scholarly journals ComF is a key mediator in single-stranded DNA transport and handling during natural transformation

2021 ◽  
Author(s):  
Prashant P Damke ◽  
Louisa Celma ◽  
Sumedha Kondekar ◽  
Anne Marie Di Guilmi ◽  
Stephanie Marsin ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Helicobacter Pylori ◽  
Natural Transformation ◽  
Bacterial Species ◽  
Zinc Finger Motif ◽  
Single Stranded Dna ◽  
C Protein ◽  
Dna Transport ◽  
Dna Capture

Natural transformation plays a major role in the spreading of antibiotic resistances and virulence factors. Whilst bacterial species display specificities in the molecular machineries allowing transforming DNA capture and integration into their genome, the ComF(C) protein is essential for natural transformation in all Gram- positive and - negative species studied. Despite this, its role remains largely unknown. Here, we show that Helicobacter pylori ComF is not only involved in DNA transport through the cell membrane, but it also required for the handling of the ssDNA once it is delivered into the cytoplasm. ComF crystal structure revealed the presence of a zinc-finger motif and a putative phosphoribosyl transferase domain, both necessary for its in vivo activity. ComF is a membrane-associated protein with affinity for single-stranded DNA. Collectively, our results suggest that ComF provides the link between the transport of the transforming DNA into the cytoplasm and its handling by the recombination machinery.

scholarly journals Structural insights into the unique single-stranded DNA-binding mode of Helicobacter pylori DprA

2013 ◽  
Vol 42 (5) ◽  
pp. 3478-3491 ◽  
Author(s):  
Wei Wang ◽  
Jingjin Ding ◽  
Ying Zhang ◽  
Yonglin Hu ◽  
Da-Cheng Wang
Keyword(s):  
Helicobacter Pylori ◽  
Bacterial Species ◽  
Complex Structure ◽  
Protein A ◽  
Binding Mode ◽  
Binding Assays ◽  
Mobility Shift ◽  
Single Stranded Dna ◽  
Exogenous Dna ◽  
Ssdna Binding

Abstract Natural transformation (NT) in bacteria is a complex process, including binding, uptake, transport and recombination of exogenous DNA into the chromosome, consequently generating genetic diversity and driving evolution. DNA processing protein A (DprA), which is distributed among virtually all bacterial species, is involved in binding to the internalized single-stranded DNA (ssDNA) and promoting the loading of RecA on ssDNA during NTs. Here we present the structures of DNA_processg_A (DprA) domain of the Helicobacter pylori DprA (HpDprA) and its complex with an ssDNA at 2.20 and 1.80 Å resolutions, respectively. The complex structure revealed for the first time how the conserved DprA domain binds to ssDNA. Based on structural comparisons and binding assays, a unique ssDNA-binding mode is proposed: the dimer of HpDprA binds to ssDNA through two small, positively charged binding pockets of the DprA domains with classical Rossmann folds and the key residue Arg52 is re-oriented to ‘open’ the pocket in order to accommodate one of the bases of ssDNA, thus enabling HpDprA to grasp substrate with high affinity. This mode is consistent with the oligomeric composition of the complex as shown by electrophoretic mobility-shift assays and static light scattering measurements, but differs from the direct polymeric complex of Streptococcus pneumoniae DprA–ssDNA.

scholarly journals Identification of the periplasmic DNA receptor for natural transformation of Helicobacter pylori

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Prashant P. Damke ◽  
Anne Marie Di Guilmi ◽  
Paloma Fernández Varela ◽  
Christophe Velours ◽  
Stéphanie Marsin ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Natural Transformation ◽  
Bacterial Species ◽  
Dna Binding Protein ◽  
Gram Negative Bacteria ◽  
Membrane Channel ◽  
Inner Membrane ◽  
Double Stranded Dna ◽  
Periplasmic Domain ◽  
H Pylori

AbstractHorizontal gene transfer through natural transformation is a major driver of antibiotic resistance spreading in many pathogenic bacterial species. In the case of Gram-negative bacteria, and in particular of Helicobacter pylori, the mechanisms underlying the handling of the incoming DNA within the periplasm are poorly understood. Here we identify the protein ComH as the periplasmic receptor for the transforming DNA during natural transformation in H. pylori. ComH is a DNA-binding protein required for the import of DNA into the periplasm. Its C-terminal domain displays strong affinity for double-stranded DNA and is sufficient for the accumulation of DNA in the periplasm, but not for DNA internalisation into the cytoplasm. The N-terminal region of the protein allows the interaction of ComH with a periplasmic domain of the inner-membrane channel ComEC, which is known to mediate the translocation of DNA into the cytoplasm. Our results indicate that ComH is involved in the import of DNA into the periplasm and its delivery to the inner membrane translocator ComEC.

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 Single stranded DNA annealing is a conserved activity of telomere resolvases

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0246212
Author(s):  
Siobhan L. McGrath ◽  
Shu Hui Huang ◽  
Kerri Kobryn
Keyword(s):  
Dna Binding ◽  
Dna Cleavage ◽  
Bacterial Species ◽  
Dna Binding Protein ◽  
Tyrosine Recombinase ◽  
Single Stranded Dna ◽  
Terminal Domain ◽  
Topoisomerase Ib ◽  
Replication Intermediates

Bacterial species of the genera Agrobacterium and Borrelia possess chromosomes terminated by hairpin telomeres. Replication produces dimeric replication intermediates fused via replicated telomere junctions. A specialized class of enzymes, referred to as telomere resolvases, promotes the resolution of the replicated intermediate into linear monomers terminated by hairpin telomeres. Telomere resolution is catalyzed via DNA cleavage and rejoining events mechanistically similar to those promoted by topoisomerase-IB and tyrosine recombinase enzymes. Examination of the borrelial telomere resolvase, ResT, revealed unanticipated multifunctionality; aside from its expected telomere resolution activity ResT possessed a singled-stranded DNA (ssDNA) annealing activity that extended to both naked ssDNA and ssDNA complexed with its cognate single-stranded DNA binding protein (SSB). At present, the role this DNA annealing activity plays in vivo remains unknown. We have demonstrated here that single-stranded DNA annealing is also a conserved property of the agrobacterial telomere resolvase, TelA. This activity in TelA similarly extends to both naked ssDNA and ssDNA bound by its cognate SSB. TelA’s annealing activity was shown to stem from the N-terminal domain; removal of this domain abolished annealing without affecting telomere resolution. Further, independent expression of the N-terminal domain of TelA produced a functional annealing protein. We suggest that the apparent conservation of annealing activity in two telomere resolvases, from distantly related bacterial species, implies a role for this activity in hairpin telomere metabolism. Our demonstration of the separation of the telomere resolution and annealing activities of TelA provides a platform for future experiments aimed at identifying the role DNA annealing performs in vivo.

scholarly journals ComM is a hexameric helicase that promotes branch migration during natural transformation in diverse Gram-negative species

2017 ◽  
Author(s):  
Thomas M. Nero ◽  
Triana N. Dalia ◽  
Joseph Che-Yen Wang ◽  
David T. Kysela ◽  
Matthew L. Bochman ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Natural Transformation ◽  
Bacterial Species ◽  
Branch Migration ◽  
Acinetobacter Baylyi ◽  
Migration Activity ◽  
Gram Negative ◽  
Aaa Protein

ABSTRACTAcquisition of foreign DNA by natural transformation is an important mechanism of adaptation and evolution in diverse microbial species. Here, we characterize the mechanism of ComM, a broadly conserved AAA+ protein previously implicated in homologous recombination of transforming DNA (tDNA) in naturally competent Gram-negative bacterial species.In vivo, we found that ComM was required for efficient comigration of linked genetic markers inVibrio choleraeandAcinetobacter baylyi, which is consistent with a role in branch migration. Also, ComM was particularly important for integration of tDNA with increased sequence heterology, suggesting that its activity promotes the acquisition of novel DNA sequences.In vitro, we showed that purified ComM binds ssDNA, oligomerizes into a hexameric ring, and has bidirectional helicase and branch migration activity. Based on these data, we propose a model for tDNA integration during natural transformation. This study provides mechanistic insight into the enigmatic steps involved in tDNA integration and uncovers the function of a protein required for this conserved mechanism of horizontal gene transfer.

scholarly journals Probiotic Lactobacillus spp. act Against Helicobacter pylori-induced Inflammation

2019 ◽  
Vol 8 (1) ◽  
pp. 90 ◽  
Author(s):  
Yi-Hsing Chen ◽  
Wan-Hua Tsai ◽  
Hui-Yu Wu ◽  
Chun-Ya Chen ◽  
Wen-Ling Yeh ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Bacterial Species ◽  
Lactobacillus Rhamnosus ◽  
Gastrointestinal Diseases ◽  
In Vivo Studies ◽  
Bacterial Strains ◽  
H Pylori ◽  
Lactobacillus Spp

The bacterial species, Helicobacter pylori, is associated with several gastrointestinal diseases, and poses serious health threats owing to its resistance to antibiotics. Lactobacillus spp., on the other hand, possess probiotic activities that have beneficial effects in humans. However, the mechanisms by which Lactobacillus spp. harbor favorable functions and act against H. pylori infection remain to be explored. The aim of this study was to investigate the ability of bacterial strains, Lactobacillus rhamnosus and Lactobacillus acidophilus, termed GMNL-74 and GMNL-185, respectively, to inhibit H. pylori growth and inflammation. Our results showed that GMNL-74 and GMNL-185 possess potent antimicrobial activity against multidrug resistant (MDR)-H. pylori. In addition, an in vitro cell-based model revealed that the inhibition of H. pylori adhesion and invasion of gastric epithelial cells and interleukin-8 production were significantly decreased by treatment with both the Lactobacillus strains. In vivo studies demonstrated that colonization of H. pylori and induced inflammation in the mouse stomach were also alleviated by these Lactobacillus strains. Furthermore, the abundance of beneficial gut bacteria, including Bifidobacterium spp. and Akkermansia muciniphilia, were significantly increased in H. pylori-infected mice treated with GMNL-74 and GMNL-185. These results demonstrate that Lactobacillus spp. ameliorate H. pylori-induced inflammation and supports beneficial gut specific bacteria that act against H. pylori infection.

scholarly journals The Molecular Basis of FimT-mediated DNA Uptake during Bacterial Natural Transformation

2021 ◽  
Author(s):  
Sebastian A.G. Braus ◽  
Francesca L. Short ◽  
Stefanie Holz ◽  
Matthew J.M. Stedman ◽  
Alvar D. Gossert ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Molecular Basis ◽  
Natural Transformation ◽  
Bacterial Species ◽  
Bacterial Genome ◽  
Dna Uptake ◽  
Exogenous Dna ◽  
Wide Range

AbstractNaturally competent bacteria encode sophisticated protein machineries for the uptake and translocation of exogenous DNA into the cell. If this DNA is integrated into the bacterial genome, the bacterium is said to be naturally transformed. Most competent bacterial species utilise type IV pili for the initial DNA uptake step. These proteinaceous cell-surface structures are composed of thousands of pilus subunits (pilins), designated as major or minor according to their relative abundance in the pilus. In this study, we show that the minor pilin FimT plays an important role in the natural transformation of Legionella pneumophila. We used NMR spectroscopy, in vitro DNA binding assays and in vivo transformation assays to understand the molecular basis of FimT’s role in this process. FimT directly interacts with DNA via an electropositive patch, rich in arginines, several of which are well-conserved and located in FimT’s conformationally flexible C-terminal tail. We also show that FimT orthologues from other γ-Proteobacteria share the ability to bind to DNA. Our functional characterisation and comprehensive bioinformatic analysis of FimT, suggest that it plays an important role for DNA uptake in a wide range of competent species.

Structural analysis of the photosynthetic membrane from Ectothiorhodospira halochloris

1983 ◽  
Vol 41 ◽  
pp. 740-741
Author(s):  
H. Engelhardt ◽  
R. Guckenberger ◽  
W. Baumeister
Keyword(s):  
Lattice Structure ◽  
Bacterial Species ◽  
Hexagonal Lattice ◽  
Reaction Centre ◽  
Photosynthetic Membrane ◽  
Rhodopseudomonas Viridis ◽  
Photosynthetic Membranes ◽  
Ectothiorhodospira Halochloris ◽  
Main Components

Bacterial photosynthetic membranes contain, apart from lipids and electron transport components, reaction centre (RC) and light harvesting (LH) polypeptides as the main components. The RC-LH complexes in Rhodopseudomonas viridis membranes are known since quite seme time to form a hexagonal lattice structure in vivo; hence this membrane attracted the particular attention of electron microscopists. Contrary to previous claims in the literature we found, however, that 2-D periodically organized photosynthetic membranes are not a unique feature of Rhodopseudomonas viridis. At least five bacterial species, all bacteriophyll b - containing, possess membranes with the RC-LH complexes regularly arrayed. All these membranes appear to have a similar lattice structure and fine-morphology. The lattice spacings of the Ectothiorhodospira haloohloris, Ectothiorhodospira abdelmalekii and Rhodopseudomonas viridis membranes are close to 13 nm, those of Thiocapsa pfennigii and Rhodopseudomonas sulfoviridis are slightly smaller (∼12.5 nm).

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