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 Type IV Pilus Biogenesis, Twitching Motility, and DNA Uptake in Thermus thermophilus: Discrete Roles of Antagonistic ATPases PilF, PilT1, and PilT2

2013 ◽  
Vol 80 (2) ◽  
pp. 644-652 ◽  
Author(s):  
Ralf Salzer ◽  
Friederike Joos ◽  
Beate Averhoff
Keyword(s):  
Natural Transformation ◽  
Thermophilic Bacteria ◽  
Bacterial Species ◽  
Thermus Thermophilus ◽  
Dna Uptake ◽  
Twitching Motility ◽  
Type Iv Pilus ◽  
Content Type ◽  
Ecological Diversification ◽  
Type Iv

ABSTRACTNatural transformation has a large impact on lateral gene flow and has contributed significantly to the ecological diversification and adaptation of bacterial species.Thermus thermophilusHB27 has emerged as the leading model organism for studies of DNA transporters in thermophilic bacteria. Recently, we identified a zinc-binding polymerization nucleoside triphosphatase (NTPase), PilF, which is essential for the transport of DNA through the outer membrane. Here, we present genetic evidence that PilF is also essential for the biogenesis of pili. One of the most challenging questions was whetherT. thermophilushas any depolymerization NTPase acting as a counterplayer of PilF. We identified two depolymerization NTPases, PilT1 (TTC1621) and PilT2 (TTC1415), both of which are required for type IV pilus (T4P)-mediated twitching motility and adhesion but dispensable for natural transformation. This suggests that T4P dynamics are not required for natural transformation. The latter finding is consistent with our suggestion that inT. thermophilus, T4P and natural transformation are linked but distinct systems.

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.

scholarly journals Native Plasmid-Encoded Mercury Resistance Genes Are Functional and Demonstrate Natural Transformation in Environmental Bacterial Isolates

mSystems ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Ankita Kothari ◽  
Drishti Soneja ◽  
Albert Tang ◽  
Hans K. Carlson ◽  
Adam M. Deutschbauer ◽  
...  
Keyword(s):  
Natural Transformation ◽  
Phylogenetic Analyses ◽  
Environmental Dna ◽  
Metal Resistance ◽  
Mercury Resistance ◽  
Bacterial Isolates ◽  
Rapid Adaptation ◽  
Bacterial Strains ◽  
Content Type ◽  
Native Plasmid

ABSTRACT Plasmid-mediated horizontal gene transfer (HGT) is a major driver of genetic diversity in bacteria. We experimentally validated the function of a putative mercury resistance operon present on an abundant 8-kbp native plasmid found in groundwater samples without detectable levels of mercury. Phylogenetic analyses of the plasmid-encoded mercury reductases from the studied groundwater site show them to be distinct from those reported in proximal metal-contaminated sites. We synthesized the entire native plasmid and demonstrated that the plasmid was sufficient to confer functional mercury resistance in Escherichia coli. Given the possibility that natural transformation is a prevalent HGT mechanism in the low-cell-density environments of groundwaters, we also assayed bacterial strains from this environment for competence. We used the native plasmid-encoded metal resistance to design a screen and identified 17 strains positive for natural transformation. We selected 2 of the positive strains along with a model bacterium to fully confirm HGT via natural transformation. From an ecological perspective, the role of the native plasmid population in providing advantageous traits combined with the microbiome’s capacity to take up environmental DNA enables rapid adaptation to environmental stresses. IMPORTANCE Horizontal transfer of mobile genetic elements via natural transformation has been poorly understood in environmental microbes. Here, we confirm the functionality of a native plasmid-encoded mercury resistance operon in a model microbe and then query for the dissemination of this resistance trait via natural transformation into environmental bacterial isolates. We identified 17 strains including Gram-positive and Gram-negative bacteria to be naturally competent. These strains were able to successfully take up the plasmid DNA and obtain a clear growth advantage in the presence of mercury. Our study provides important insights into gene dissemination via natural transformation enabling rapid adaptation to dynamic stresses in groundwater environments.

scholarly journals 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

2018 ◽  
Vol 84 (18) ◽  
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.

scholarly journals ZpdN, a Plasmid-Encoded Sigma Factor Homolog, Induces pBS32-Dependent Cell Death in Bacillus subtilis

2016 ◽  
Vol 198 (21) ◽  
pp. 2975-2984 ◽  
Author(s):  
B.-E. Myagmarjav ◽  
M. A. Konkol ◽  
J. Ramsey ◽  
S. Mukhopadhyay ◽  
D. B. Kearns
Keyword(s):  
Cell Death ◽  
Bacillus Subtilis ◽  
Mitomycin C ◽  
Sigma Factor ◽  
Natural Transformation ◽  
Potent Inhibitor ◽  
Dna Uptake ◽  
Content Type ◽  
Dependent Cell ◽  
Necessary And Sufficient

ABSTRACTThe ancestralBacillus subtilisstrain 3610 contains an 84-kb plasmid called pBS32 that was lost during domestication of commonly used laboratory derivatives. Here we demonstrate that pBS32, normally present at 1 or 2 copies per cell, increases in copy number nearly 100-fold when cells are treated with the DNA-damaging agent mitomycin C. Mitomycin C treatment also caused cell lysis dependent on pBS32-borne prophage genes. ZpdN, a sigma factor homolog encoded by pBS32, was required for the plasmid response to DNA damage, and artificial expression of ZpdN was sufficient to induce pBS32 hyperreplication and cell death. Plasmid DNA released by cell death was protected by the capsid protein ZpbH, suggesting that the plasmid was packaged into a phagelike particle. The putative particles were further indicated by CsCl sedimentation but were not observed by electron microscopy and were incapable of killingB. subtiliscells extracellularly. We hypothesize that pBS32-mediated cell death releases a phagelike particle that is defective and unstable.IMPORTANCEProphages are phage genomes stably integrated into the host bacterium's chromosome and less frequently are maintained as extrachromosomal plasmids. Here we report that the extrachromosomal plasmid pBS32 ofBacillus subtilisencodes a prophage that, when activated, kills the host. pBS32 also encodes both the sigma factor homolog ZpdN that is necessary and sufficient for prophage induction and the protein ComI, which is a potent inhibitor of DNA uptake by natural transformation. We provide evidence that the entire pBS32 sequence may be part of the prophage and thus that competence inhibition may be linked to lysogeny.

scholarly journals CryoEM structure of the Vibrio cholerae Type IV competence pilus secretin PilQ

2020 ◽  
Author(s):  
Sara J. Weaver ◽  
Matthew H. Sazinsky ◽  
Triana N. Dalia ◽  
Ankur B. Dalia ◽  
Grant J. Jensen
Keyword(s):  
Antibiotic Resistance ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Vibrio Cholerae ◽  
Natural Transformation ◽  
Structural Information ◽  
Genetic Material ◽  
Surface Binding ◽  
Exogenous Dna ◽  
Type Iv

AbstractNatural 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, the Type IV competence pilus 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. A lack of structural information has hindered our understanding of this process, however. Here, we solved the first ever high-resolution structure of a Type IV competence pilus secretin. A functional tagged allele of VcPilQ purified from native V. cholerae cells was used to determine the cryoEM structure of the PilQ secretin in amphipol to ∼2.7 Å. This structure highlights for the first time key differences in the architecture of the Type IV competence pilus secretin from the Type II and Type III Secretin System secretins. Based on our cryoEM structure, we designed a series of mutants to interrogate the mechanism of PilQ. These experiments provide insight into the channel that DNA likely traverses to promote the spread of antibiotic resistance via horizontal gene transfer by natural transformation. We prove that it is possible to reduce pilus biogenesis and natural transformation by sealing the gate, suggesting VcPilQ as a new drug target.

scholarly journals Natural DNA Transformation Is Functional in Lactococcus lactis subsp. cremoris KW2

2017 ◽  
Vol 83 (16) ◽  
Author(s):  
Blandine David ◽  
Amandine Radziejwoski ◽  
Frédéric Toussaint ◽  
Laetitia Fontaine ◽  
Marie Henry de Frahan ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactococcus Lactis ◽  
Natural Transformation ◽  
Single Point ◽  
Adaptor Protein ◽  
Single Point Mutation ◽  
Dna Transformation ◽  
Dna Uptake ◽  
Genetic Traits ◽  
Content Type

ABSTRACT Lactococcus lactis is one of the most commonly used lactic acid bacteria in the dairy industry. Activation of competence for natural DNA transformation in this species would greatly improve the selection of novel strains with desired genetic traits. Here, we investigated the activation of natural transformation in L. lactis subsp. cremoris KW2, a strain of plant origin whose genome encodes the master competence regulator ComX and the complete set of proteins usually required for natural transformation. In the absence of knowledge about competence regulation in this species, we constitutively overproduced ComX in a reporter strain of late competence phase activation and showed, by transcriptomic analyses, a ComX-dependent induction of all key competence genes. We further demonstrated that natural DNA transformation is functional in this strain and requires the competence DNA uptake machinery. Since constitutive ComX overproduction is unstable, we alternatively expressed comX under the control of an endogenous xylose-inducible promoter. This regulated system was used to successfully inactivate the adaptor protein MecA and subunits of the Clp proteolytic complex, which were previously shown to be involved in ComX degradation in streptococci. In the presence of a small amount of ComX, the deletion of mecA, clpC, or clpP genes markedly increased the activation of the late competence phase and transformability. Altogether, our results report the functionality of natural DNA transformation in L. lactis and pave the way for the identification of signaling mechanisms that trigger the competence state in this species. IMPORTANCE Lactococcus lactis is a lactic acid bacterium of major importance, which is used as a starter species for milk fermentation, a host for heterologous protein production, and a delivery platform for therapeutic molecules. Here, we report the functionality of natural transformation in L. lactis subsp. cremoris KW2 by the overproduction of the master competence regulator ComX. The developed procedure enables a flexible approach to modify the chromosome with single point mutation, sequence insertion, or sequence replacement. These results represent an important step for the genetic engineering of L. lactis that will facilitate the design of strains optimized for industrial applications. This will also help to discover natural regulatory mechanisms controlling competence in the genus Lactococcus.

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 Requirement of Novel Competence Genes pilT andpilU of Pseudomonas stutzeri for Natural Transformation and Suppression of pilT Deficiency by a Hexahistidine Tag on the Type IV Pilus Protein PilAI

2001 ◽  
Vol 183 (16) ◽  
pp. 4694-4701 ◽  
Author(s):  
Stefan Graupner ◽  
Nicole Weger ◽  
Monika Sohni ◽  
Wilfried Wackernagel
Keyword(s):  
Amino Acid ◽  
Natural Transformation ◽  
Pseudomonas Stutzeri ◽  
Amino Acid Identity ◽  
Binding Motif ◽  
Dna Uptake ◽  
Twitching Motility ◽  
Type Iv Pilus ◽  
Type Iv ◽  
Insertional Inactivation

ABSTRACT The ubiquitous species Pseudomonas stutzeri has type IV pili, and these are essential for the natural transformation of the cells. An absolute transformation-deficient mutant obtained after transposon mutagenesis had an insertion in a gene which was termedpilT. The deduced amino acid sequence has identity with PilT of Pseudomonas aeruginosa (94%), Neisseria gonorrhoeae (67%), and other gram-negative species and it contains a nucleotide-binding motif. The mutant was hyperpiliated but defective for further pilus-associated properties, such as twitching motility and plating of pilus-specific phage PO4. [3H]thymidine-labeled DNA was bound by the mutant but not taken up. Downstream of pilT a gene, termedpilU, coding for a putative protein with 88% amino acid identity with PilU of P. aeruginosa was identified. Insertional inactivation did not affect piliation, twitching motility, or PO4 infection but reduced transformation to about 10%. The defect was fully complemented by PilU of nontransformable P. aeruginosa. When thepilAI gene (coding for the type IV pilus prepilin) was manipulated to code for a protein in which the six C-terminal amino acids were replaced by six histidine residues and then expressed from a plasmid, it gave a nonpiliated and twitching motility-defective phenotype in pilAI::Gmr cells but allowed transformability. Moreover, the mutant allele suppressed the absolute transformation deficiency caused by the pilT mutation. Considering the hypothesized role of pilT + in pilus retraction and the presumed requirement of retraction for DNA uptake, it is proposed that the pilT-independent transformation is promoted by PilA mutant protein either as single molecules or as minimal pilin assembly structures in the periplasm which may resemble depolymerized pili and that these cause the outer membrane pores to open for DNA entry.

scholarly journals Loss of Meningococcal PilU Delays Microcolony Formation and Attenuates VirulenceIn Vivo

2012 ◽  
Vol 80 (7) ◽  
pp. 2538-2547 ◽  
Author(s):  
Jens Eriksson ◽  
Olaspers Sara Eriksson ◽  
Ann-Beth Jonsson
Keyword(s):  
Dna Uptake ◽  
Wild Type ◽  
Content Type ◽  
Type Iv ◽  
Hexameric Atpase ◽  
Normal Human ◽  
Bacterial Aggregates ◽  
High Degree ◽  
Type Infection

ABSTRACTNeisseria meningitidisis a major cause of sepsis and bacterial meningitis worldwide. This bacterium expresses type IV pili (Tfp), which mediate important virulence traits such as the formation of bacterial aggregates, host cell adhesion, twitching motility, and DNA uptake. The meningococcal PilT protein is a hexameric ATPase that mediates pilus retraction. The PilU protein is produced from thepilT-pilUoperon and shares a high degree of homology with PilT. The function of PilT in Tfp biology has been studied extensively, whereas the role of PilU remains poorly understood. Here we show thatpilUmutants have delayed microcolony formation on host epithelial cells compared to the wild type, indicating that bacterium-bacterium interactions are affected. In normal human serum, thepilUmutant survived at a higher rate than that for wild-type bacteria. However, in a murine model of disease, mice infected with thepilTmutant demonstrated significantly reduced bacterial blood counts and survived at a higher rate than that for mice infected with the wild type. Infection of mice with thepilUmutant resulted in a trend of lower bacteremia, and still a significant increase in survival, than that of the wild type. In conclusion, these data suggest that PilU promotes timely microcolony formation and that both PilU and PilT are required for full bacterial virulence.

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