scholarly journals A Cotransformation Method To Identify a Restriction-Modification Enzyme That Reduces Conjugation Efficiency inCampylobacter jejuni

2018 ◽  
Vol 84 (23) ◽  
Author(s):  
Ximin Zeng ◽  
Zuowei Wu ◽  
Qijing Zhang ◽  
Jun Lin
Keyword(s):  
Comparative Genomics ◽  
Heat Shock ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Campylobacter Jejuni ◽  
Selection Marker ◽  
Sequencing Analysis ◽  
Content Type ◽  
Restriction Modification ◽  
Modification Enzyme

ABSTRACTConjugation is an important mechanism for horizontal gene transfer inCampylobacter jejuni, the leading cause of human bacterial gastroenteritis in developed countries. However, to date, the factors that significantly influence conjugation efficiency inCampylobacterspp. are still largely unknown. Given that multiple recombinant loci could independently occur within one recipient cell during natural transformation, the genetic materials from a high-frequency conjugation (HFC)C. jejunistrain may be cotransformed with a selection marker into a low-frequency conjugation (LFC) recipient strain, creating new HFC transformants suitable for the identification of conjugation factors using a comparative genomics approach. To test this, an erythromycin resistance selection marker was created in an HFCC. jejunistrain; subsequently, the DNA of this strain was naturally transformed into NCTC 11168, an LFCC. jejunistrain, leading to the isolation of NCTC 11168-derived HFC transformants. Whole-genome sequencing analysis and subsequent site-directed mutagenesis identified Cj1051c, a putative restriction-modification enzyme (akaCjeI) that could drastically reduce the conjugation efficiency of NCTC 11168 (>5,000-fold). Chromosomal complementation of three diverse HFCC. jejunistrains with CjeI also led to a dramatic reduction in conjugation efficiency (∼1,000-fold). The purified recombinant CjeI could effectively digest theEscherichia coli-derived shuttle vector pRY107. The endonuclease activity of CjeI was abolished upon short heat shock treatment at 50°C, which is consistent with our previous observation that heat shock enhanced conjugation efficiency inC. jejuni. Together, in this study, we successfully developed and utilized a unique cotransformation strategy to identify a restriction-modification enzyme that significantly influences conjugation efficiency inC. jejuni.IMPORTANCEConjugation is an important horizontal gene transfer mechanism contributing to the evolution of bacterial pathogenesis and antimicrobial resistance.Campylobacter jejuni, the leading foodborne bacterial organism, displays significant strain diversity due to horizontal gene transfer; however, the molecular components influencing conjugation efficiency inC. jejuniare still largely unknown. In this study, we developed a cotransformation strategy for comparative genomics analysis and successfully identified a restriction-modification enzyme that significantly influences conjugation efficiency inC. jejuni. The new cotransformation strategy developed in this study is also expected to be broadly applied in other naturally competent bacteria for functional comparative genomics research.

scholarly journals Heat Shock-Enhanced Conjugation Efficiency in Standard Campylobacter jejuni Strains

2015 ◽  
Vol 81 (13) ◽  
pp. 4546-4552 ◽  
Author(s):  
Ximin Zeng ◽  
Devarshi Ardeshna ◽  
Jun Lin
Keyword(s):  
Heat Shock ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Campylobacter Jejuni ◽  
Recipient Cell ◽  
The United States ◽  
Donor Strain ◽  
Content Type ◽  
Conjugative Gene Transfer ◽  
Molecular Manipulation

ABSTRACTCampylobacter jejuni, the leading bacterial cause of human gastroenteritis in the United States, displays significant strain diversity due to horizontal gene transfer. Conjugation is an important horizontal gene transfer mechanism contributing to the evolution of bacterial pathogenesis and antimicrobial resistance. It has been observed that heat shock could increase transformation efficiency in some bacteria. In this study, the effect of heat shock onC. jejuniconjugation efficiency and the underlying mechanisms were examined. With a modifiedEscherichia colidonor strain, differentC. jejunirecipient strains displayed significant variation in conjugation efficiency ranging from 6.2 × 10−8to 6.0 × 10−3CFU per recipient cell. Despite reduced viability, heat shock of standardC. jejuniNCTC 11168 and 81-176 strains (e.g., 48 to 54°C for 30 to 60 min) could dramatically enhanceC. jejuniconjugation efficiency up to 1,000-fold. The phenotype of the heat shock-enhanced conjugation inC. jejunirecipient cells could be sustained for at least 9 h. Filtered supernatant from the heat shock-treatedC. jejunicells could not enhance conjugation efficiency, which suggests that the enhanced conjugation efficiency is independent of secreted substances. Mutagenesis analysis indicated that the clustered regularly interspaced short palindromic repeats system and the selected restriction-modification systems (Cj0030/Cj0031, Cj0139/Cj0140, Cj0690c, and HsdR) were dispensable for heat shock-enhanced conjugation inC. jejuni. Taking all results together, this study demonstrated a heat shock-enhanced conjugation efficiency in standardC. jejunistrains, leading to an optimized conjugation protocol for molecular manipulation of this organism. The findings from this study also represent a significant step toward elucidation of the molecular mechanism of conjugative gene transfer inC. jejuni.

scholarly journals Is the Evolution of Salmonella enterica subsp. enterica Linked to Restriction-Modification Systems?

mSystems ◽  
2016 ◽  
Vol 1 (3) ◽  
Author(s):  
Louise Roer ◽  
Rene S. Hendriksen ◽  
Pimlapas Leekitcharoenphon ◽  
Oksana Lukjancenko ◽  
Rolf Sommer Kaas ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Salmonella Enterica ◽  
Phylogenetic Trees ◽  
Bacterial Species ◽  
Content Type ◽  
Restriction Modification ◽  
Plasmid Replicons ◽  
Epidemiology And Public Health ◽  
Restriction Modification Systems

ABSTRACT The evolution of bacterial pathogens, their plasticity and ability to rapidly change and adapt to new surroundings are crucial for understanding the epidemiology and public health. With the application of genomics, it became clear that horizontal gene transfer played a key role in evolution. To understand the evolution and diversification of pathogens, we need to understand the processes that drive the horizontal gene transfer. Restriction-modification systems are thought to cause rearrangements within the chromosome, as well as act as a barrier to horizontal gene transfer. However, here we show that the correlation between restriction-modification systems and evolution in other bacterial species does not apply to Salmonella enterica subsp. enterica. In summary, from this work, we conclude that other mechanisms might be involved in controlling and shaping the evolution of Salmonella enterica subsp. enterica. Salmonella enterica subsp. enterica bacteria are highly diverse foodborne pathogens that are subdivided into more than 1,500 serovars. The diversity is believed to result from mutational evolution, as well as intra- and interspecies recombination that potentially could be influenced by restriction-modification (RM) systems. The aim of this study was to investigate whether RM systems were linked to the evolution of Salmonella enterica subsp. enterica. The study included 221 Salmonella enterica genomes, of which 68 were de novo sequenced and 153 were public available genomes from ENA. The data set covered 97 different serovars of Salmonella enterica subsp. enterica and an additional five genomes from four other Salmonella subspecies as an outgroup for constructing the phylogenetic trees. The phylogenetic trees were constructed based on multiple alignment of core genes, as well as the presence or absence of pangenes. The topology of the trees was compared to the presence of RM systems, antimicrobial resistance (AMR) genes, Salmonella pathogenicity islands (SPIs), and plasmid replicons. We did not observe any correlation between evolution and the RM systems in S. enterica subsp. enterica. However, sublineage correlations and serovar-specific patterns were observed. Additionally, we conclude that plasmid replicons, SPIs, and AMR were all better correlated to serovars than to RM systems. This study suggests a limited influence of RM systems on the evolution of Salmonella enterica subsp. enterica, which could be due to the conjugational mode of horizontal gene transfer in Salmonella. Thus, we conclude that other factors must be involved in shaping the evolution of bacteria. IMPORTANCE The evolution of bacterial pathogens, their plasticity and ability to rapidly change and adapt to new surroundings are crucial for understanding the epidemiology and public health. With the application of genomics, it became clear that horizontal gene transfer played a key role in evolution. To understand the evolution and diversification of pathogens, we need to understand the processes that drive the horizontal gene transfer. Restriction-modification systems are thought to cause rearrangements within the chromosome, as well as act as a barrier to horizontal gene transfer. However, here we show that the correlation between restriction-modification systems and evolution in other bacterial species does not apply to Salmonella enterica subsp. enterica. In summary, from this work, we conclude that other mechanisms might be involved in controlling and shaping the evolution of Salmonella enterica subsp. enterica.

scholarly journals Complete Sequences of Two Plasmids Found in a Brazilian Bacillus thuringiensis Serovar israelensis Strain

2019 ◽  
Vol 8 (9) ◽  
Author(s):  
Fabrício S. Campos ◽  
Fernando B. Cerqueira ◽  
Gil R. Santos ◽  
Eliseu J. G. Pereira ◽  
Roberto F. T. Corrêia ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Crucial Role ◽  
Bacterial Genomes ◽  
Content Type ◽  
Complete Sequences

Plasmids play a crucial role in the evolution of bacterial genomes by mediating horizontal gene transfer. In this work, we sequenced two plasmids found in a Brazilian Bacillus thuringiensis serovar israelensis strain which showed 100% nucleotide identities with Bacillus thuringiensis serovar kurstaki plasmids.

scholarly journals An Evolutionary Link between Natural Transformation and CRISPR Adaptive Immunity

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Peter Jorth ◽  
Marvin Whiteley
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Bacterial Diversity ◽  
Natural Transformation ◽  
Bacterial Species ◽  
Comparative Genomic ◽  
Content Type ◽  
Immune Systems ◽  
Adaptive Immune ◽  
Adaptive Immune Systems

ABSTRACTNatural transformation by competent bacteria is a primary means of horizontal gene transfer; however, evidence that competence drives bacterial diversity and evolution has remained elusive. To test this theory, we used a retrospective comparative genomic approach to analyze the evolutionary history ofAggregatibacter actinomycetemcomitans, a bacterial species with both competent and noncompetent sister strains. Through comparative genomic analyses, we reveal that competence is evolutionarily linked to genomic diversity and speciation. Competence loss occurs frequently during evolution and is followed by the loss of clustered regularly interspaced short palindromic repeats (CRISPRs), bacterial adaptive immune systems that protect against parasitic DNA. Relative to noncompetent strains, competent bacteria have larger genomes containing multiple rearrangements. In contrast, noncompetent bacterial genomes are extremely stable but paradoxically susceptible to infective DNA elements, which contribute to noncompetent strain genetic diversity. Moreover, incomplete noncompetent strain CRISPR immune systems are enriched for self-targeting elements, which suggests that the CRISPRs have been co-opted for bacterial gene regulation, similar to eukaryotic microRNAs derived from the antiviral RNA interference pathway.IMPORTANCEThe human microbiome is rich with thousands of diverse bacterial species. One mechanism driving this diversity is horizontal gene transfer by natural transformation, whereby naturally competent bacteria take up environmental DNA and incorporate new genes into their genomes. Competence is theorized to accelerate evolution; however, attempts to test this theory have proved difficult. Through genetic analyses of the human periodontal pathogenAggregatibacter actinomycetemcomitans, we have discovered an evolutionary connection between competence systems promoting gene acquisition and CRISPRs (clustered regularly interspaced short palindromic repeats), adaptive immune systems that protect bacteria against genetic parasites. We show that competentA. actinomycetemcomitansstrains have numerous redundant CRISPR immune systems, while noncompetent bacteria have lost their CRISPR immune systems because of inactivating mutations. Together, the evolutionary data linking the evolution of competence and CRISPRs reveals unique mechanisms promoting genetic heterogeneity and the rise of new bacterial species, providing insight into complex mechanisms underlying bacterial diversity in the human body.

scholarly journals Acquisition of Beta-Lactamase by Neisseria meningitidis through Possible Horizontal Gene Transfer

2018 ◽  
Vol 62 (9) ◽  
Author(s):  
Eva Hong ◽  
Ala-Eddine Deghmane ◽  
Muhamed-Kheir Taha
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Genome Sequencing ◽  
Meningococcal Disease ◽  
Bacterial Species ◽  
Whole Genome ◽  
Beta Lactamase ◽  
Content Type ◽  
Beta Lactamases ◽  
Lactamase Gene

ABSTRACT We report the detection in France of a beta-lactamase-producing invasive meningococcal isolate. Whole-genome sequencing of the isolate revealed a ROB-1-type beta-lactamase gene that is frequently encountered in Haemophilus influenzae, suggesting horizontal transfer between isolates of these bacterial species. Beta-lactamases are exceptional in meningococci, with no reports for more than 2 decades. This report is worrying, as the expansion of such isolates may jeopardize the effective treatment against invasive meningococcal disease.

scholarly journals Trasposon Mediated Horizontal Gene Transfer (T-HGT) of Circulating Tumor DNA Reshapes MM Clonal Architecture

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3065-3065
Author(s):  
Munevver Cinar ◽  
Steven Flygare ◽  
Marina Mosunjac ◽  
Ganji Nagaraju ◽  
Dongkyoo Park ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Drug Sensitivity ◽  
Genetic Material ◽  
Host Cells ◽  
Response To Treatment ◽  
Hierarchical Architecture ◽  
Tumor Evolution ◽  
Sequencing Analysis

Spatial genetic heterogeneity is a characteristic phenomenon that influences multiple myeloma's (MM) phenotype and drug sensitivity (Rasche L. et al and Bolli N et al.). Hence, the branch model of tumor evolution is not sufficient to explain the disorganized architecture observed in MM. In this study, we investigated whether MM ctDNA horizontal gene transfer (HGT) affect tumor genetic architecture and drug sensitivity, resembling what is seen in prokaryotes, and elucidated the mechanisms involved in the mobilization of genetic material from one cell to another. We identified that plasma from patients with MM transmits drug sensitivity or resistance to cells in culture. This transmission of drug sensitivity is mediated by ctDNA transfer of oncogenes to a host cell. Importantly, in vitro and in vivo demonstrated that ctDNA mainly targets cells resembling the cell of origin (tropism). Karyotype spreads and whole genome sequencing demonstrated that once patients ctDNA encounters host cells, it migrates into the nucleus where it ultimately integrates into the cell's genome. Integration to the genome was confirmed to be targeted to myeloma cells. Further sequencing analysis of multiple MM samples identified ctDNA tropism and integration is dependent on the 5' and 3' end presence of transposable elements (TE), particularly of the MIR and ALUsq family. These results were further validated by TE mediated delivery of GFP into MM cells in vitro and HSVTK in tumors of mouse xenografts. In conclusion, this data indicates for the first time that TE mediates MM ctDNA HGT into homologous tumor cells shaping the hierarchical architecture of tumor clones and affecting tumor response to treatment. Therapeutically, this unique quality of ctDNA can be exploited for targeted gene therapeutic approaches in MM and potentially other cancers. Disclosures Bernal-Mizrachi: Kodikas Therapeutic Solutions, Inc: Equity Ownership; TAKEDA: Research Funding; Winship Cancer Institute: Employment, Patents & Royalties.

scholarly journals Complete Genome Sequence of the Naphthalene-Degrading Bacterium Pseudomonas stutzeri AN10 (CCUG 29243)

2012 ◽  
Vol 194 (23) ◽  
pp. 6642-6643 ◽  
Author(s):  
Isabel Brunet-Galmés ◽  
Antonio Busquets ◽  
Arantxa Peña ◽  
Margarita Gomila ◽  
Balbina Nogales ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Pseudomonas Stutzeri ◽  
Content Type ◽  
Naphthalene Degradation ◽  
Degrading Bacterium ◽  
Model Strain

ABSTRACTPseudomonas stutzeriAN10 (CCUG 29243) can be considered a model strain for aerobic naphthalene degradation. We report the complete genome sequence of this bacterium. Its 4.71-Mb chromosome provides insights into other biodegradative capabilities of strain AN10 (i.e., benzoate catabolism) and suggests a high number of horizontal gene transfer events.

scholarly journals Biosynthesis of the Unique Wall Teichoic Acid of Staphylococcus aureus Lineage ST395

mBio ◽  
2014 ◽  
Vol 5 (2) ◽  
Author(s):  
Volker Winstel ◽  
Patricia Sanchez-Carballo ◽  
Otto Holst ◽  
Guoqing Xia ◽  
Andreas Peschel
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Biosynthesis Pathway ◽  
Glycerol Phosphate ◽  
Coagulase Negative Staphylococci ◽  
Content Type ◽  
Gram Positive Bacteria ◽  
Wall Teichoic Acids

ABSTRACT The major clonal lineages of the human pathogen Staphylococcus aureus produce cell wall-anchored anionic poly-ribitol-phosphate (RboP) wall teichoic acids (WTA) substituted with d-Alanine and N-acetyl-d-glucosamine. The phylogenetically isolated S. aureus ST395 lineage has recently been found to produce a unique poly-glycerol-phosphate (GroP) WTA glycosylated with N-acetyl-d-galactosamine (GalNAc). ST395 clones bear putative WTA biosynthesis genes on a novel genetic element probably acquired from coagulase-negative staphylococci (CoNS). We elucidated the ST395 WTA biosynthesis pathway and identified three novel WTA biosynthetic genes, including those encoding an α-O-GalNAc transferase TagN, a nucleotide sugar epimerase TagV probably required for generation of the activated sugar donor substrate for TagN, and an unusually short GroP WTA polymerase TagF. By using a panel of mutants derived from ST395, the GalNAc residues carried by GroP WTA were found to be required for infection by the ST395-specific bacteriophage Φ187 and to play a crucial role in horizontal gene transfer of S. aureus pathogenicity islands (SaPIs). Notably, ectopic expression of ST395 WTA biosynthesis genes rendered normal S. aureus susceptible to Φ187 and enabled Φ187-mediated SaPI transfer from ST395 to regular S. aureus. We provide evidence that exchange of WTA genes and their combination in variable, mosaic-like gene clusters have shaped the evolution of staphylococci and their capacities to undergo horizontal gene transfer events. IMPORTANCE The structural highly diverse wall teichoic acids (WTA) are cell wall-anchored glycopolymers produced by most Gram-positive bacteria. While most of the dominant Staphylococcus aureus lineages produce poly-ribitol-phosphate WTA, the recently described ST395 lineage produces a distinct poly-glycerol-phosphate WTA type resembling the WTA backbone of coagulase-negative staphylococci (CoNS). Here, we analyzed the ST395 WTA biosynthesis pathway and found new types of WTA biosynthesis genes along with an evolutionary link between ST395 and CoNS, from which the ST395 WTA genes probably originate. The elucidation of ST395 WTA biosynthesis will help to understand how Gram-positive bacteria produce highly variable WTA types and elucidate functional consequences of WTA variation.

scholarly journals The Protease ClpXP and the PAS Domain Protein DivL Regulate CtrA and Gene Transfer Agent Production inRhodobacter capsulatus

2018 ◽  
Vol 84 (11) ◽  
Author(s):  
Alexander B. Westbye ◽  
Lukas Kater ◽  
Christina Wiesmann ◽  
Hao Ding ◽  
Calvin K. Yip ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Rhodobacter Capsulatus ◽  
Caulobacter Crescentus ◽  
Response Regulator ◽  
Pas Domain ◽  
Content Type ◽  
Regulatory Systems ◽  
Gene Transfer Agent ◽  
Domain Protein

ABSTRACTSeveral members of theRhodobacterales(Alphaproteobacteria) produce a conserved horizontal gene transfer vector, called the gene transfer agent (GTA), that appears to have evolved from a bacteriophage. The model system used to study GTA biology is theRhodobacter capsulatusGTA (RcGTA), a small, tailed bacteriophage-like particle produced by a subset of the cells in a culture. The response regulator CtrA is conserved in theAlphaproteobacteriaand is an essential regulator of RcGTA production: it controls the production and maturation of the RcGTA particle and RcGTA release from cells. CtrA also controls the natural transformation-like system required for cells to receive RcGTA-donated DNA. Here, we report that dysregulation of the CckA-ChpT-CtrA phosphorelay either by the loss of the PAS domain protein DivL or by substitution of the autophosphorylation residue of the hybrid histidine kinase CckA decreased CtrA phosphorylation and greatly increased RcGTA protein production inR. capsulatus. We show that the loss of the ClpXP protease or the three C-terminal residues of CtrA results in increased CtrA levels inR. capsulatusand identify ClpX(P) to be essential for the maturation of RcGTA particles. Furthermore, we show that CtrA phosphorylation is important for head spike production. Our results provide novel insight into the regulation of CtrA and GTAs in theRhodobacterales.IMPORTANCEMembers of theRhodobacteralesare abundant in ocean and freshwater environments. The conserved GTA produced by manyRhodobacteralesmay have an important role in horizontal gene transfer (HGT) in aquatic environments and provide a significant contribution to their adaptation. GTA production is controlled by bacterial regulatory systems, including the conserved CckA-ChpT-CtrA phosphorelay; however, several questions about GTA regulation remain. Our identification that a short DivL homologue and ClpXP regulate CtrA inR. capsulatusextends the model of CtrA regulation fromCaulobacter crescentusto a member of theRhodobacterales. We found that the magnitude of RcGTA production greatly depends on DivL and CckA kinase activity, adding yet another layer of regulatory complexity to RcGTA. RcGTA is known to undergo CckA-dependent maturation, and we extend the understanding of this process by showing that the ClpX chaperone is required for formation of tailed, DNA-containing particles.

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