In Vivo Himar1 Transposon Mutagenesis of Burkholderia pseudomallei

Burkholderia psedudomallei is the etiologic agent of melioidosis, and the bacterium is listed as a potential agent of bioterrorism because of its low infectious dose, multiple infectious routes, and intrinsic antibiotic resistance. To further accelerate research with this understudied bacterium, we developed a Himar1-based random mutagenesis system for B. pseudomallei (HimarBP). The transposons contain a Flp recombinase-excisable, approved kanamycin resistance selection marker and an R6K origin of replication for transposon rescue. In vivo mutagenesis of virulent B. pseudomallei strain 1026b was highly efficient, with up to 44% of cells transformed with the delivery plasmid harboring chromosomal HimarBP insertions. Southern analyses revealed single insertions with no evidence of delivery plasmid maintenance. Sequence analysis of rescued HimarBP insertions revealed random insertions on both chromosomes within open reading frames and intergenic regions and that the orientation of insertions was largely unbiased. Auxotrophic mutants were obtained at a frequency of 0.72%, and nutritional supplementation experiments supported the functional assignment of genes within the respective biosynthetic pathways. HimarBP insertions were stable in the absence of selection and could be readily transferred between naturally transformable strains. Experiments with B. thailandensis suggest that the newly developed HimarBP transposons can also be used for random mutagenesis of other Burkholderia spp., especially the closely related species B. mallei. Our results demonstrate that comprehensive transposon libraries of B. pseudomallei can be generated, providing additional tools for the study of the biology, pathogenesis, and antibiotic resistance of this pathogen.     

In vivo Himar1-based transposon mutagenesis of Francisella tularensis

Francisella tularensis is the intracellular pathogen that causes human tularemia. It is recognized as a potential agent of bioterrorism due to its low infectious dose and multiple routes of entry. We report the development of a Himar1-based random mutagenesis system for F. tularensis (HimarFT). In vivo mutagenesis of F. tularensis live vaccine strain (LVS) with HimarFT occurs at high efficiency. Approximately 12 to 15% of cells transformed with the delivery plasmid result in transposon insertion into the genome. Results from Southern blot analysis of 33 random isolates suggest that single insertions occurred, accompanied by the loss of the plasmid vehicle in most cases. Nucleotide sequence analysis of rescued genomic DNA with HimarFT indicates that the orientation of integration was unbiased and that insertions occurred in open reading frames and intergenic and repetitive regions of the chromosome. To determine the utility of the system, transposon mutagenesis was performed, followed by a screen for growth on Chamberlain's chemically defined medium (CDM) to isolate auxotrophic mutants. Several mutants were isolated that grew on complex but not on the CDM. We genetically complemented two of the mutants for growth on CDM with a newly constructed plasmid containing a nourseothricin resistance marker. In addition, uracil or aromatic amino acid supplementation of CDM supported growth of isolates with insertions in pyrD, carA, or aroE1 supporting the functional assignment of genes within each biosynthetic pathway. A mutant containing an insertion in aroE1 demonstrated delayed replication in macrophages and was restored to the parental growth phenotype when provided with the appropriate plasmid in trans. Our results suggest that a comprehensive library of mutants can be generated in F. tularensis LVS, providing an additional genetic tool to identify virulence determinants required for survival within the host.     

A mariner-Based Transposon System for In Vivo Random Mutagenesis of Clostridium difficile

Understanding the molecular basis of Clostridium difficile infection is a prerequisite to the development of effective countermeasures. Although there are methods for constructing gene-specific mutants of C. difficile, currently there is no effective method for generating libraries of random mutants. In this study, we developed a novel mariner-based transposon system for in vivo random mutagenesis of C. difficile {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291, the BI/NAP1/027 epidemic strain at the center of the C. difficile outbreaks in Stoke Mandeville, United Kingdom, in 2003 to 2004 and 2004 to 2005. Transposition occurred at a frequency of 4.5 (±0.4) × 10⁻⁴ per cell to give stable insertions at random genomic loci, which were defined only by the nucleotide sequence TA. Furthermore, mutants with just a single transposon insertion were generated in an overwhelming majority (98.3% in this study). Phenotypic screening of a C. difficile {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 random mutant library yielded a sporulation/germination-defective clone with an insertion in the germination-specific protease gene cspBA and an auxotroph with an insertion in the pyrimidine biosynthesis gene pyrB. These results validate our mariner-based transposon system for use in forward genetic studies of C. difficile.Clostridium difficile infection is widely recognized as the leading cause of health care-associated diarrhea in North America and Europe. Infection usually follows antibiotic treatment, which disrupts the native gastrointestinal microflora and thus allows C. difficile to proliferate. The emergence of so-called “epidemic” or “hypervirulent” strains of C. difficile over the last 5 to 10 years has compounded an already serious problem. Classed as BI/NAP1/027, these epidemic strains are believed to cause a more severe disease and lead to increased mortality and relapse rates (11, 20, 24).Understanding the genetic and molecular basis of C. difficile infection will be a crucial step in the development of effective countermeasures. Methods for directed gene inactivation in C. difficile have recently been described (7, 21). This has opened the way for reverse genetic studies, in which the exact role of a specific gene, hypothesized to be important in a given phenotype, can be elucidated experimentally. By way of contrast, forward genetic studies aim to identify the genetic basis of a particular phenotype without making any assumptions about the genes involved. In forward genetic studies, transposons are often used to generate libraries of random insertion mutants. Libraries are then screened to identify mutants that are defective in a particular phenotype. Identification of the gene or genes which have been inactivated by transposon insertion then implicates them as having a role in that particular phenotype. Recently, just such an approach was used to identify a novel toxin-regulatory locus in Clostridium perfringens (29). This study elegantly demonstrated the power of forward genetic studies in bacterial pathogens.A number of transposon mutagenesis systems have been described for Gram-positive bacteria (2, 3, 15, 16, 29, 32). Two different systems have recently been developed for use in C. perfringens (15, 29). Both are in vitro mutagenesis systems which rely on being able to transform the recipient organism. As such, they are not suitable for use in C. difficile because in the laboratory at present, recombinant DNA can be transferred into C. difficile only via conjugation. The conjugative transposons Tn916 and Tn5397 have been studied in C. difficile, but both have been found either to have a strong target site preference or to yield multiple insertions in individual clones (9, 30). Therefore, neither is well suited to generating libraries of random C. difficile mutants.We reasoned that a mariner-based transposon mutagenesis system would be an effective tool for generating libraries of random C. difficile mutants. The mariner-transposable element Himar1 has been shown to insert randomly into the genomes of many bacterial species (3, 6, 16, 17, 32). The cognate Himar1 transposase is the only factor required for transposition, which occurs via a cut-and-paste mechanism (13, 14). The transposon itself is defined by inverted terminal repeats (ITRs) at either end and inserts into a TA target site. This is highly appropriate for an organism with a low-GC content such as C. difficile. In this study, we have developed a novel mariner-based transposon system for in vivo random mutagenesis of C. difficile. Moreover, we have demonstrated the system in C. difficile {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291, the BI/NAP1/027 epidemic strain at the center of the C. difficile outbreaks in Stoke Mandeville, United Kingdom, in 2003 to 2004 and 2004 to 2005. This new genetic tool opens the way for forward genetic studies of C. difficile.     

In Vivo Random Mutagenesis of Bacillus subtilis by Use of TnYLB-1, a mariner-Based Transposon

This report describes the construction and characterization of a mariner-based transposon system designed to be used in Bacillus subtilis, but potentially applicable to other gram-positive bacteria. Two pUC19-derived plasmids were created that contain the mariner-Himar1 transposase gene, modified for expression in B. subtilis, under the control of either σ^A- or σ^B-dependent promoters. Both plasmids also contain a transposable element (TnYLB-1) consisting of a Kan^r cassette bracketed by the Himar1-recognized inverse terminal repeats, as well as the temperature-sensitive replicon and Erm^r gene of pE194ts. TnYLB-1 transposes into the B. subtilis chromosome with high frequency (10⁻²) from either plasmid. Southern hybridization analyses of 15 transposants and sequence analyses of the insertion sites of 10 of these are consistent with random transposition, requiring only a “TA” dinucleotide as the essential target in the recipient DNA. Two hundred transposants screened for sporulation proficiency and auxotrophy yielded five Spo⁻ clones, three with insertions in known sporulation genes (kinA, spoVT, and yqfD) and two in genes (ybaN and yubB) with unknown functions. Two auxotrophic mutants were identified among the 200 transposants, one with an insertion in lysA and another in a gene (yjzB) whose function is unknown.     

Establishment of Tn5096-Based Transposon Mutagenesis in Gordonia polyisoprenivorans

The transposons Tn5, Tn10, Tn611, and Tn5096 were characterized regarding transposition in Gordonia polyisoprenivorans strain VH2. No insertional mutants were obtained employing Tn5 or Tn10. The thermosensitive plasmid pCG79 harboring Tn611 integrated into the chromosome of G. polyisoprenivorans; however, the insertional mutants were fairly unstable und reverted frequently to the wild-type phenotype. In contrast, various stable mutants were obtained employing Tn5096-mediated transposon mutagenesis. Auxotrophic mutants, mutants defective or deregulated in carotenoid biosynthesis, and mutants defective in utilization of rubber and/or highly branched isoprenoid hydrocarbons were obtained by integration of plasmid pMA5096 harboring Tn5096 as a whole into the genome. From about 25,000 isolated mutants, the insertion loci of pMA5096 were subsequently mapped in 20 independent mutants in genes which could be related to the above-mentioned metabolic pathways or to putative regulation proteins. Analyses of the genotypes of pMA5096-mediated mutants defective in biodegradation of poly(cis-1,4-isoprene) did not reveal homologues to recently identified genes coding for enzymes catalyzing the initial cleavage of poly(cis-1,4-isoprene). One rubber-negative mutant was disrupted in mcr, encoding an α-methylacyl-coenzyme A racemase. This mutant was defective in degradation of poly(cis-1,4-isoprene) and also of highly branched isoprenoid hydrocarbons.     

Discordant Results Obtained with Francisella tularensis during In Vitro and In Vivo Immunological Studies Are Attributable to Compromised Bacterial Structural Integrity

Francisella tularensis (Ft) is a highly infectious intracellular pathogen and the causative agent of tularemia. Because Ft can be dispersed via small droplet-aerosols and has a very low infectious dose it is characterized as a category A Select Agent of biological warfare. Respiratory infection with the attenuated Live Vaccine Strain (LVS) and the highly virulent SchuS4 strain of Ft engenders intense peribronchiolar and perivascular inflammation, but fails to elicit select pro-inflammatory mediators (e.g., TNF, IL-1β, IL-6, IL-12, and IFN-γ) within the first ∼72 h. This in vivo finding is discordant with the principally T_H1-oriented response to Ft frequently observed in cell-based studies wherein the aforementioned cytokines are produced. An often overlooked confounding factor in the interpretation of experimental results is the influence of environmental cues on the bacterium''s capacity to elicit certain host responses. Herein, we reveal that adaptation of Ft to its mammalian host imparts an inability to elicit select pro-inflammatory mediators throughout the course of infection. Furthermore, in vitro findings that non-host adapted Ft elicits such a response from host cells reflect aberrant recognition of the DNA of structurally-compromised bacteria by AIM2-dependent and -independent host cell cytosolic DNA sensors. Growth of Ft in Muller-Hinton Broth or on Muller-Hinton-based chocolate agar plates or genetic mutation of Ft was found to compromise the structural integrity of the bacterium thus rendering it capable of aberrantly eliciting pro-inflammatory mediators (e.g., TNF, IL-1β, IL-6, IL-12, and IFN-γ). Our studies highlight the profound impact of different growth conditions on host cell response to infection and demonstrate that not all in vitro-derived findings may be relevant to tularemia pathogenesis in the mammalian host. Rational development of a vaccine and immunotherapeutics can only proceed from a foundation of knowledge based upon in vitro findings that recapitulate those observed during natural infection.     

Population structure of Francisella tularensis

We have sequenced fragments of five metabolic housekeeping genes and two genes encoding outer membrane proteins from 81 isolates of Francisella tularensis, representing all four subspecies. Phylogenetic clustering of gene sequences from F. tularensis subsp. tularensis and F. tularensis subsp. holarctica aligned well with subspecies affiliations. In contrast, F. tularensis subsp. novicida and F. tularensis subsp. mediasiatica were indicated to be phylogenetically incoherent taxa. Incongruent gene trees and mosaic structures of housekeeping genes provided evidence for genetic recombination in F. tularensis.     

Persistence factors of Francisella tularensis

The study of the persistence potential of 64 F. tularensis strains isolated from different sources was carried out. The wide spread of the antilysozyme, antilactoferrin and anticomplementory activities of F. tularensis were detected. F. tularensis, isolated from ticks and water, were characterized by the highest level of the expression of antilysozyme activity, while anticomplementory and antilactoferrin activities of the infective agents were characteristic of those microorganisms which were isolated from rodents and their excrements.     

Noncultivatable forms of Francisella tularensis

Conditions for the appearance of F. tularensis uncultivated forms and for their reversion into the initial state have been studied. As revealed in this study, the combined influence of stress factors (starvation and low temperature) may result in the transition of F. tularensis into the uncultivated state in which it persists in the environment during the period between epidemics. The reversion of F. tularensis uncultivated forms into the initial state has been carried out with the use of sensitive animals. The uncultivated state of F. tularensis should be regarded as the actual form of the existence of the causative agent of tularemia in soil and water ecosystems.     

Chlorine disinfection of Francisella tularensis

Aims: To determine the range of free available chlorine (FAC) required for disinfection of the live vaccine strain (LVS) and wild‐type strains of Francisella tularensis. Methods and Results: Seven strains of planktonic F. tularensis were exposed to 0·5 mg·l^?1 FAC for two pH values, 7 and 8, at 5 and 25°C. LVS was inactivated 2 to 4 times more quickly than any of the wild‐type F. tularensis strains at pH 8 and 5°C. Conclusions: Free available chlorine residual concentrations routinely maintained in drinking water distribution systems would require up to two hours to reduce all F. tularensis strains by 4 log₁₀. LVS was inactivated most quickly of the tested strains. Significance and Impact of the Study: This work provides contact time (CT) values that are useful for drinking water risk assessment and also suggests that LVS may not be a good surrogate in disinfection studies.     

Mutagenesis of Neisseria meningitidis by in vitro transposition of Himar1 mariner

Now that the meningococcal genome sequence has been completed, the lack of a suitable method for saturation mutagenesis remains a major obstacle to the unraveling of the pathogenic propensity of Neisseria meningitidis. Here, we demonstrate that in vitro Himar1 mariner transposition on chromosomal or PCR-amplified meningococcal DNA, which is subsequently reintroduced into N. meningitidis by natural transformation, is an extremely efficient mutagenesis method. Southern blot analysis, sequencing the Himar1 insertion point in numerous transposition mutants, and a limited screening of the mutant libraries for clones impaired in maltose catabolism confirmed that Himar1 transposed randomly in N. meningitidis. Taken together, these data demonstrate that Himar1 in vitro transposition can lead to the exhaustive mutagenesis of N. meningitidis, allowing for the first time a genomic-scale mutational analysis of this important human pathogen.     

Glycosylation of DsbA in Francisella tularensis subsp. tularensis

In Francisella tularensis subsp. tularensis, DsbA has been shown to be an essential virulence factor and has been observed to migrate to multiple protein spots on two-dimensional electrophoresis gels. In this work, we show that the protein is modified with a 1,156-Da glycan moiety in O-linkage. The results of mass spectrometry studies suggest that the glycan is a hexasaccharide, comprised of N-acetylhexosamines, hexoses, and an unknown monosaccharide. Disruption of two genes within the FTT0789-FTT0800 putative polysaccharide locus, including a galE homologue (FTT0791) and a putative glycosyltransferase (FTT0798), resulted in loss of glycan modification of DsbA. The F. tularensis subsp. tularensis ΔFTT0798 and ΔFTT0791::Cm mutants remained virulent in the murine model of subcutaneous tularemia. This indicates that glycosylation of DsbA does not play a major role in virulence under these conditions. This is the first report of the detailed characterization of the DsbA glycan and putative role of the FTT0789-FTT0800 gene cluster in glycan biosynthesis.     

Evolution of subspecies of Francisella tularensis

Analysis of unidirectional genomic deletion events and single nucleotide variations suggested that the four subspecies of Francisella tularensis have evolved by vertical descent. The analysis indicated an evolutionary scenario where the highly virulent F. tularensis subsp. tularensis (type A) appeared before the less virulent F. tularensis subsp. holarctica (type B). Compared to their virulent progenitors, attenuated strains of F. tularensis exhibited specific unidirectional gene losses.     

Natural penicillin resistance of Francisella tularensis

Altered viable forms of F. tularensis with spheroplast specific damages of the surface structures were isolated after the culture exposure to lithium chloride (0.5 and 1%). Study of natural penicillin resistance in the spheroplasts and bacterial forms of F. tularensis revealed their difference: the spheroplasts of the strains tested had a lower resistance to beta-lactam antibiotics than the bacterial forms while the activity of spheroplast beta-lactamase did not differ from that of the enzyme of the bacterial form and equalled 224 to 252 U/ml of the cell suspension. Therefore, on the model of the lithium-induced spheroplasts it appeared possible to show that the damages of the surface structures of the cell walls of F. tularensis changed the penicillin resistance level which was indicative of involvement of the F. tularensis cell walls in the phenomenon of the natural resistance to beta-lactams.     

Towards proteome database of Francisella tularensis

The accessibility of the partial genome sequence of Francisella tularensis strain Schu 4 was the starting point for a comprehensive proteome analysis of the intracellular pathogen F. tularensis. The main goal of this study is identification of protein candidates of value for the development of diagnostics, therapeutics and vaccines. In this review, the current status of 2-DE F. tularensis database building, approaches used for identification of biologically important subsets of F. tularensis proteins, and functional and topological assignments of identified proteins using various prediction programs and database homology searches are presented.     

Multilocus VNTR-typing of Francisella tularensis strains

In the analysis of F. tularensis genome with the use of the specially developed program "DNA" a great number of loci containing tandem repeats were found. For analysis, 3 of them were selected and designated as FtA, FtB, FtC. The study of DNA of 40 F. tularensis strains in the polymerase chain reaction with specific primers to these loci a great variability in the number of repeats was established, the presence of 17 alleles being found in locus FtA, 5 alleles in locus FtB and 5 alleles in locus FtC. The strains under study formed 24 variants of genotypes, whose occurrence varied from 0.025 to 0.125. Taking into account the variability of the detected loci and a great number of potential loci VNTR in the genome, further development of this method will facilitate the creation of local and general data bases of the strains, thus ensuring more effective genetic typing of F. tularensis.     

Properties of Aldolase from Francisella tularensis

The aldolase of Francisella tularensis resembles Class II aldolases in its requirement for divalent ions and its inactivation by metal chelating agents. Cysteine and other reducing agents stimulated the activity of the enzyme.