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

Golz, Julia C.; Stingl, Kerstin

doi:10.3390/ijms221810169

Cited by 5 publications

(12 citation statements)

References 42 publications

(57 reference statements)

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“…C. jejuni can enter a competence phase, in which nearly half of the cells were capable to take up external DNA at one to four distinct locations within a cell [ 8 ]. Here, we wanted to know how much DNA was imported at distinct DNA uptake locations and if multiple DNA molecules were imported at one location.…”

Section: Resultsmentioning

confidence: 99%

“…The DNA uptake assay was performed as described in [ 8 ]. In short, genomic C. jejuni DNA was extracted using the PureLink Genomic DNA Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) and fluorescently labelled with Label IT reagent (Mirus Label IT Fluorescein or Cy3, Mirus Bio LLC, Madison, WI, USA) in a volume:weight ratio of 1:1.…”

Section: Methodsmentioning

confidence: 99%

“…In short, genomic C. jejuni DNA was extracted using the PureLink Genomic DNA Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) and fluorescently labelled with Label IT reagent (Mirus Label IT Fluorescein or Cy3, Mirus Bio LLC, Madison, WI, USA) in a volume:weight ratio of 1:1. After pre-culturing bacteria on ColbA and, subsequently, in Bolton broth, C. jejuni were subcultured in the same liquid medium but with pH adjustment using 175 µL 1 M sodium hydroxide per 5 mL Bolton broth in order to reach the “ON-status” for natural transformation/competence (pH~7.5 after growth in exponential phase) [ 8 ]. H. pylori were grown in TSB without pH adjustment.…”

Section: Methodsmentioning

confidence: 99%

“…Cell suspensions were incubated for 30 min under aerobic ( C. jejuni ) or microaerobic ( H. pylori ) conditions (5% O 2 , 10% CO 2 , rest N 2 ) at 37 °C before centrifugation at 16,000× g for 5 min and resuspension in 15 µL BHI supplemented with 10 U DNase (Roche, Rotkreuz, Schweiz). After the digestion of external DNA for 5 min at 37 °C, cells were immobilized on a 1.5% agarose surface and mounted on an Axio Observer ZI (Zeiss, Jena, Germany) microscope as described previously [ 8 ], using 25–100 ms of exposure time. Cells harboring at least one fluorescent focus were considered competent.…”

Section: Methodsmentioning

confidence: 99%

“…A prerequisite of natural transformation is the capacity to take up DNA from the environment. Competence development in C. jejuni occurred at neutral pH under microaerobic conditions and was shut-down at slight acidity as well as under atmospheric oxygen levels [ 8 ]. This suggested that natural transformation in C. jejuni was most active in its natural habitat, the intestinal tract of warm-blooded animals.…”

Section: Introductionmentioning

confidence: 99%

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Cj0683 Is a Competence Protein Essential for Efficient Initialization of DNA Uptake in Campylobacter jejuni

et al. 2023

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C. jejuni is an important food-borne pathogen displaying high genetic diversity, substantially based on natural transformation. The mechanism of DNA uptake from the environment depends on a type II secretion/type IV pilus system, whose components are partially known. Here, we quantified DNA uptake in C. jejuni at the single cell level and observed median transport capacities of approximately 30 kb per uptake location. The process appeared to be limited by the initialization of DNA uptake, was finite, and, finalized within 30 min of contact to DNA. Mutants lacking either the outer membrane pore PilQ or the inner membrane channel ComEC were deficient in natural transformation. The periplasmic DNA binding protein ComE was negligible for DNA uptake, which is in contrast to its proposed function. Intriguingly, a mutant lacking the unique periplasmic protein Cj0683 displayed rare but fully functional DNA uptake events. We conclude that Cj0683 was essential for the efficient initialization of DNA uptake, consistent with the putative function as a competence pilus protein. Unravelling features important in natural transformation might lead to target identification, reducing the adaptive potential of pathogens.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cj0683 Is a Competence Protein Essential for Efficient Initialization of DNA Uptake in Campylobacter jejuni

et al. 2023

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The point mutation A1387G in the 16S rRNA gene confers aminoglycoside resistance in Campylobacter jejuni and Campylobacter coli

Zarske,

Werckenthin,

Golz

et al. 2024

Antimicrob Agents Chemother

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Thermotolerant Campylobacter spp. are the most frequent cause of foodborne bacterial diarrhea and high-priority antibiotic-resistant pathogens, according to the World Health Organization (WHO). Monitoring revealed current low prevalence of gentamicin resistance in European Campylobacter spp. isolates but substantial presence of gentamicin modifying genes circulating globally. Using a combined approach of natural transformation and whole-genome sequencing, we revealed a novel gentamicin resistance mechanism, namely the point mutation A1387G in the 16S rRNA gene, originally identified in a C. coli isolate from turkey caecal content. The transformation rate of the resistance using genomic DNA of the resistant donor to sensitive recipient C. jejuni and C. coli was ~2.5 log 10 lower compared to the control rpsL -A128G point mutation conferring streptomycin resistance. Antimicrobial susceptibility tests showed cross-resistance to apramycin, kanamycin, and tobramycin, with transformants exhibiting more than 4- to 8-fold increased MICs to apramycin and tobramycin and over 64-fold higher MICs to kanamycin compared to wild-type isolates. Although transformants showed 177–1,235 variations relative to the recipient, only the A1387G point mutation in the 16S rRNA was in common. This mutation was causal for resistance, as transformation of a 16S rRNA_A1387G PCR fragment into susceptible isolates also led to resistant transformants. Sanger sequencing of the 16S rRNA genes and Oxford nanopore whole-genome sequencing of transformants identified clones harboring either all three copies with A1387G or a mixed population of wild-type and mutated 16S rRNA gene alleles. Within 15 passages on non-selective medium, transformants with mixed populations of the 16S rRNA gene copies partially reverted to wild type, both geno- and phenotypically. In contrast, transformants harboring the A1387G point mutation in all three 16S rRNA gene copies kept full resistance within at least 45 passages. We speculate that partial acquisition and rapid loss of the point mutation limited its spread among C . spp. isolates. In-depth knowledge on resistance mechanisms contributes to optimal diagnosis and preventative measures.

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Identification of knowledge gaps in whole-genome sequence analysis of multi-resistant thermotolerant Campylobacter spp.

Zarske,

Luu,

Deneke

et al. 2024

BMC Genomics

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Background Campylobacter spp. is the most frequent cause of bacterial food-borne gastroenteritis and a high priority antibiotic resistant bacterium according to the World Health Organization (WHO). European monitoring of thermotolerant Campylobacter spp. does not reflect the global burden of resistances already circulating within the bacterial population worldwide. Methods We systematically compared whole genome sequencing with comprehensive phenotypic antimicrobial susceptibility, analyzing 494 thermotolerant Campylobacter poultry isolates from Vietnam and Germany. Any discrepancy was checked by repeating the wet lab and improving the dry lab part. Selected isolates were additionally analyzed via long-read Oxford Nanopore technology, leading to closed chromosomes and plasmids. Results Overall, 22 different resistance genes and gene variants (e. g. erm(B), aph(3’)-IIIa, aph(2’’)-If, catA, lnu(C), blaOXA, sat4) and point mutations in three distinct genes (gyrA, 23S rRNA, rpsL) associated with AMR were present in the Campylobacter isolates. Two AMR genes were missing in the database and one falsely associated with resistance. Bioinformatic analysis based on short-read data partly failed to identify tet(O) and aadE, when the genes were present as duplicate or homologous gene variants. Intriguingly, isolates also contained different determinants, redundantly conferring resistance to chloramphenicol, gentamicin, kanamycin, lincomycin and streptomycin. We found a novel tet(W) in tetracycline sensitive strains, harboring point mutations. Furthermore, analysis based on assemblies from short-read data was impaired to identify full length phase variable aad9, due to variations of the poly-C tract within the gene. The genetic determinant responsible for gentamicin resistance of one isolate from Germany could not be identified. GyrT86I, presenting the main determinant for (fluoro-)quinolone resistance led to a rare atypical phenotype of ciprofloxacin resistance but nalidixic acid sensitivity. Long-read sequencing predicted AMR genes were mainly located on the chromosome, and rarely on plasmids. Predictions from long- and short-read sequencing, respectively, often differed. AMR genes were often organized in multidrug resistance islands (MDRI) and partially located in proximity to transposase genes, suggesting main mobilization of resistance determinants is via natural transformation and transposition in Campylobacter. Conclusions The results of this study suggest that there is frequent resistance gene duplication, mosaicism, and mutation leading to gene variation and truncation in Campylobacter strains that have not been reported in previous studies and are missing from databases. Furthermore, there is a need for deciphering yet unknown resistance mechanisms and resistance spread in thermotolerant Campylobacter spp. that may pose a challenge to global food safety.

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

Cited by 5 publications

References 42 publications

Cj0683 Is a Competence Protein Essential for Efficient Initialization of DNA Uptake in Campylobacter jejuni

Cj0683 Is a Competence Protein Essential for Efficient Initialization of DNA Uptake in Campylobacter jejuni

The point mutation A1387G in the 16S rRNA gene confers aminoglycoside resistance in Campylobacter jejuni and Campylobacter coli

Identification of knowledge gaps in whole-genome sequence analysis of multi-resistant thermotolerant Campylobacter spp.

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