scholarly journals Proteases as Secreted Exoproteins in Mycoplasmas from Ruminant Lungs and Their Impact on Surface-Exposed Proteins

2019 ◽  
Vol 85 (23) ◽  
Author(s):  
Sarah Ganter ◽  
Guylaine Miotello ◽  
Lucía Manso-Silván ◽  
Jean Armengaud ◽  
Florence Tardy ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Serine Protease ◽  
Extracellular Proteins ◽  
Mycoplasma Species ◽  
Ectodomain Shedding ◽  
Content Type ◽  
Caseinolytic Activity ◽  
Mycoplasma Mycoides

ABSTRACT Many mycoplasma species are isolated from the ruminant lungs as either saprophytes or true pathogens. These wall-less bacteria possess a minimal genome and reduced metabolic capabilities. Accordingly, they rely heavily on their hosts for the supply of essential metabolites and, notably, peptides. Seven of 13 ruminant lung-associated Mycoplasma (sub)species were shown to possess caseinolytic activity when grown in rich media and assessed with a quantitative fluorescence test. For some species, this activity was detected in spent medium, an indication that proteases were secreted outside the mycoplasma cells. To identify these proteases, we incubated concentrated washed cell pellets in a defined medium and analyzed the supernatants by tandem mass spectrometry. Secreted-protease activity was detected mostly in the species belonging to the Mycoplasma mycoides cluster (MMC) and, to a lesser extent, in Mycoplasma bovirhinis. Analyzing a Mycoplasma mycoides subsp. capri strain, chosen as a model, we identified 35 expressed proteases among 55 predicted coding genes, of which 5 were preferentially found in the supernatant. Serine protease S41, acquired by horizontal gene transfer, was responsible for the caseinolytic activity, as demonstrated by zymography and mutant analysis. In an M. capricolum mutant, inactivation of the S41 protease resulted in marked modification of the expression or secretion of 17 predicted surface-exposed proteins. This is an indication that the S41 protease could have a role in posttranslational cleavage of surface-exposed proteins and ectodomain shedding, whose physiological impacts still need to be explored. IMPORTANCE Few studies pertaining to proteases in ruminant mycoplasmas have been reported. Here, we focus on proteases that are secreted outside the mycoplasma cell using a mass spectrometry approach. The most striking result is the identification, within the Mycoplasma mycoides cluster, of a serine protease that is exclusively detected outside the mycoplasma cells and is responsible for casein digestion. This protease may also be involved in the posttranslational processing of surface proteins, as suggested by analysis of mutants showing a marked reduction in the secretion of extracellular proteins. By analogy, this finding may help increase understanding of the mechanisms underlying this ectodomain shedding in other mycoplasma species. The gene encoding this protease is likely to have been acquired via horizontal gene transfer from Gram-positive bacteria and sortase-associated surface proteases. Whether this protease and the associated ectodomain shedding are related to virulence has yet to be ascertained.

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 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 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.

scholarly journals Dissemination of the blaNDM-5 Gene via IncX3-Type Plasmid among Enterobacteriaceae in Children

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Dongxing Tian ◽  
Bingjie Wang ◽  
Hong Zhang ◽  
Fen Pan ◽  
Chun Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Drug Resistance ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Klebsiella Pneumoniae ◽  
Pediatric Patients ◽  
Transmission Mechanism ◽  
Klebsiella Aerogenes ◽  
Content Type ◽  
E Coli

ABSTRACT The continuous emergence of novel New Delhi metallo-β-lactamase-5 (NDM-5)-producing Enterobacteriaceae isolates is receiving more and more public attention. Twenty-two NDM-5-producing strains were identified from 146 carbapenemase-producing Enterobacteriaceae (CRE) strains isolated from pediatric patients between January and March 2017, indicating that the blaNDM-5 gene has spread to children. All 22 isolates, including 16 Klebsiella pneumoniae strains, four Klebsiella aerogenes strains, and two Escherichia coli strains, showed significantly high resistance to β-lactam antibiotics (except aztreonam) but remained susceptible to tigecycline and colistin. K. pneumoniae and K. aerogenes strains were respectively defined as homologous clonal isolates by pulsed-field gel electrophoresis (PFGE). Multilocus sequence typing (MLST) results confirmed the genetic relatedness with all K. pneumoniae strains belonging to sequence type (ST) 48. Two E. coli isolates (ST617 and ST1236) were considered genetically unrelated. Twenty-two blaNDM-5 plasmids were positive for the IncX3 amplicon and showed almost identical profiles after digestion with HindIII and EcoRI. Four representative strains (K. pneumoniae K725, K. aerogenes CR33, E. coli Z214, and E. coli Z244) were selected for further study. Plasmids harboring blaNDM-5 showed strong stability in both clinical isolates and transconjugants, without apparent plasmid loss after 100 serial generations. S1-PFGE followed by Southern blot analysis demonstrated that the blaNDM-5 gene was located on an ∼46-kb plasmid. Plasmid sequences of pNDM-K725, pNDM-CR33, and pNDM-Z214 were almost identical but were slightly different from that of pNDM-Z244. Compared with pNDM-Z244, ΔISAba125 and partial copies of IS3000 were missing. The genetic backgrounds of the blaNDM-5 gene in four strains were slightly different from that of the typical pNDM_MGR194. This study comprehensively characterized the horizontal gene transfer of the blaNDM-5 gene among different Enterobacteriaceae isolates in pediatric patients, and the IncX3-type plasmid was responsible for the spread. IMPORTANCE The emergence of CRE strains resistant to multiple antibiotics is considered a substantial threat to human health. Therefore, all the efforts to provide a detailed molecular transmission mechanism of specific drug resistance can contribute positively to prevent the further spread of multidrug-resistant bacteria. Although the new superbug harboring blaNDM-5 has been reported in many countries, it was mostly identified among E. coli strains, and the gene transfer mechanism has not been fully recognized and studied. In this work, we identified 22 blaNDM-5-positive strains in different species of Enterobacteriaceae, including 16 Klebsiella pneumoniae strains, four Klebsiella aerogenes strains, and two Escherichia coli strains, which indicated the horizontal gene transfer of blaNDM-5 among Enterobacteriaceae strains in pediatric patients. Moreover, blaNDM-5 was located on a 46-kb IncX3 plasmid, which is possibly responsible for this widespread horizontal gene transfer. The different genetic contexts of the blaNDM-5 gene indicated some minor evolutions of the plasmid, based on the complete sequences of the blaNDM-5 plasmids. These findings are of great significance to understand the transmission mechanism of drug resistance genes, develop anti-infection treatment, and take effective infection control measures.

scholarly journals Homologues of Genetic Transformation DNA Import Genes Are Required for Rhodobacter capsulatus Gene Transfer Agent Recipient Capability Regulated by the Response Regulator CtrA

2015 ◽  
Vol 197 (16) ◽  
pp. 2653-2663 ◽  
Author(s):  
Cedric A. Brimacombe ◽  
Hao Ding ◽  
Jeanette A. Johnson ◽  
J. Thomas Beatty
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Rhodobacter Capsulatus ◽  
Natural Transformation ◽  
Response Regulator ◽  
Content Type ◽  
Gene Acquisition ◽  
Metabolic Properties ◽  
Cell Genome ◽  
Key Aspects

ABSTRACTGene transfer agents (GTAs) morphologically resemble small, double-stranded DNA (dsDNA) bacteriophages; however, their only known role is to package and transfer random pieces of the producing cell genome to recipient cells. The best understood GTA is that ofRhodobacter capsulatus, termed RcGTA. We discovered that homologues of three genes involved in natural transformation in other bacteria,comEC,comF, andcomM, are essential for RcGTA-mediated gene acquisition. This paper gives genetic and biochemical evidence that RcGTA-borne DNA entry into cells requires the ComEC and ComF putative DNA transport proteins and genetic evidence that putative cytoplasmic ComM protein of unknown function is required for recipient capability. Furthermore, the master regulator of RcGTA production in <1% of a cell population, CtrA, which is also required for gene acquisition in recipient cells, is expressed in the vast majority of the population. Our results indicate that RcGTA-mediated gene transfer combines key aspects of two bacterial horizontal gene transfer mechanisms, where donor DNA is packaged in transducing phage-like particles and recipient cells take up DNA using natural transformation-related machinery. Both of these differentiated subsets of a culture population, donors and recipients, are dependent on the same response regulator, CtrA.IMPORTANCEHorizontal gene transfer (HGT) is a major driver of bacterial evolution and adaptation to environmental stresses. Traits such as antibiotic resistance or metabolic properties can be transferred between bacteria via HGT; thus, HGT can have a tremendous effect on the fitness of a bacterial population. The three classically described HGT mechanisms are conjugation, transformation, and phage-mediated transduction. More recently, the HGT factor GTA was described, where random pieces of producing cell genome are packaged into phage-like particles that deliver DNA to recipient cells. In this report, we show that transport of DNA borne by theR. capsulatusRcGTA into recipient cells requires key genes previously thought to be specific to natural transformation pathways. These findings indicate that RcGTA combines central aspects of phage-mediated transduction and natural transformation in an efficient, regulated mode of HGT.

scholarly journals Transposon Insertion Sequencing in a Clinical Isolate of Legionella pneumophila Identifies Essential Genes and Determinants of Natural Transformation

2020 ◽  
Vol 203 (3) ◽  
Author(s):  
Léo Hardy ◽  
Pierre-Alexandre Juan ◽  
Bénédicte Coupat-Goutaland ◽  
Xavier Charpentier
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Legionella Pneumophila ◽  
Genetic Basis ◽  
Natural Transformation ◽  
Essential Genes ◽  
Content Type ◽  
Transposon Insertion ◽  
Life Traits ◽  
Transposon Insertion Sequencing

ABSTRACT Legionella pneumophila is a Gram-negative bacterium ubiquitous in freshwater environments which, if inhaled, can cause a severe pneumonia in humans. The emergence of L. pneumophila is linked to several traits selected in the environment, the acquisition of some of which involved intra- and interkingdom horizontal gene transfer events. Transposon insertion sequencing (TIS) is a powerful method to identify the genetic basis of selectable traits as well as to identify fitness determinants and essential genes, which are possible antibiotic targets. TIS has not yet been used to its full power in L. pneumophila, possibly because of the difficulty of obtaining a high-saturation transposon insertion library. Indeed, we found that isolates of sequence type 1 (ST1), which includes the commonly used laboratory strains, are poorly permissive to saturating mutagenesis by conjugation-mediated transposon delivery. In contrast, we obtained high-saturation libraries in non-ST1 clinical isolates, offering the prospect of using TIS on unaltered L. pneumophila strains. Focusing on one of them, we then used TIS to identify essential genes in L. pneumophila. We also revealed that TIS could be used to identify genes controlling vertical transmission of mobile genetic elements. We then applied TIS to identify all the genes required for L. pneumophila to develop competence and undergo natural transformation, defining the set of major and minor type IV pilins that are engaged in DNA uptake. This work paves the way for the functional exploration of the L. pneumophila genome by TIS and the identification of the genetic basis of other life traits of this species. IMPORTANCE Legionella pneumophila is the etiologic agent of a severe form of nosocomial and community-acquired pneumonia in humans. The environmental life traits of L. pneumophila are essential to its ability to accidentally infect humans. A comprehensive identification of their genetic basis could be obtained through the use of transposon insertion sequencing. However, this powerful approach had not been fully implemented in L. pneumophila. Here, we describe the successful implementation of the transposon-sequencing approach in a clinical isolate of L. pneumophila. We identify essential genes, potential drug targets, and genes required for horizontal gene transfer by natural transformation. This work represents an important step toward identifying the genetic basis of the many life traits of this environmental and pathogenic species.

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