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

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.

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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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Complete Genome Sequence of Psittacine Adenovirus 1, Identified from Poicephalus senegalus in Italy

Microbiology Resource Announcements ◽

10.1128/mra.01037-18 ◽

2018 ◽

Vol 7 (11) ◽

Cited By ~ 5

Author(s):

Adelaide Milani ◽

Gianpiero Zamperin ◽

Alice Fusaro ◽

Annalisa Salviato ◽

Luca Bano ◽

...

Keyword(s):

Genetic Diversity ◽

Genome Sequence ◽

Complete Genome Sequence ◽

Complete Genome ◽

Full Genome Sequence ◽

Full Genome ◽

High Genetic Diversity ◽

Content Type ◽

First Time

Using a metagenomics approach, we were able to determine for the first time the full-genome sequence of a psittacine adenovirus 1 isolate that was recovered from the liver of a dead Senegal parrot (Poicephalus senegalus) in Italy. The results of the phylogenetic investigations revealed the existence of high genetic diversity among adenoviruses circulating in psittacine birds.

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Population Polymorphism of Nuclear Mitochondrial DNA Insertions Reveals Widespread Diploidy Associated with Loss of Heterozygosity in Debaryomyces hansenii

Eukaryotic Cell ◽

10.1128/ec.00263-09 ◽

2010 ◽

Vol 9 (3) ◽

pp. 449-459 ◽

Cited By ~ 20

Author(s):

Noémie Jacques ◽

Christine Sacerdot ◽

Meriem Derkaoui ◽

Bernard Dujon ◽

Odile Ozier-Kalogeropoulos ◽

...

Keyword(s):

Genetic Diversity ◽

Flow Cytometry ◽

Mitochondrial Dna ◽

Loss Of Heterozygosity ◽

Debaryomyces Hansenii ◽

Intraspecific Variability ◽

Content Type ◽

Population Polymorphism ◽

First Time

ABSTRACT Debaryomyces hansenii, a yeast that participates in the elaboration of foodstuff, displays important genetic diversity. Our recent phylogenetic classification of this species led to the subdivision of the species into three distinct clades. D. hansenii harbors the highest number of nuclear mitochondrial DNA (NUMT) insertions known so far for hemiascomycetous yeasts. Here we assessed the intraspecific variability of the NUMTs in this species by testing their presence/absence first in 28 strains, with 21 loci previously detected in the completely sequenced strain CBS 767T, and second in a larger panel of 77 strains, with 8 most informative loci. We were able for the first time to structure populations in D. hansenii, although we observed little NUMT insertion variability within the clades. We determined the chronology of the NUMT insertions, which turned out to correlate with the previously defined taxonomy and provided additional evidence that colonization of nuclear genomes by mitochondrial DNA is a dynamic process in yeast. In combination with flow cytometry experiments, the NUMT analysis revealed the existence of both haploid and diploid strains, the latter being heterozygous and resulting from at least four crosses among strains from the various clades. As in the diploid pathogen Candida albicans, to which D. hansenii is phylogenetically related, we observed a differential loss of heterozygosity in the diploid strains, which can explain some of the large genetic diversity found in D. hansenii over the years.

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A Short Protocol for Gene Knockout and Complementation in Xylella fastidiosa Shows that One of the Type IV Pilin Paralogs (PD1926) Is Needed for Twitching while Another (PD1924) Affects Pilus Number and Location

Applied and Environmental Microbiology ◽

10.1128/aem.01167-18 ◽

2018 ◽

Vol 84 (18) ◽

Cited By ~ 7

Author(s):

Prem P. Kandel ◽

Hongyu Chen ◽

Leonardo De La Fuente

Keyword(s):

Natural Transformation ◽

Gene Knockout ◽

Xylella Fastidiosa ◽

Twitching Motility ◽

Wild Type ◽

Content Type ◽

Overlap Extension Pcr ◽

Type Iv ◽

Overlap Extension ◽

Pilin Subunit

ABSTRACT Twitching motility is one of the major virulence factors of the plant-pathogenic bacterium Xylella fastidiosa, and it is mediated by type IV pili (TFP) that are present at one of the cell poles. Genome analysis of X. fastidiosa showed the presence of at least four paralogs of the gene pilA, which encodes the TFP major pilin subunit. However, whether all of these paralogs have a functional role in TFP structure and function is unknown. Here, using a short and reliable protocol based on overlap extension PCR and natural transformation, deletion mutants of two pilA paralogs (pilA1 PD1924 and pilA2 PD1926) were generated in two X. fastidiosa subsp. fastidiosa strains, WM1-1 and TemeculaL, followed by assessment of twitching motility and biofilm formation. Deletion of pilA2 caused loss of twitching motility, whereas deletion of pilA1 did not influence twitching motility but caused hyperpiliation and extended distribution of TFP along the sides of the cell. Loss of twitching motility due to pilA2 deletion was restored when a wild-type copy of the pilA2 gene was added at a neutral site in the genome of mutants in both wild-type backgrounds. This study demonstrates that PCR templates generated by overlap extension PCR can be successfully used to rapidly generate gene knockouts and perform genetic complementation in X. fastidiosa, and that twitching motility in X. fastidiosa is controlled by regulating the transcription of the major pilin subunit, pilA2. IMPORTANCE The bacterial plant pathogen Xylella fastidiosa causes incurable diseases in multiple hosts, including grape, citrus, and blueberry. Historically restricted to the Americas, it was recently found to cause epidemics in olives in Italy and to infect other hosts in Europe and Asia. In this study, we report a short protocol to create deletion and complemented mutants using fusion PCR and natural transformation. We also determined the distinct function of two pilin paralogs, the main structural component of TFP involved in twitching motility, which allows this bacterium to move inside the xylem vessels against the flow. One of the paralogs is needed for twitching movement, whereas the other does not have an effect on motility but influences the number and position of TFP. Since twitching motility is fundamental for the virulence of this xylem-limited bacterium, this study contributes to the understanding of the regulation of virulence by this pathogen.

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Lausannevirus Encodes a Functional Dihydrofolate Reductase Susceptible to Proguanil

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02573-16 ◽

2017 ◽

Vol 61 (4) ◽

Author(s):

L. Mueller ◽

P. M. Hauser ◽

F. Gauye ◽

G. Greub

Keyword(s):

Dihydrofolate Reductase ◽

Expression System ◽

Open Reading Frames ◽

Dna Viruses ◽

Content Type ◽

The Family ◽

Nucleocytoplasmic Large Dna Viruses ◽

First Time ◽

Reading Frames

ABSTRACT Lausannevirus belongs to the family Marseilleviridae within the group of nucleocytoplasmic large DNA viruses (NCLDVs). These giant viruses exhibit unique features, including a large genome, ranging from 100 kb to 2.5 Mb and including from 150 to more than 2,500 genes, as well as the presence of genes coding for proteins involved in transcription and translation. The large majority of Lausannevirus open reading frames have unknown functions. Interestingly, a bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is encoded in the Lausannevirus genome. The enzyme plays central roles in DNA precursor biosynthesis. DHFR is the pharmacological target of antifolates, such as trimethoprim, pyrimethamine, and proguanil. First, the functionality of Lausannevirus DHFR-TS was demonstrated by the successful complementation of a DHFR-deficient Saccharomyces cerevisiae strain with a plasmid expressing the heterologous gene. Additionally, using this heterologous expression system, we demonstrated the in vitro susceptibility of Lausannevirus DHFR-TS to proguanil and its resistance to pyrimethamine and trimethoprim. Proguanil may provide a unique and useful treatment if Lausannevirus proves to be a human pathogen. To our knowledge, this is the first time that a DHFR-TS has been described and characterized in an NCLDV.

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

mBio ◽

10.1128/mbio.00231-11 ◽

2012 ◽

Vol 3 (1) ◽

Cited By ~ 40

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.00455-16 ◽

2016 ◽

Vol 82 (10) ◽

pp. 3070-3081 ◽

Cited By ~ 10

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.

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Large Sequence Diversity within the Biosynthesis Locus and Common Biochemical Features of Campylobacter coli Lipooligosaccharides

Journal of Bacteriology ◽

10.1128/jb.00347-16 ◽

2016 ◽

Vol 198 (20) ◽

pp. 2829-2840 ◽

Cited By ~ 11

Author(s):

Alejandra Culebro ◽

Joana Revez ◽

Ben Pascoe ◽

Yasmin Friedmann ◽

Matthew D. Hitchings ◽

...

Keyword(s):

Genetic Diversity ◽

Population Dynamics ◽

Chemical Composition ◽

Phenotypic Diversity ◽

Host Adaptation ◽

Campylobacter Coli ◽

Genomic Information ◽

Content Type ◽

Species Specific ◽

First Time

ABSTRACTDespite the importance of lipooligosaccharides (LOSs) in the pathogenicity of campylobacteriosis, little is known about the genetic and phenotypic diversity of LOS inCampylobacter coli. In this study, we investigated the distribution of LOS locus classes among a large collection of unrelatedC. coliisolates sampled from several different host species. Furthermore, we pairedC. coligenomic information and LOS chemical composition for the first time to investigate possible associations between LOS locus class sequence diversity and biochemical heterogeneity. After identifying three new LOS locus classes, only 85% of the 144 isolates tested were assigned to a class, suggesting higher genetic diversity than previously thought. This genetic diversity is at the basis of a completely unexplored LOS structural heterogeneity. Mass spectrometry analysis of the LOSs of nine isolates, representing four different LOS classes, identified two features distinguishingC. coliLOS from that ofCampylobacter jejuni. 2-Amino-2-deoxy-d-glucose (GlcN)–GlcN disaccharides were present in the lipid A backbone, in contrast to the β-1′-6-linked 3-diamino-2,3-dideoxy-d-glucopyranose (GlcN3N)–GlcN backbone observed inC. jejuni. Moreover, despite the fact that many of the genes putatively involved in 3-acylamino-3,6-dideoxy-d-glucose (Quip3NAcyl) were apparently absent from the genomes of various isolates, this rare sugar was found in the outer core of allC. coliisolates. Therefore, regardless of the high genetic diversity of the LOS biosynthesis locus inC. coli, we identified species-specific phenotypic features ofC. coliLOS that might explain differences betweenC. jejuniandC. coliin terms of population dynamics and host adaptation.IMPORTANCEDespite the importance ofC. colito human health and its controversial role as a causative agent of Guillain-Barré syndrome, little is known about the genetic and phenotypic diversity ofC. coliLOSs. Therefore, we pairedC. coligenomic information and LOS chemical composition for the first time to address this paucity of information. We identified two species-specific phenotypic features ofC. coliLOS, which might contribute to elucidating the reasons behind the differences betweenC. jejuniandC. coliin terms of population dynamics and host adaptation.

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Effect of Nutritional Determinants and TonB on the Natural Transformation of Riemerella anatipestifer

Frontiers in Microbiology ◽

10.3389/fmicb.2021.644868 ◽

2021 ◽

Vol 12 ◽

Author(s):

Li Zhang ◽

Li Huang ◽

Mi Huang ◽

Mengying Wang ◽

Dekang Zhu ◽

...

Keyword(s):

Natural Transformation ◽

Iron Acquisition ◽

Vitamin B1 ◽

Underlying Mechanism ◽

Iron Chelator ◽

Transformation Frequency ◽

Nutritional Factors ◽

Riemerella Anatipestifer ◽

The Family ◽

Competent Bacterium

Riemerella anatipestifer is a gram-negative bacterium that is the first naturally competent bacterium identified in the family Flavobacteriaceae. However, the determinants that influence the natural transformation and the underlying mechanism remain unknown. In this study, we evaluated the effects of various nutritional factors of the GCB medium [glucose, L-glutamine, vitamin B1, Fe (NO3)3, NaCl, phosphate, and peptone], on the natural transformation of R. anatipestifer ATCC 11845. Among the assayed nutrients, peptone and phosphate affected the natural transformation of R. anatipestifer ATCC 11845, and the transformation frequency was significantly decreased when phosphate or peptone was removed from the GCB medium. When the iron chelator 2,2′-dipyridyl (Dip) was added, the transformation frequency was decreased by approximately 100-fold and restored gradually when Fe (NO3)3 was added, suggesting that the natural transformation of R. anatipestifer ATCC 11845 requires iron. Given the importance of TonB in nutrient transportation, we further identified whether TonB is involved in the natural transformation of R. anatipestifer ATCC 11845. Mutation of tonBA or tonBB, but not tbfA, was shown to inhibit the natural transformation of R. anatipestifer ATCC 11845 in the GCB medium. In parallel, it was shown that the tonBB mutant, but not the tonBA mutant, decreased iron acquisition in the GCB medium. This result suggested that the tonBB mutant affects the natural transformation frequency due to the deficiency of iron utilization.

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Draft Genome Sequence of Riemerella anatipestifer Isolate 17CS0503

Genome Announcements ◽

10.1128/genomea.00274-18 ◽

2018 ◽

Vol 6 (20) ◽

Author(s):

Anne Busch ◽

Martin Ryll ◽

Alexander Immel ◽

Sabin Kornell ◽

Ben Krause-Kyora ◽

...

Keyword(s):

Genome Sequence ◽

Anas Platyrhynchos ◽

Draft Genome ◽

Draft Genome Sequence ◽

Negative Bacterium ◽

Gram Negative ◽

Content Type ◽

Riemerella Anatipestifer ◽

Peking Duck ◽

The Family

ABSTRACT Riemerella anatipestifer is a Gram-negative bacterium belonging to the family Flavobacteriaceae. It is primarily associated with acute septicemia in younger birds. The R. anatipestifer isolate 17CS0503 described here was isolated from a Peking duck (Anas platyrhynchos domesticus) in Hannover, Germany, in 1999.

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