Natural Transformation Frequency Research Articles

Natural Organic Matter Enhances Natural Transformation of Extracellular Antibiotic Resistance Genes in Sunlit Water.

Antibiotic resistance genes (ARGs) as emerging environmental contaminants exacerbate the risk of spreading antibiotic resistance. Natural organic matter (NOM) is ubiquitous in aquatic environments and plays a crucial role in biogeochemical cycles. However, its impact on the dissemination of extracellular antibiotic resistance genes (eARGs) under sunlight exposure remains elusive. This study reveals that environmentally relevant levels of NOM (0.1-20 mg/L) can significantly enhance the natural transformation frequency of the model bacterium Acinetobacter baylyi ADP1 by up to 7.6-fold under simulated sunlight. Similarly, this enhancement was consistently observed in natural water and wastewater systems. Further mechanism analysis revealed that reactive oxygen species (ROS) generated by NOM under sunlight irradiation, primarily singlet oxygen and hydroxyl radicals, play a crucial role in this process. These ROS induce intracellular oxidative stress and elevated cellular membrane permeability, thereby indirectly boosting ATP production and enhancing cell competence of extracellular DNA uptake and integration. Our findings highlight a previously underestimated role of natural factors in the dissemination of eARGs within aquatic ecosystems and deepen our understanding of the complex interplay between NOM, sunlight, and microbes in environmental water bodies. This underscores the importance of developing comprehensive strategies to mitigate the spread of antibiotic resistance in aquatic environments.

Effect of chemotherapeutic agents on natural transformation frequency in Acinetobacter baylyi.

Natural transformation is the ability of a bacterial cell to take up extracellular DNA which is subsequently available for recombination into the chromosome (or maintenance as an extrachromosomal element). Like other mechanisms of horizontal gene transfer, natural transformation is a significant driver for the dissemination of antimicrobial resistance. Recent studies have shown that many pharmaceutical compounds such as antidepressants and anti-inflammatory drugs can upregulate transformation frequency in the model species Acinetobacter baylyi. Chemotherapeutic compounds have been shown to increase the abundance of antimicrobial resistance genes and increase colonization rates of potentially pathogenic bacteria in patient gastrointestinal tracts, indicating an increased risk of infection and providing a pool of pathogenicity or resistance genes for transformable commensal bacteria. We here test for the effect of six cancer chemotherapeutic compounds on A. baylyi natural transformation frequency, finding two compounds, docetaxel and daunorubicin, to significantly decrease transformation frequency, and daunorubicin to also decrease growth rate significantly. Enhancing our understanding of the effect of chemotherapeutic compounds on the frequency of natural transformation could aid in preventing the horizontal spread of antimicrobial resistance genes.

Impact of chemicals and physical stressors on horizontal gene transfer via natural transformation

Contaminants in the environment can increase natural transformation rates in bacteria. However, the co-occurrence of a large suite of contaminants may result in final transformation rates that are not based on the geometric addition of individual responses. Here we show that the combination of different chemicals and physical stressors results in natural transformation that do not always follow geometric additive responses. Specifically, some combinations increased transformation rates synergistically, while others decreased rates antagonistically. Unpredictability in the natural transformation outcome was also observed when Acinetobacter baylyi ADP1 was exposed to chlorinated reclaimed water. We determined that the stimulatory effect of contaminants on natural transformation can be compensated when extracellular DNA concentrations fall below 0.1 ng l−1 per 2 × 107 cells. Thus, wastewater treatment process that can minimize concentrations of extracellular DNA and cell load would be a key intervention strategy to minimize natural transformation frequency arising from the use of treated wastewater.

Chlorination (but Not UV Disinfection) Generates Cell Debris that Increases Extracellular Antibiotic Resistance Gene Transfer via Proximal Adsorption to Recipients and Upregulated Transformation Genes.

To advance the understanding of antibiotic resistance propagation from wastewater treatment plants, it is important to elucidate how different effluent disinfection processes affect the dissemination of predominantly extracellular antibiotic resistance genes (eARGs). Here, we show that, by facilitating proximal adsorption to recipient cells, bacterial debris generated by chlorination (but not by UV irradiation) increases the natural transformation frequency of their adsorbed eARG by 2.9 to 7.2-fold relative to free eARGs. This is because chlorination increases the bacterial surface roughness by 1.1 to 6.7-fold and the affinity toward eARGs by 1.6 to 5.8-fold, and 98% of the total eARGs released after chlorination were adsorbed to cell debris. In contrast, UV irradiation released predominantly free eARGs with 18% to 56% lower transformation frequency. The collision theory indicates that the ARG donor-recipient collision frequency increased by 35.1-fold for eARGs adsorbed onto chlorination-generated bacterial debris, and the xDLVO model infers a 29% lower donor-recipient contact energy barrier for these ARGs. Exposure to chlorination-generated bacterial debris also upregulated genes associated with natural transformation in Vibrio vulnificus (e.g., tfoX encoding the major activator of natural transformation) by 2.6 to 5.2-fold, likely due to the generation of chlorinated molecules (5.1-fold higher Cl content after chlorination) and persistent reactive species (e.g., carbon-centered radicals) on bacterial debris. Increased proximal eARG adsorption to bacterial debris was also observed in the secondary effluent after chlorination; this decreased eARG decay by 64% and increased the relative abundance of ARGs by 7.2-fold. Overall, this study highlights that different disinfection approaches can result in different physical states of eARGs that affect their resulting dissemination potential via transformation.

Acinetobacter baylyi Strain BD413 Can Acquire an Antibiotic Resistance Gene by Natural Transformation on Lettuce Phylloplane and Enter the Endosphere.

Antibiotic resistance spread must be considered in a holistic framework which comprises the agri-food ecosystems, where plants can be considered a bridge connecting water and soil habitats with the human microbiome. However, the study of horizontal gene transfer events within the plant microbiome is still overlooked. Here, the environmental strain Acinetobacter baylyi BD413 was used to study the acquisition of extracellular DNA (exDNA) carrying an antibiotic resistance gene (ARG) on lettuce phylloplane, performing experiments at conditions (i.e., plasmid quantities) mimicking those that can be found in a water reuse scenario. Moreover, we assessed how the presence of a surfactant, a co-formulant widely used in agriculture, affected exDNA entry in bacteria and plant tissues, besides the penetration and survival of bacteria into the leaf endosphere. Natural transformation frequency in planta was comparable to that occurring under optimal conditions (i.e., temperature, nutrient provision, and absence of microbial competitors), representing an entrance pathway of ARGs into an epiphytic bacterium able to penetrate the endosphere of a leafy vegetable. The presence of the surfactant determined a higher presence of culturable transformant cells in the leaf tissues but did not significantly increase exDNA entry in A. baylyi BD413 cells and lettuce leaves. More research on HGT (Horizontal Gene Transfer) mechanisms in planta should be performed to obtain experimental data on produce safety in terms of antibiotic resistance.

Identification of the Natural Transformation Genes in Riemerella anatipestifer by Random Transposon Mutagenesis.

In our previous study, it was shown that Riemerella anatipestifer, a Gram-negative bacterium, is naturally competent, but the genes involved in the process of natural transformation remain largely unknown. In this study, a random transposon mutant library was constructed using the R. anatipestifer ATCC11845 strain to screen for the genes involved in natural transformation. Among the 3000 insertion mutants, nine mutants had completely lost the ability of natural transformation, and 14 mutants showed a significant decrease in natural transformation frequency. We found that the genes RA0C_RS04920, RA0C_RS04915, RA0C_RS02645, RA0C_RS04895, RA0C_RS05130, RA0C_RS05105, RA0C_RS09020, and RA0C_RS04870 are essential for the occurrence of natural transformation in R. anatipestifer ATCC11845. In particular, RA0C_RS04895, RA0C_RS05130, RA0C_RS05105, and RA0C_RS04870 were putatively annotated as ComEC, DprA, ComF, and RecA proteins, respectively, in the NCBI database. However, RA0C_RS02645, RA0C_RS04920, RA0C_RS04915, and RA0C_RS09020 were annotated as proteins with unknown function, with no homology to any well-characterized natural transformation machinery proteins. The homologs of these proteins are mainly distributed in the members of Flavobacteriaceae. Taken together, our results suggest that R. anatipestifer encodes a unique natural transformation machinery.

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

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.

Natural Transformation of Riemerella columbina and Its Determinants.

In a previous study, it was shown that Riemerella anatipestifer, a member of Flavobacteriaceae, is naturally competent. However, whether natural competence is universal in Flavobacteriaceae remains unknown. In this study, it was shown for the first time that Riemerella columbina was naturally competent in the laboratory condition; however, Flavobacterium johnsoniae was not naturally competent under the same conditions. The competence of R. columbina was maintained throughout the growth phases, and the transformation frequency was highest during the logarithmic phase. A competition assay revealed that R. columbina preferentially took up its own genomic DNA over heterologous DNA. The natural transformation frequency of R. columbina was significantly increased in GCB medium without peptone or phosphate. Furthermore, natural transformation of R. columbina was inhibited by 0.5 mM EDTA, but could be restored by the addition of CaCl2, MgCl2, ZnCl2, and MnCl2, suggesting that these divalent cations promote the natural transformation of R. columbina. Overall, this study revealed that natural competence is not universal in Flavobacteriaceae members and triggering of competence differs from species to species.

1249. Genetic Evidence That Gepotidacin Shows Well-balanced Dual Targeting against DNA Gyrase And Topoisomerase IV in Neisseria gonorrhoeae

BackgroundGepotidacin (GEP) is a novel triazaacenaphthylene bacterial type II topoisomerase inhibitor targeting both bacterial DNA gyrase and topoisomerase IV by a different mechanism from fluoroquinolone antibiotics. Although in vitro frequency of resistance to GEP in Neisseria gonorrhoeae (NG) is low, during a phase 2 trial, clinical resistance to gepotidacin in NG emerged in a subset of fluoroquinolone-resistant NG isolates that contained a pre-existing ParC D86N mutation by introduction of a new GyrA A92T mutation. The objective of this study was to evaluate the role of GyrA A92T & Parc D86N mutations in resistance to GEP.MethodsWe utilized the high frequency of natural transformation to introduce GyrA A92T and ParC D86N mutations, individually and in combination, into NG isolates either with GyrA S91F D95G mutations or with wild type (WT) GyrA by selection on ciprofloxacin (CIP) or GEP to generate isogenic strains for susceptibility evaluation.ResultsResults are summarized in enclosed table. Overall, GyrA A92T and ParC D86N mutations alone did not confer a significant (>4-fold) increase in GEP MIC; whereas together they gave >16-fold increases in GEP MIC. Importantly, quinolone target mutations (GyrA S91F D95G and ParC D86N) together showed no significant effect on the GEP MIC; while they gave >1000-fold increase in CIP MIC. As expected, GyrA A92T and ParC D86N mutations alone or together in WT GyrA background had no significant effect on CIP susceptibility.Susceptibility of isogenic NG strains to gepotidacin and ciprofloxacin ConclusionOur results indicated that unlike fluoroquinolones that primarily target DNA gyrase in NG, there is no obvious primary target for GEP, supporting well-balanced dual targeting of DNA gyrase and topoisomerase IV by GEP in NG. Though, the pre-existing ParC D86N mutation is a potential risk marker for clinical resistance development, as this mutation compromises dual targeting of GEP, our studies provide mechanistic insight for appropriate clinical dose selection to potentially suppress further resistance development in this subset of clinical isolates.DisclosuresPan Chan, PhD, GlaxoSmithKline (Employee, Shareholder) Karen Ingraham, MS, GlaxoSmithKline (Employee, Shareholder) Sharon Min, MS, GlaxoSmithKline (Employee, Shareholder) Nicole Scangarella-Oman, MS, GlaxoSmithKline plc. (Employee, Shareholder) Steve Rittenhouse, PhD, GlaxoSmithKline (Employee, Shareholder) Jianzhong Huang, PhD, GlaxoSmithKline (Employee, Shareholder)

Calcium transcriptionally regulates movement, recombination and other functions of Xylella fastidiosa under constant flow inside microfluidic chambers.

Summary Xylella fastidiosa is a xylem‐limited bacterial pathogen causing devastating diseases in many economically important crops. Calcium (Ca) is a major inorganic nutrient in xylem sap that influences virulence‐related traits of this pathogen, including biofilm formation and twitching motility. This study aimed to adapt a microfluidic system, which mimics the natural habitat of X. fastidiosa, for whole transcriptome analysis under flow conditions. A microfluidic chamber with two parallel channels was used, and RNA isolated from cells grown inside the system was analysed by RNA‐Seq. Ca transcriptionally regulated the machinery of type IV pili and other genes related to pathogenicity and host adaptation. Results were compared to our previous RNA‐Seq study in biofilm cells in batch cultures (Parker et al., 2016, Environ Microbiol 18, 1620). Ca‐regulated genes in both studies belonged to similar functional categories, but the number and tendencies (up‐/downregulation) of regulated genes were different. Recombination‐related genes were upregulated by Ca, and we proved experimentally that 2 mM Ca enhances natural transformation frequency. Taken together, our results suggest that the regulatory role of Ca in X. fastidiosa acts differently during growth in flow or batch conditions, and this can correlate to the different phases of growth (planktonic and biofilm) during the infection process.

Acquisition of Extracellular DNA by Acinetobacter baylyi ADP1 in Response to Solar and UV-C254nm Disinfection.

Extracellular DNA (eDNA) cannot be effectively removed by most of the existing wastewater treatment technologies and can contribute to the gain of new functional traits when transformed into competent bacteria present in downstream environments. This study evaluates the contributions of solar and UV-C254nm irradiation to the transformation of eDNA in Acinetobacter baylyi ADP1. Solar irradiation was evaluated because it is a natural environmental stressor to which eDNA would be exposed during wastewater reuse. UV-C254nm was evaluated as an alternative to a chlorine-based disinfection strategy. Our findings showed that solar disinfection increased the natural transformation frequency by up to 2.0-fold after irradiance at 153 mJ/cm2. This was largely mediated by reactive oxygen species generation, which was correlated with an upregulation of both DNA repair (recA and ddrR) and competence (comA and pilX) genes. In contrast, even though UV-C254nm exposure was accompanied by the upregulation of DNA repair (recA, ddrR, and uvrB) genes and, hence, possibly higher integration rates of eDNA, we observed a concentration-dependent decrease in transformation rates. This decrease in transformation was likely due to the UV dimerization of eDNA, which resulted in the integration of damaged genes that cannot be transcribed into any functional gene products. These results imply that even though sunlight stimulates eDNA uptake and integration in the natural environment, UV disinfection implemented at a treatment plant can potentially minimize subsequent detrimental effects by damaging the extracellular genetic material and ensuring that there is no substantial expression of these transformed genes.

Human serum albumin alters specific genes that can play a role in survival and persistence in Acinetobacter baumannii

In the past few decades Acinetobacter baumannii has emerged as a notorious nosocomial pathogen because of its ability to acquire genetic material and persist in extreme environments. Recently, human serum albumin (HSA) was shown to significantly increase natural transformation frequency in A. baumannii. This observation led us to perform transcriptomic analysis of strain A118 under HSA induction to identify genes that are altered by HSA. Our results revealed the statistically significant differential expression of 296 protein-coding genes, including those associated with motility, biofilm formation, metabolism, efflux pumps, capsule synthesis, and transcriptional regulation. Phenotypic analysis of these traits showed an increase in surface-associated motility, a decrease in biofilm formation, reduced activity of a citric acid cycle associated enzyme, and increased survival associated with zinc availability. Furthermore, the expression of genes known to play a role in pathogenicity and antibiotic resistance were altered. These genes included those associated with RND-type efflux pumps, the type VI secretion system, iron acquisition/metabolism, and ß-lactam resistance. Together, these results illustrate how human products, in particular HSA, may play a significant role in both survival and persistence of A. baumannii.

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

The 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 baumannii is 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 in A. baumannii are unknown. In this study, we demonstrate that naturally competent strain A118 increases its natural transformation frequency upon the addition of Ca(2+)or albumin. We show that comEA and pilQ are 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 in A. baumannii Overall, our results suggest that the main protein in blood enhances HGT in A. baumannii, contributing to the increase of AMR in this threatening human pathogen.

Environmental factors affecting the expression of type IV pilus genes as well as piliation of Thermus thermophilus.

The thermophilic bacterium Thermus thermophilus HB27 is known for its highly efficient natural transformation system, which has become a model system to study the structure and function of DNA transporter in thermophilic bacteria. The DNA transporter is functionally linked to type IV pili (T4P), which are essential for twitching motility and adhesion to solid surfaces. However, the pilus structures themselves are dispensable for natural transformation. Here, we report that the cellular mRNA levels of the major structural subunit of the T4P, PilA4, are regulated by environmental factors. Growth of T. thermophilus in minimal medium or low temperature (55 °C) leads to a significant increase in pilA4 transcripts. In contrast, the transcript levels of the minor pilin pilA1 as well as other T4P genes are nearly unaffected. The elevated pilA4 mRNA levels are accompanied by an increase in piliation of the cells but not by elevated natural transformation frequencies. Hyperpiliation leads to increased adhesion to plastic surfaces. The increased cell-surface interactions are suggested to represent an adaptive response to temperature stress and may be advantageous for survival of T. thermophilus.

Characterization of Nontypable Haemophilus influenzae Isolates Recovered from Adult Patients with Underlying Chronic Lung Disease Reveals Genotypic and Phenotypic Traits Associated with Persistent Infection

Nontypable Haemophilus influenzae (NTHi) has emerged as an important opportunistic pathogen causing infection in adults suffering obstructive lung diseases. Existing evidence associates chronic infection by NTHi to the progression of the chronic respiratory disease, but specific features of NTHi associated with persistence have not been comprehensively addressed. To provide clues about adaptive strategies adopted by NTHi during persistent infection, we compared sequential persistent isolates with newly acquired isolates in sputa from six patients with chronic obstructive lung disease. Pulse field gel electrophoresis (PFGE) identified three patients with consecutive persistent strains and three with new strains. Phenotypic characterisation included infection of respiratory epithelial cells, bacterial self-aggregation, biofilm formation and resistance to antimicrobial peptides (AMP). Persistent isolates differed from new strains in showing low epithelial adhesion and inability to form biofilms when grown under continuous-flow culture conditions in microfermenters. Self-aggregation clustered the strains by patient, not by persistence. Increasing resistance to AMPs was observed for each series of persistent isolates; this was not associated with lipooligosaccharide decoration with phosphorylcholine or with lipid A acylation. Variation was further analyzed for the series of three persistent isolates recovered from patient 1. These isolates displayed comparable growth rate, natural transformation frequency and murine pulmonary infection. Genome sequencing of these three isolates revealed sequential acquisition of single-nucleotide variants in the AMP permease sapC, the heme acquisition systems hgpB, hgpC, hup and hxuC, the 3-deoxy-D-manno-octulosonic acid kinase kdkA, the long-chain fatty acid transporter ompP1, and the phosphoribosylamine glycine ligase purD. Collectively, we frame a range of pathogenic traits and a repertoire of genetic variants in the context of persistent infection by NTHi.

The pga gene cluster in Aggregatibacter actinomycetemcomitans is necessary for the development of natural competence in Ca2+‐promoted biofilms

Natural competence is the ability of bacteria to incorporate extracellular DNA into their genomes. This competence is affected by a number of factors, including Ca(2+) utilization and biofilm formation. As bacteria can form thick biofilms in the presence of extracellular Ca(2+) , the additive effects of Ca(2+) -promoted biofilm formation on natural competence should be examined. We evaluated natural competence in Aggregatibacter actinomycetemcomitans, an important periodontal pathogen, in the context of Ca(2+) -promoted biofilms, and examined whether the pga gene cluster, required for bacterial cell aggregation, is necessary for competence development. The A.actinomycetemcomitans cells grown in the presence of 1mm CaCl2 exhibited enhanced cell aggregation and increased levels of cell-associated Ca(2+) . Biofilm-derived cells grown in the presence of Ca(2+) exhibited the highest levels of natural transformation frequency and enhanced expression of the competence regulator gene, tfoX. Natural competence was enhanced by the additive effects of Ca(2+) -promoted biofilms, in which high levels of pga gene expression were also detected. Mutation of the pga gene cluster disrupted biofilm formation and competence development, suggesting that these genes play a critical role in the ability of A.actinomycetemcomitans to adapt to its natural environment. The Ca(2+) -promoted biofilms may enhance the ability of bacteria to acquire extracellular DNA.

Analysis of Vibration and Noise in Transformer under DC Bias

DC bias would cause the half-wave saturation of transformer, distortion of the exaction current, generation of high harmonic and the vibration and noise increases rapidly, which harm the safe operation of the transformers. In this paper, simulation of magnetic vibration and noise of a 160KVA dry-type transformer with DC bias and modal analysis with the finite element software comsol multiphysics is obtained. The software conduct multi-physical field coupling analysis to electric field, magnetic field, structural stress field and acoustic field, calculate the distribution of the noise and the natural frequency of transformer and propose effective measures to reduce the noise and vibration of transformer with DC bias.

Dramatic scenario of urogenital Candida albicans in presence of Lactic acid bacteria (LAB Effects of different light treatments on the natural transformation of Synechocystis sp. strain PCC 6803)

Although many factors that affect the frequency of natural transformation of the unicellular cyanobacterium Synechocystis have been extensively reported, little is known regarding the effects of changes in the spectral quality and intensity of light on its natural transformation. The frequency of natural transformation was significantly enhanced or inhibited by the treatments with low light (LL) or high light (HL) under the incubation condition of cells and DNA before plating and/or on the plates in comparison with that by the treatment with growth light (GL); the changes in the spectral quality of light did not remarkably affect the transformation efficiency of Synechocystis. Further, the lengths of the appearance time of transformants were shortened or retarded by HL or LL illumination when cells and DNA were incubated on the plates relative to that by GL illumination. Further, the transformation efficiency of Synechocystis was closely associated with the permeability of the cell membranes. Treatment with LL significantly enhances the frequency of natural transformation whereas HL illumination remarkably shortens the appearance time of Synechocystis transformants. These phenomena can be extensively applied to future studies according to the specific demands of the transformation experiments. Possible mechanisms underlying these phenomena are discussed. Key words: Appearance time of transformants, light treatments, natural transformation,Synechocystis, transformation efficiency.

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

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 Deltappk1 mutant was deficient in poly-P accumulation, which was associated with a decreased ability to form viable-but-nonculturable cells under acid stress. The Deltappk1 mutant also showed a decreased frequency of natural transformation and an increased susceptibility to various antimicrobials. Furthermore, the Deltappk1 mutant was characterized by a dose-dependent deficiency in chicken colonization. Complementation of the Deltappk1 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.

Influence of DNA conformation and role ofcomAandrecAon natural transformation inRalstonia solanacearum

Naturally competent bacteria such as the plant pathogen Ralstonia solanacearum are characterized by their ability to take up free DNA from their surroundings. In this study, we investigated the efficiency of various DNA types including chromosomal linear DNA and circular or linearized integrative and (or) replicative plasmids to naturally transform R. solanacearum. To study the respective regulatory role of DNA transport and maintenance in the definite acquisition of new DNA by bacteria, the natural transformation frequencies were compared with those obtained when the bacterial strain was transformed by electroporation. An additional round of electrotransformation and natural transformation was carried out with the same set of donor DNAs and with R. solanacearum disrupted mutants that were potentially affected in competence (comA gene) and recombination (recA gene) functions. Our results confirmed the critical role of the comA gene for natural transformation and that of recA for recombination and, more surprisingly, for the maintenance of an autonomous plasmid in the host cell. Finally, our results showed that homologous recombination of chromosomal linear DNA fragments taken up by natural transformation was the most efficient way for R. solanacearum to acquire new DNA, in agreement with previous data showing competence development and natural transformation between R. solanacearum cells in plant tissues.