scholarly journals “Take It or Leave It”—Factors Regulating Competence Development and DNA Uptake in Campylobacter jejuni

2021 ◽  
Vol 22 (18) ◽  
pp. 10169
Author(s):  
Julia C. Golz ◽  
Kerstin Stingl
Keyword(s):  
Campylobacter Jejuni ◽  
Natural Transformation ◽  
Genetic Material ◽  
Competence Development ◽  
Dna Uptake ◽  
State Transformation ◽  
Sustained Reduction ◽  
Reduction Strategies ◽  
Uptake Assay ◽  
Microaerobic Conditions

Campylobacter jejuni has a large adaptive potential due to enormous genetic exchange. Factors regulating natural transformation in this food-borne pathogen are largely unknown but of interest for the application of sustained reduction strategies in the food-processing industry. Using a single cell DNA uptake assay, we visualized that recognition of methylated C. jejuni DNA was essential for the first step of DNA uptake into a DNase resistant state. Transformation rates using a resistance marker correlated with the fraction of competent bacteria, harboring one to maximally four locations of active DNA uptake, not necessarily being located at the cell pole. Competence developed with rising pH between 6.5 and 7.5 under microaerobic conditions and was nearly insensitive towards growth temperatures between 32 °C and 42 °C, CO2 concentrations ranging from 0 to 50% and growth rates. However, competence development was abolished at pH 5 or under aerobic stress conditions, in which the bacteria ceased growth but fully survived. The DNA uptake machinery in competent bacteria shut down at slightly acidic pH and was reversibly switched on upon neutralization. It was dependent on the proton motive force and, in contrast to competence development, slightly enhanced under aerobic conditions. The results suggest that natural transformation in C. jejuni occurs in the neutral and microaerobic intestinal environment for enhanced genetic diversity and pre-adaption before host switch. In addition, highly competent bacteria might be shed into the environment, still able to acquire genetic material for increased survival.

scholarly journals A DNase Encoded by Integrated Element CJIE1 Inhibits Natural Transformation of Campylobacter jejuni

2009 ◽  
Vol 191 (7) ◽  
pp. 2296-2306 ◽  
Author(s):  
Esther J. Gaasbeek ◽  
Jaap A. Wagenaar ◽  
Magalie R. Guilhabert ◽  
Marc M. S. M. Wösten ◽  
Jos P. M. van Putten ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Molecular Mechanism ◽  
Campylobacter Jejuni ◽  
Natural Transformation ◽  
Dnase Activity ◽  
Comparative Genome Hybridization ◽  
Dna Uptake ◽  
Knockout Mutant ◽  
Gene Encoding ◽  
Plasmid Analysis

ABSTRACT The species Campylobacter jejuni is considered naturally competent for DNA uptake and displays strong genetic diversity. Nevertheless, nonnaturally transformable strains and several relatively stable clonal lineages exist. In the present study, the molecular mechanism responsible for the nonnatural transformability of a subset of C. jejuni strains was investigated. Comparative genome hybridization indicated that C. jejuni Mu-like prophage integrated element 1 (CJIE1) was more abundant in nonnaturally transformable C. jejuni strains than in naturally transformable strains. Analysis of CJIE1 indicated the presence of dns (CJE0256), which is annotated as a gene encoding an extracellular DNase. DNase assays using a defined dns mutant and a dns-negative strain expressing Dns from a plasmid indicated that Dns is an endogenous DNase. The DNA-hydrolyzing activity directly correlated with the natural transformability of the knockout mutant and the dns-negative strain expressing Dns from a plasmid. Analysis of a broader set of strains indicated that the majority of nonnaturally transformable strains expressed DNase activity, while all naturally competent strains lacked this activity. The inhibition of natural transformation in C. jejuni via endogenous DNase activity may contribute to the formation of stable lineages in the C. jejuni population.

scholarly journals Natural Transformation of Campylobacter jejuni Requires Components of a Type II Secretion System

2003 ◽  
Vol 185 (18) ◽  
pp. 5408-5418 ◽  
Author(s):  
Rebecca S. Wiesner ◽  
David R. Hendrixson ◽  
Victor J. DiRita
Keyword(s):  
Campylobacter Jejuni ◽  
Natural Transformation ◽  
Bacterial Species ◽  
Type Ii Secretion ◽  
Dna Uptake ◽  
Type Ii ◽  
Secretion Systems ◽  
Dna Transport ◽  
Type Iv ◽  
Type Ii Secretion System

ABSTRACT The human pathogen Campylobacter jejuni is one of more than 40 naturally competent bacterial species able to import macromolecular DNA from the environment and incorporate it into their genomes. However, in C. jejuni little is known about the genes involved in this process. We used random transposon mutagenesis to identify genes that are required for the transformation of this organism. We isolated mutants with insertions in 11 different genes; most of the mutants are affected in the DNA uptake stage of transformation, whereas two mutants are affected in steps subsequent to DNA uptake, such as recombination into the chromosome or in DNA transport across the inner membrane. Several of these genes encode proteins homologous to those involved in type II secretion systems, biogenesis of type IV pili, and competence for natural transformation in gram-positive and gram-negative species. Other genes identified in our screen encode proteins unique to C. jejuni or are homologous to proteins that have not been shown to play a role in the transformation in other bacteria.

scholarly journals Nucleases Encoded by the Integrated Elements CJIE2 and CJIE4 Inhibit Natural Transformation of Campylobacter jejuni

2009 ◽  
Vol 192 (4) ◽  
pp. 936-941 ◽  
Author(s):  
Esther J. Gaasbeek ◽  
Jaap A. Wagenaar ◽  
Magalie R. Guilhabert ◽  
Jos P. M. van Putten ◽  
Craig T. Parker ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Campylobacter Jejuni ◽  
Genetic Factors ◽  
Natural Transformation ◽  
Homologous Gene ◽  
Dna Uptake ◽  
Dna Arrays ◽  
Genetic Transfer ◽  
Hydrolysis Of ◽  
Hydrolysis Of Dna

ABSTRACT The species Campylobacter jejuni is naturally competent for DNA uptake; nevertheless, nonnaturally transformable strains do exist. For a subset of strains we previously showed that a periplasmic DNase, encoded by dns, inhibits natural transformation in C. jejuni. In the present study, genetic factors coding for DNase activity in the absence of dns were identified. DNA arrays indicated that nonnaturally transformable dns-negative strains contain putative DNA/RNA nonspecific endonucleases encoded by CJE0566 and CJE1441 of strain RM1221. These genes are located on C. jejuni integrated elements 2 and 4. Expression of CJE0566 and CJE1441 from strain RM1221 and a homologous gene from strain 07479 in DNase-negative Escherichia coli and C. jejuni strains indicated that these genes code for DNases. Genetic transfer of the genes to a naturally transformable C. jejuni strain resulted in a decreased efficiency of natural transformation. Modeling suggests that the C. jejuni DNases belong to the Serratia nuclease family. Overall, the data indicate that the acquisition of prophage-encoded DNA/RNA nonspecific endonucleases inhibits the natural transformability of C. jejuni through hydrolysis of DNA.

scholarly journals Type IV-Like Pili Facilitate Transformation in Naturally Competent Archaea

2020 ◽  
Vol 202 (21) ◽  
Author(s):  
Dallas R. Fonseca ◽  
Mohd Farid Abdul Halim ◽  
Matthew P. Holten ◽  
Kyle C. Costa
Keyword(s):  
Natural Transformation ◽  
Genetic Material ◽  
Environmental Dna ◽  
Microbial Evolution ◽  
Dna Uptake ◽  
Content Type ◽  
Average Efficiency ◽  
Type Iv ◽  
Integrative Vectors ◽  
Microbial Organisms

ABSTRACT Naturally competent organisms are capable of DNA uptake directly from the environment through the process of transformation. Despite the importance of transformation to microbial evolution, DNA uptake remains poorly characterized outside of the bacterial domain. Here, we identify the pilus as a necessary component of the transformation machinery in archaea. We describe two naturally competent organisms, Methanococcus maripaludis and Methanoculleus thermophilus. In M. maripaludis, replicative vectors were transferred with an average efficiency of 2.4 × 103 transformants μg−1 DNA. In M. thermophilus, integrative vectors were transferred with an average efficiency of 2.7 × 103 transformants μg−1 DNA. Additionally, natural transformation of M. thermophilus could be used to introduce chromosomal mutations. To our knowledge, this is the first demonstration of a method to introduce targeted mutations in a member of the order Methanomicrobiales. For both organisms, mutants lacking structural components of the type IV-like pilus filament were defective for DNA uptake, demonstrating the importance of pili for natural transformation. Interestingly, competence could be induced in a noncompetent strain of M. maripaludis by expressing pilin genes from a replicative vector. These results expand the known natural competence pili to include examples from the archaeal domain and highlight the importance of pili for DNA uptake in diverse microbial organisms. IMPORTANCE Microbial organisms adapt and evolve by acquiring new genetic material through horizontal gene transfer. One way that this occurs is natural transformation, the direct uptake and genomic incorporation of environmental DNA by competent organisms. Archaea represent up to a third of the biodiversity on Earth, yet little is known about transformation in these organisms. Here, we provide the first characterization of a component of the archaeal DNA uptake machinery. We show that the type IV-like pilus is essential for natural transformation in two archaeal species. This suggests that pili are important for transformation across the tree of life and further expands our understanding of gene flow in archaea.

scholarly journals Noncanonical crRNAs derived from host transcripts enable multiplexable RNA detection by Cas9

Science ◽  
2021 ◽  
pp. eabe7106
Author(s):  
Chunlei Jiao ◽  
Sahil Sharma ◽  
Gaurav Dugar ◽  
Natalia L. Peeck ◽  
Thorsten Bischler ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Genetic Material ◽  
Simultaneous Detection ◽  
Type Ii ◽  
Rna Detection ◽  
Single Base ◽  
Dna Targeting ◽  
Single Base Resolution ◽  
Rna Complexes

CRISPR-Cas systems recognize foreign genetic material using CRISPR RNAs (crRNAs). In Type II systems, a trans-activating crRNA (tracrRNA) hybridizes to crRNAs to drive their processing and utilization by Cas9. While analyzing Cas9-RNA complexes from Campylobacter jejuni, we discovered tracrRNA hybridizing to cellular RNAs, leading to formation of “noncanonical” crRNAs capable of guiding DNA targeting by Cas9. Our discovery inspired the engineering of reprogrammed tracrRNAs that link the presence of any RNA-of-interest to DNA targeting with different Cas9 orthologs. This capability became the basis for a multiplexable diagnostic platform termed LEOPARD (Leveraging Engineered tracrRNAs and On-target DNAs for PArallel RNA Detection). LEOPARD allowed simultaneous detection of RNAs from different viruses in one test and distinguished SARS-CoV-2 and its D614G variant with single-base resolution in patient samples.

scholarly journals Biofilm Formation by Campylobacter jejuni Is Increased under Aerobic Conditions

2010 ◽  
Vol 76 (7) ◽  
pp. 2122-2128 ◽  
Author(s):  
Mark Reuter ◽  
Arthur Mallett ◽  
Bruce M. Pearson ◽  
Arnoud H. M. van Vliet
Keyword(s):  
Campylobacter Jejuni ◽  
Food Chain ◽  
Biofilm Formation ◽  
Planktonic Cells ◽  
Aerobic Conditions ◽  
Food Borne ◽  
Passive Release ◽  
Bacterial Gastroenteritis ◽  
Microaerobic Conditions ◽  
Developed World

ABSTRACT The microaerophilic human pathogen Campylobacter jejuni is the leading cause of food-borne bacterial gastroenteritis in the developed world. During transmission through the food chain and the environment, the organism must survive stressful environmental conditions, particularly high oxygen levels. Biofilm formation has been suggested to play a role in the environmental survival of this organism. In this work we show that C. jejuni NCTC 11168 biofilms developed more rapidly under environmental and food-chain-relevant aerobic conditions (20% O2) than under microaerobic conditions (5% O2, 10% CO2), although final levels of biofilms were comparable after 3 days. Staining of biofilms with Congo red gave results similar to those obtained with the commonly used crystal violet staining. The level of biofilm formation by nonmotile aflagellate strains was lower than that observed for the motile flagellated strain but nonetheless increased under aerobic conditions, suggesting the presence of flagellum-dependent and flagellum-independent mechanisms of biofilm formation in C. jejuni. Moreover, preformed biofilms shed high numbers of viable C. jejuni cells into the culture supernatant independently of the oxygen concentration, suggesting a continuous passive release of cells into the medium rather than a condition-specific active mechanism of dispersal. We conclude that under aerobic or stressful conditions, C. jejuni adapts to a biofilm lifestyle, allowing survival under detrimental conditions, and that such a biofilm can function as a reservoir of viable planktonic cells. The increased level of biofilm formation under aerobic conditions is likely to be an adaptation contributing to the zoonotic lifestyle of C. jejuni.

scholarly journals The major subunit of widespread competence pili exhibits a novel and conserved type IV pilin fold

2020 ◽  
Vol 295 (19) ◽  
pp. 6594-6604 ◽  
Author(s):  
Devon Sheppard ◽  
Jamie-Lee Berry ◽  
Rémi Denise ◽  
Eduardo P. C. Rocha ◽  
Steve Matthews ◽  
...  
Keyword(s):  
Natural Transformation ◽  
Dna Uptake ◽  
Human Pathogens ◽  
Unusual Structure ◽  
Widespread Distribution ◽  
Solution Structures ◽  
Type Iv ◽  
Filament Assembly ◽  
Pilin Subunit ◽  
Type Iv Pilin

Type IV filaments (T4F), which are helical assemblies of type IV pilins, constitute a superfamily of filamentous nanomachines virtually ubiquitous in prokaryotes that mediate a wide variety of functions. The competence (Com) pilus is a widespread T4F, mediating DNA uptake (the first step in natural transformation) in bacteria with one membrane (monoderms), an important mechanism of horizontal gene transfer. Here, we report the results of genomic, phylogenetic, and structural analyses of ComGC, the major pilin subunit of Com pili. By performing a global comparative analysis, we show that Com pili genes are virtually ubiquitous in Bacilli, a major monoderm class of Firmicutes. This also revealed that ComGC displays extensive sequence conservation, defining a monophyletic group among type IV pilins. We further report ComGC solution structures from two naturally competent human pathogens, Streptococcus sanguinis (ComGCSS) and Streptococcus pneumoniae (ComGCSP), revealing that this pilin displays extensive structural conservation. Strikingly, ComGCSS and ComGCSP exhibit a novel type IV pilin fold that is purely helical. Results from homology modeling analyses suggest that the unusual structure of ComGC is compatible with helical filament assembly. Because ComGC displays such a widespread distribution, these results have implications for hundreds of monoderm species.

Production and Characterization of a Monoclonal Antibody to Campylobacter jejuni

2009 ◽  
Vol 72 (4) ◽  
pp. 870-875 ◽  
Author(s):  
S. A. HEO ◽  
R. NANNAPANENI ◽  
M. G. JOHNSON ◽  
J. S. PARK ◽  
K. H. SEO
Keyword(s):  
Monoclonal Antibody ◽  
Campylobacter Jejuni ◽  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Campylobacter Coli ◽  
Carbonate Buffer ◽  
Horse Blood ◽  
Microaerobic Conditions ◽  
Immunoglobulin Subclass

Campylobacter species are a group of spiral-shaped bacteria that can cause disease in humans and animals. We developed a high-affinity monoclonal antibody (MAb) probe that recognizes Campylobacter jejuni cells. Cell suspensions grown under microaerobic conditions at 42°C for 20 h on Bolton agar plates with lysed horse blood were used as live and heat-killed preparations, centrifuged at 8,000 × g for 20 min, and resuspended in carbonate buffer (pH 9.6) for coating on the enzyme-linked immunosorbent assay plates. BALB/c mice were immunized with C. jejuni sonicated cells at 107 CFU/ml to generate MAb-producing hybridoma clones. Of about 500 initial hybridoma clones, MAb 33D2, which reacted with C. jejuni and Campylobacter coli, was selected for further evaluation. MAb 33D2 is in the immunoglobulin subclass G2a and had relatively weaker reactivity with the C. coli strains tested. MAb 33D2 did not show any cross-reactions with the nine non-Campylobacter bacteria tested in the enzyme-linked immunosorbent assay and had a stronger affinity for C. jejuni as live versus heat-killed cells. In Western blot assays, MAb 33D2 recognized two major antigens of 62 and 43 kDa in extracts from C. jejuni cells but only one antigen of 62 kDa in extracts from C. coli cells.

scholarly journals CryoEM structure of the type IVa pilus secretin required for natural competence in Vibrio cholerae

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Sara J. Weaver ◽  
Davi R. Ortega ◽  
Matthew H. Sazinsky ◽  
Triana N. Dalia ◽  
Ankur B. Dalia ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Vibrio Cholerae ◽  
Natural Transformation ◽  
Domain Architecture ◽  
Genetic Material ◽  
Surface Binding ◽  
Exogenous Dna ◽  
Insight Into

Abstract Natural transformation is the process by which bacteria take up genetic material from their environment and integrate it into their genome by homologous recombination. It represents one mode of horizontal gene transfer and contributes to the spread of traits like antibiotic resistance. In Vibrio cholerae, a type IVa pilus (T4aP) is thought to facilitate natural transformation by extending from the cell surface, binding to exogenous DNA, and retracting to thread this DNA through the outer membrane secretin, PilQ. Here, we use a functional tagged allele of VcPilQ purified from native V. cholerae cells to determine the cryoEM structure of the VcPilQ secretin in amphipol to ~2.7 Å. We use bioinformatics to examine the domain architecture and gene neighborhood of T4aP secretins in Proteobacteria in comparison with VcPilQ. This structure highlights differences in the architecture of the T4aP secretin from the type II and type III secretion system secretins. Based on our cryoEM structure, we design a series of mutants to reversibly regulate VcPilQ gate dynamics. These experiments support the idea of VcPilQ as a potential druggable target and provide insight into the channel that DNA likely traverses to promote the spread of antibiotic resistance via horizontal gene transfer by natural transformation.

scholarly journals Importance of Polyphosphate Kinase 1 for Campylobacter jejuni Viable-but-Nonculturable Cell Formation, Natural Transformation, and Antimicrobial Resistance

2009 ◽  
Vol 75 (24) ◽  
pp. 7838-7849 ◽  
Author(s):  
Dharanesh Gangaiah ◽  
Issmat I. Kassem ◽  
Zhe Liu ◽  
Gireesh Rajashekara
Keyword(s):  
Antimicrobial Resistance ◽  
Campylobacter Jejuni ◽  
Stress Responses ◽  
Natural Transformation ◽  
Cell Formation ◽  
Acid Stress ◽  
Therapeutic Interventions ◽  
Wild Type ◽  
Viable But Nonculturable ◽  
Stress Survival

ABSTRACT Campylobacter jejuni, a gram-negative, microaerophilic bacterium, is a predominant cause of bacterial gastroenteritis in humans. Although considered fragile and fastidious and lacking many classical stress response mechanisms, C. jejuni exhibits a remarkable capacity for survival and adaptation, successfully infecting humans and persisting in the environment. Consequently, understanding the physiological and genetic properties that allow C. jejuni to survive and adapt to various stress conditions is crucial for therapeutic interventions. Of importance is polyphosphate (poly-P) kinase 1 (PPK1), which is a key enzyme mediating the synthesis of poly-P, an essential molecule for survival, mediating stress responses, host colonization, and virulence in many bacteria. Therefore, we investigated the role of PPK1 in C. jejuni pathogenesis, stress survival, and adaptation. Our findings demonstrate that a C. jejuni Δppk1 mutant was deficient in poly-P accumulation, which was associated with a decreased ability to form viable-but-nonculturable cells under acid stress. The Δppk1 mutant also showed a decreased frequency of natural transformation and an increased susceptibility to various antimicrobials. Furthermore, the Δppk1 mutant was characterized by a dose-dependent deficiency in chicken colonization. Complementation of the Δppk1 mutant with the wild-type copy of ppk1 restored the deficient phenotypes to levels similar to those of the wild type. Our results suggest that poly-P plays an important role in stress survival and adaptation and might contribute to genome plasticity and the spread and development of antimicrobial resistance in C. jejuni. These findings highlight the potential of PPK1 as a novel target for therapeutic interventions.

Sign in / Sign up

Export Citation Format

Share Document