manipulation ◽  
Opportunistic Pathogen ◽  
Type Iv Pili ◽  
Clinical Isolates ◽  
Twitching Motility ◽  
Content Type ◽  
Type Iv

ABSTRACTAcinetobacter baumanniiis a Gram-negative, opportunistic pathogen. Recently, multipleA. baumanniigenomes have been sequenced; these data have led to the identification of many genes predicted to encode proteins required for the biogenesis of type IV pili (TFP). However, there is no experimental evidence demonstrating thatA. baumanniistrains actually produce functional TFP. Here, we demonstrated thatA. baumanniistrain M2 is naturally transformable and capable of twitching motility, two classical TFP-associated phenotypes. Strains were constructed with mutations inpilA,pilD, andpilT, genes whose products have been well characterized in other systems. These mutants were no longer naturally transformable and did not exhibit twitching motility. These TFP-associated phenotypes were restored when these mutations were complemented. More PilA was detected on the surface of thepilTmutant than the parental strain, and TFP were visualized on thepilTmutant by transmission electron microscopy. Thus,A. baumanniiproduces functional TFP and utilizes TFP for both natural transformation and twitching motility. Several investigators have hypothesized that TFP might be responsible, in part, for the flagellum-independent surface-associated motility exhibited by manyA. baumanniiclinical isolates. We demonstrated that surface-associated motility was not dependent on the products of thepilA,pilD, andpilTgenes and, by correlation, TFP. The identification of functional TFP inA. baumanniilays the foundation for future work determining the role of TFP in models of virulence that partially recapitulate human disease.IMPORTANCESeveral investigators have documented the presence of genes predicted to encode proteins required for the biogenesis of TFP in manyA. baumanniigenomes. Furthermore, some have speculated that TFP may play a role in the unique surface-associated motility phenotype exhibited by manyA. baumanniiclinical isolates, yet there has been no experimental evidence to prove this. Unfortunately, progress in understanding the biology and virulence ofA. baumanniihas been slowed by the difficulty of constructing and complementing mutations in this species. Strain M2, a recently characterized clinical isolate, is amenable to genetic manipulation. We have established a reproducible system for the generation of marked and/or unmarked mutations using a modified recombineering strategy as well as a genetic complementation system utilizing a modified mini-Tn7element in strain M2. Using this strategy, we demonstrated that strain M2 produces TFP and that TFP are not required for surface-associated motility exhibited by strain M2.

scholarly journals Development of a Modified Gentamicin Resistance Cassette for Genetic Manipulation of the Oral Spirochete Treponema denticola

2012 ◽  
Vol 78 (6) ◽  
pp. 2059-2062 ◽  
Author(s):  
Jiang Bian ◽  
J. Christopher Fenno ◽  
Chunhao Li
Keyword(s):  
Antibiotic Resistance ◽  
Genetic Manipulation ◽  
Targeted Mutagenesis ◽  
Treponema Denticola ◽  
Content Type ◽  
Reliable Tool ◽  
Gentamicin Resistance

ABSTRACTHerein, we report that a modified gentamicin cassette and a PCR-based method can be used for targeted mutagenesis of the oral spirocheteTreponema denticola. This approach minimizes polar effects and spontaneous antibiotic resistance. Therefore, it can serve as a reliable tool for genetic manipulation ofT. denticola.

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