Characterization of an atypical but widespread type IV secretion system for transfer of the integrative and conjugative element (ICEclc) in Pseudomonas putida

Abstract Conjugation of DNA relies on multicomponent protein complexes bridging two bacterial cytoplasmic compartments. Whereas plasmid conjugation systems have been well documented, those of integrative and conjugative elements (ICEs) have remained poorly studied. We characterize here the conjugation system of the ICEclc element in Pseudomonas putida UWC1 that is a model for a widely distributed family of ICEs. By in frame deletion and complementation, we show the importance on ICE transfer of 22 genes in a 20-kb conserved ICE region. Protein comparisons recognized seven homologs to plasmid type IV secretion system components, another six homologs to frequent accessory proteins, and the rest without detectable counterparts. Stationary phase imaging of P. putida ICEclc with in-frame fluorescent protein fusions to predicted type IV components showed transfer-competent cell subpopulations with multiple fluorescent foci, largely overlapping in dual-labeled subcomponents, which is suggestive for multiple conjugation complexes per cell. Cross-dependencies between subcomponents in ICE-type IV secretion system assembly were revealed by quantitative foci image analysis in a variety of ICEclc mutant backgrounds. In conclusion, the ICEclc family presents an evolutionary distinct type IV conjugative system with transfer competent cells specialized in efficient transfer.

[1]  J. R. van der Meer,et al.  A bistable prokaryotic differentiation system underlying development of conjugative transfer competence , 2022, PLoS genetics.

[2]  Oriol Vinyals,et al.  Highly accurate protein structure prediction with AlphaFold , 2021, Nature.

[3]  Silvio C. E. Tosatto,et al.  Pfam: The protein families database in 2021 , 2020, Nucleic Acids Res..

[4]  Tiago R D Costa,et al.  Type IV secretion systems: Advances in structure, function, and activation , 2020, Molecular microbiology.

[5]  D. Lacy,et al.  Structural analysis of the Legionella pneumophila Dot/Icm type IV secretion system core complex , 2020, bioRxiv.

[6]  C. Mazza,et al.  An analog to digital converter controls bistable transfer competence development of a widespread bacterial integrative and conjugative element , 2020, bioRxiv.

[7]  Jun Liu,et al.  Analysis of Dot/Icm Type IVB Secretion System Subassemblies by Cryoelectron Tomography Reveals Conformational Changes Induced by DotB Binding , 2020, mBio.

[8]  Liisa Holm,et al.  DALI and the persistence of protein shape , 2019, Protein science : a publication of the Protein Society.

[9]  P. Christie,et al.  Structural bases for F plasmid conjugation and F pilus biogenesis in Escherichia coli , 2019, Proceedings of the National Academy of Sciences.

[10]  J. R. van der Meer,et al.  Transient Replication in Specialized Cells Favors Transfer of an Integrative and Conjugative Element , 2019, mBio.

[11]  A. B. Dalia,et al.  PilT and PilU are homohexameric ATPases that coordinate to retract type IVa pili , 2019, bioRxiv.

[12]  B. Maier,et al.  Type IV pili: dynamics, biophysics and functional consequences , 2019, Nature Reviews Microbiology.

[13]  G. Jensen,et al.  Molecular architecture, polar targeting and biogenesis of the Legionella Dot/Icm T4SS , 2019, Nature Microbiology.

[14]  C. Baron,et al.  VirB8 homolog TraE from plasmid pKM101 forms a hexameric ring structure and interacts with the VirB6 homolog TraD , 2018, Proceedings of the National Academy of Sciences.

[15]  Jun Liu,et al.  A Unique Cytoplasmic ATPase Complex Defines the Legionella pneumophila Type IV Secretion Channel , 2018, Nature Microbiology.

[16]  E. Orlova,et al.  Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery , 2017, The EMBO journal.

[17]  J. R. van der Meer,et al.  The hidden life of integrative and conjugative elements , 2017, FEMS microbiology reviews.

[18]  Paul A. Wiggins,et al.  SuperSegger: robust image segmentation, analysis and lineage tracking of bacterial cells , 2016, Molecular Microbiology.

[19]  Eun-Jeong Yoon,et al.  Clonal Dissemination of Pseudomonas aeruginosa Sequence Type 235 Isolates Carrying blaIMP-6 and Emergence of blaGES-24 and blaIMP-10 on Novel Genomic Islands PAGI-15 and -16 in South Korea , 2016, Antimicrobial Agents and Chemotherapy.

[20]  Sara Mitri,et al.  Highly variable individual donor cell fates characterize robust horizontal gene transfer of an integrative and conjugative element , 2016, Proceedings of the National Academy of Sciences.

[21]  I. Charles,et al.  Genomic islands 1 and 2 play key roles in the evolution of extensively drug-resistant ST235 isolates of Pseudomonas aeruginosa , 2016, Open Biology.

[22]  H. Nagai,et al.  The Type IVB secretion system: an enigmatic chimera. , 2016, Current opinion in microbiology.

[23]  A. Grossman,et al.  Integrative and Conjugative Elements (ICEs): What They Do and How They Work. , 2015, Annual review of genetics.

[24]  Michael J E Sternberg,et al.  The Phyre2 web portal for protein modeling, prediction and analysis , 2015, Nature Protocols.

[25]  A. Goesmann,et al.  Comparative genome analysis of Pseudomonas knackmussii B13, the first bacterium known to degrade chloroaromatic compounds. , 2015, Environmental microbiology.

[26]  E. Rocha,et al.  Key components of the eight classes of type IV secretion systems involved in bacterial conjugation or protein secretion , 2014, Nucleic acids research.

[27]  J. R. van der Meer,et al.  Improved Statistical Analysis of Low Abundance Phenomena in Bimodal Bacterial Populations , 2013, PloS one.

[28]  Friedrich B M Reinhard,et al.  Cell Differentiation to “Mating Bodies” Induced by an Integrating and Conjugative Element in Free-Living Bacteria , 2013, Current Biology.

[29]  J. R. van der Meer,et al.  Cellular Variability of RpoS Expression Underlies Subpopulation Activation of an Integrative and Conjugative Element , 2012, PLoS genetics.

[30]  P. Zambryski,et al.  Membrane and Core Periplasmic Agrobacterium tumefaciens Virulence Type IV Secretion System Components Localize to Multiple Sites around the Bacterial Perimeter during Lateral Attachment to Plant Cells , 2011, mBio.

[31]  V. de Lorenzo,et al.  Engineering multiple genomic deletions in Gram-negative bacteria: analysis of the multi-resistant antibiotic profile of Pseudomonas putida KT2440. , 2011, Environmental microbiology.

[32]  J. R. van der Meer,et al.  A dual functional origin of transfer in the ICEclc genomic island of Pseudomonas knackmussii B13 , 2011, Molecular microbiology.

[33]  Víctor de Lorenzo,et al.  pBAM1: an all-synthetic genetic tool for analysis and construction of complex bacterial phenotypes , 2011, BMC Microbiology.

[34]  K. Imada,et al.  Crystal Structure of Legionella DotD: Insights into the Relationship between Type IVB and Type II/III Secretion Systems , 2010, PLoS pathogens.

[35]  Matthew K. Waldor,et al.  Integrative and conjugative elements: mosaic mobile genetic elements enabling dynamic lateral gene flow , 2010, Nature Reviews Microbiology.

[36]  J. R. van der Meer,et al.  Transcriptome analysis of the mobile genome ICEclc in Pseudomonas knackmussii B13 , 2010, BMC Microbiology.

[37]  G. Rivas,et al.  The Conjugative DNA Translocase TrwB Is a Structure-specific DNA-binding Protein* , 2010, The Journal of Biological Chemistry.

[38]  A. Das,et al.  Agrobacterium tumefaciens Type IV Secretion Protein VirB3 Is an Inner Membrane Protein and Requires VirB4, VirB7, and VirB8 for Stabilization , 2010, Journal of bacteriology.

[39]  P. Zambryski,et al.  Agrobacterium type IV secretion system and its substrates form helical arrays around the circumference of virulence-induced cells , 2010, Proceedings of the National Academy of Sciences.

[40]  G. Waksman,et al.  The structural biology of type IV secretion systems , 2009, Nature Reviews Microbiology.

[41]  J. R. van der Meer,et al.  Intracellular excision and reintegration dynamics of the ICEclc genomic island of Pseudomonas knackmussii sp. strain B13 , 2009, Molecular microbiology.

[42]  D. Crook,et al.  Genomic islands: tools of bacterial horizontal gene transfer and evolution , 2008, FEMS microbiology reviews.

[43]  J. R. van der Meer,et al.  Stochasticity and bistability in horizontal transfer control of a genomic island in Pseudomonas , 2008, Proceedings of the National Academy of Sciences.

[44]  J. Claverys,et al.  RadC, a Misleading Name? , 2008, Journal of bacteriology.

[45]  D. Ferguson,et al.  Novel Type IV Secretion System Involved in Propagation of Genomic Islands , 2006, Journal of bacteriology.

[46]  A. Pühler,et al.  The clc Element of Pseudomonas sp. Strain B13, a Genomic Island with Various Catabolic Properties , 2006, Journal of bacteriology.

[47]  L. Frost,et al.  F-Like Type IV Secretion Systems Encode Proteins with Thioredoxin Folds That Are Putative DsbC Homologues , 2005, Journal of bacteriology.

[48]  E. Cascales,et al.  Biogenesis, architecture, and function of bacterial type IV secretion systems. , 2005, Annual review of microbiology.

[49]  A. Grossman,et al.  Regulation of a Bacillus subtilis mobile genetic element by intercellular signaling and the global DNA damage response. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[50]  C. Baron,et al.  Identification of the VirB4-VirB8-VirB5-VirB2 Pilus Assembly Sequence of Type IV Secretion Systems* , 2005, Journal of Biological Chemistry.

[51]  Dieter Jahn,et al.  JCat: a novel tool to adapt codon usage of a target gene to its potential expression host , 2005, Nucleic Acids Res..

[52]  J. Hinds,et al.  A novel CRP-dependent regulon controls expression of competence genes in Haemophilus influenzae. , 2005, Journal of molecular biology.

[53]  E. Cascales,et al.  Agrobacterium VirB10, an ATP energy sensor required for type IV secretion. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[54]  K Henrick,et al.  Electronic Reprint Biological Crystallography Secondary-structure Matching (ssm), a New Tool for Fast Protein Structure Alignment in Three Dimensions Biological Crystallography Secondary-structure Matching (ssm), a New Tool for Fast Protein Structure Alignment in Three Dimensions , 2022 .

[55]  E. Cascales,et al.  Agrobacterium tumefaciens VirB6 domains direct the ordered export of a DNA substrate through a type IV secretion System. , 2004, Journal of molecular biology.

[56]  Vincent Burrus,et al.  Shaping bacterial genomes with integrative and conjugative elements. , 2004, Research in microbiology.

[57]  C. Baron,et al.  VirB1 Orthologs from Brucella suis and pKM101 Complement Defects of the Lytic Transglycosylase Required for Efficient Type IV Secretion from Agrobacterium tumefaciens , 2004, Journal of bacteriology.

[58]  John W. Beaber,et al.  SOS response promotes horizontal dissemination of antibiotic resistance genes , 2004, Nature.

[59]  J. R. van der Meer,et al.  Characterization of two alternative promoters for integrase expression in the clc genomic island of Pseudomonas sp. strain B13 , 2003, Molecular microbiology.

[60]  P. Zambryski,et al.  Peptide linkage mapping of the Agrobacterium tumefaciens vir-encoded type IV secretion system reveals protein subassemblies , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[61]  Naotake Ogasawara,et al.  Whole-Genome Analysis of Genes Regulated by the Bacillus subtilis Competence Transcription Factor ComK , 2002, Journal of bacteriology.

[62]  Mark Albano,et al.  Microarray analysis of the Bacillus subtilis K‐state: genome‐wide expression changes dependent on ComK , 2002, Molecular microbiology.

[63]  A. Krogh,et al.  Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. , 2001, Journal of molecular biology.

[64]  R. Rimini,et al.  Global analysis of transcription kinetics during competence development in Streptococcus pneumoniae using high density DNA arrays , 2000, Molecular microbiology.

[65]  S. Lovett,et al.  Tandem repeat recombination induced by replication fork defects in Escherichia coli requires a novel factor, RadC. , 1999, Genetics.

[66]  Søren Brunak,et al.  A Neural Network Method for Identification of Prokaryotic and Eukaryotic Signal Peptides and Prediction of their Cleavage Sites , 1997, Int. J. Neural Syst..

[67]  E. Koonin,et al.  A family of lysozyme-like virulence factors in bacterial pathogens of plants and animals. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[68]  K. Shirasu,et al.  The product of the virB4 gene of Agrobacterium tumefaciens promotes accumulation of VirB3 protein , 1994, Journal of bacteriology.

[69]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[70]  D. E. Bradley,et al.  Characterization of the F-plasmid conjugative transfer gene traU , 1990, Journal of bacteriology.

[71]  Dm Jones.  Manual of Methods for General Bacteriology , 1981 .