Ehrlichia Isolate from a Minnesota Tick: Characterization and Genetic Transformation.

Ehrlichia muris subsp. eauclairensis is recognized as the etiological agent of human ehrlichiosis in Minnesota and Wisconsin. We describe the culture isolation of this organism from a field-collected tick and detail its relationship to other species of Ehrlichia The isolate could be grown in a variety of cultured cell lines and was effectively transmitted between Ixodes scapularis ticks and rodents, with PCR and microscopy demonstrating a broad pattern of dissemination in arthropod and mammalian tissues. Conversely, Amblyomma americanum ticks were not susceptible to infection by the Ehrlichia Histologic sections further revealed that the wild-type isolate was highly virulent for mice and hamsters, causing severe systemic disease that was frequently lethal. A Himar1 transposase system was used to create mCherry- and mKate-expressing EmCRT mutants, which retained the ability to infect rodents and ticks.IMPORTANCE Ehrlichioses are zoonotic diseases caused by intracellular bacteria that are transmitted by ixodid ticks. Here we report the culture isolation of bacteria which are closely related to, or the same as the Ehrlichia muris subsp. eauclairensis, a recently recognized human pathogen. EmCRT, obtained from a tick removed from deer at Camp Ripley, MN, is the second isolate of this subspecies described and is distinctive in that it was cultured directly from a field-collected tick. The isolate's cellular tropism, pathogenic changes caused in rodent tissues, and tick transmission to and from rodents are detailed in this study. We also describe the genetic mutants created from the EmCRT isolate, which are valuable tools for the further study of this intracellular pathogen.

Identification and Analysis of Essential Genes in Streptococcus mutans with Transposon Sequencing.

Transposon sequencing (Tn-seq) has greatly accelerated the rate at which gene function can be profiled in microbial organisms. This technique has been applied to the study of the dental caries pathogen Streptococcus mutans where it has been used to generate large transposon mutant libraries. Coupled with high-throughput sequencing and bioinformatics tools, culture of these transposon mutant libraries has facilitated the identification of essential and conditional essential genes. In this chapter, we describe a procedure for performing Tn-seq studies in S. mutans that covers pooled transposon mutant construction, in vitro culture, and DNA library sequencing and data analysis.

Progress towards an inducible, replication-proficient transposon delivery vector for Chlamydia trachomatis.

Background Genetic systems have been developed for Chlamydia but the extremely low transformation frequency remains a significant bottleneck.  Our goal is to develop a self-replicating transposon delivery vector for C. trachomatis which can be expanded prior to transposase induction. Methods We made E. coli/ C. trachomatis shuttle vectors bearing the Himar1 C9  transposase under control of the tet promoter and a novel rearrangement of the Himar1 transposon with the ß-lactamase gene.  Activity of the transposase was monitored by immunoblot and by DNA sequencing. Results We constructed pSW2-mCh-C9, a C. trachomatis plasmid designed to act as a self-replicating vector carrying both the Himar1 C9  transposase under tet promoter control and its transposon.  However, we were unable to recover this plasmid in C. trachomatis following multiple attempts at transformation. Therefore, we assembled two new deletion plasmids pSW2-mCh-C9-ΔTpon carrying only the Himar1 C9  transposase (under tet promoter control) and a sister vector (same sequence backbone) pSW2-mCh-C9-ΔTpase carrying its cognate transposon.  We demonstrated that the biological components that make up both pSW2-mCh-C9-ΔTpon and pSW2-mCh-C9-ΔTpase are active in E. coli.  Both these plasmids could be independently recovered in C. trachomatis. We attempted to perform lateral gene transfer by transformation and mixed infection with C. trachomatis strains bearing pSW2-mCh-C9-ΔTpon and pSW2-RSGFP-Tpon (a green fluorescent version of pSW2-mCh-C9-ΔTpase).  Despite success in achieving mixed infections, it was not possible to recover progeny bearing both versions of these plasmids. Conclusions We have designed a self-replicating plasmid vector pSW2-mCh-C9 for C. trachomatis carrying the Himar1 C9  transposase under tet promoter control.  Whilst this can be transformed into E. coli it cannot be recovered in C. trachomatis.  Based on selected deletions and phenotypic analyses we conclude that low level expression from the tet inducible promoter is responsible for premature transposition and hence plasmid loss early on in the transformation process.

Modeling Site-Specific Nucleotide Biases Affecting Himar1 Transposon Insertion Frequencies in TnSeq Data Sets.

TnSeq is a widely used methodology for determining gene essentiality, conditional fitness, and genetic interactions in bacteria. The Himar1 transposon is restricted to insertions at TA dinucleotides, but otherwise, few site-specific biases have been identified. As a result, most analytical approaches assume that insertions are expected to be randomly distributed among TA sites in nonessential regions. However, through analysis of Himar1 transposon libraries in Mycobacterium tuberculosis, we demonstrate that there are site-specific biases that affect the frequency of insertion of the Himar1 transposon at different TA sites. We use machine learning and statistical models to characterize patterns in the nucleotides surrounding TA sites that correlate with high or low insertion counts. We then develop a quantitative model based on these patterns that can be used to predict the expected counts at each TA site based on nucleotide context, which can explain up to half of the variance in insertion counts. We show that these insertion preferences exist in Himar1 TnSeq data sets from other mycobacterial and nonmycobacterial species. We present an improved method for identification of essential genes, called TTN-Fitness, that can better distinguish true biological fitness effects by comparing observed counts to expected counts based on our site-specific model of insertion preferences. Compared to previous essentiality methods, TTN-Fitness can make finer distinctions among genes whose disruption causes a fitness defect (or advantage), separating them out from the large pool of nonessentials, and is able to classify many smaller genes (with few TA sites) that were previously characterized as uncertain. IMPORTANCE When using the Himar1 transposon to create transposon insertion mutant libraries, it is known that the transposon is restricted to insertions at TA dinucleotide sites throughout the genome, and the absence of insertions is used to infer which genes are essential (or conditionally essential) in a bacterial organism. It is widely assumed that insertions in nonessential regions are otherwise random, and this assumption is used as the basis of several methods for statistical analysis of TnSeq data. In this paper, we show that the nucleotide sequence surrounding TA sites influences the magnitude of insertions, and these Himar1 insertion preferences (sequence biases) can partially explain why some sites have higher counts than others. We use this predictive model to make improved estimates of the fitness effects of genes, which help make finer distinctions of the phenotype and biological consequences of disruption of nonessential genes.

Genome-Wide Essentiality Analysis of Mycobacterium abscessus by Saturated Transposon Mutagenesis and Deep Sequencing.

Mycobacterium abscessus is an emerging opportunistic human pathogen that naturally resists most major classes of antibiotics, making infections difficult to treat. Thus far, little is known about M. abscessus physiology, pathogenesis, and drug resistance. Genome-wide analyses have comprehensively catalogued genes with essential functions in Mycobacterium tuberculosis and Mycobacterium avium subsp. hominissuis (here, M. avium) but not in M. abscessus. By optimizing transduction conditions, we achieved full saturation of TA insertion sites with Himar1 transposon mutagenesis in the M. abscessus ATCC 19977T genome, as confirmed by deep sequencing prior to essentiality analyses of annotated genes and other genomic features. The overall densities of inserted TA sites (85.7%), unoccupied TA sites (14.3%), and nonpermissive TA sites (8.1%) were similar to results in M. tuberculosis and M. avium. Of the 4,920 annotated genes, 326 were identified as essential, 269 (83%) of which have mutual homology with essential M. tuberculosis genes, while 39 (12%) are homologous to genes that are not essential in M. tuberculosis and M. avium, and 11 (3.4%) only have homologs in M. avium. Interestingly, 7 (2.1%) essential M. abscessus genes have no homologs in either M. tuberculosis or M. avium, two of which were found in phage-like elements. Most essential genes are involved in DNA replication, RNA transcription and translation, and posttranslational events to synthesize important macromolecules. Some essential genes may be involved in M. abscessus pathogenesis and antibiotics response, including certain essential tRNAs and new short open reading frames. Our findings will help to pave the way for better understanding of M. abscessus and benefit development of novel bactericidal drugs against M. abscessus. IMPORTANCE Limited knowledge regarding Mycobacterium abscessus pathogenesis and intrinsic resistance to most classes of antibiotics is a major obstacle to developing more effective strategies to prevent and mitigate disease. Using optimized procedures for Himar1 transposon mutagenesis and deep sequencing, we performed a comprehensive analysis to identify M. abscessus genetic elements essential for in vitro growth and compare them to similar data sets for M. tuberculosis and M. avium subsp. hominissuis. Most essential M. abscessus genes have mutual homology with essential M. tuberculosis genes, providing a foundation for leveraging available knowledge from M. tuberculosis to develop more effective drugs and other interventions against M. abscessus. A small number of essential genes unique to M. abscessus deserve further attention to gain insights into what makes M. abscessus different from other mycobacteria. The essential genes and other genomic features such as short open reading frames and noncoding RNA identified here will provide useful information for future study of M. abscessus pathogenicity and new drug development.

Clinical and immunological responses in sheep after inoculation with Himar1-transformed Anaplasma phagocytophilum and subsequent challenge with a virulent strain of the bacterium.

In Norway, the tick-transmitted bacterium Anaplasma phagocytophilum is estimated to cause tick-borne fever (TBF) in 300 000 lambs on pastures each year, resulting in economic and animal welfare consequences. Today, prophylactic measures mainly involve the use of acaricides, but a vaccine has been requested by farmers and veterinarians for decades. Several attempts have been made to produce a vaccine against A. phagocytophilum including antigenic surface proteins, inactivated whole cell vaccines and challenge followed by treatment. In the current study, a virulent wild type strain of A. phagocytophilum named Ap.Norvar1 (16S rRNA sequence partial identical to sequence in GenBank acc.no M73220) was subject to genetic transformation with a Himar1-transposon, which resulted in three bacterial mutants, capable of propagation in a tick cell line (ISE6). In order to test the immunogenicity and pathogenicity of the live, mutated bacteria, these were clinically tested in an inoculation- and challenge study in sheep. One group was inoculated with the Ap.Norvar1 as an infection control. After inoculation, the sheep inoculated with mutated bacteria and the Ap.Norvar1 developed typical clinical signs of infection and humoral immune response. After challenge with Ap.Norvar1, 28 days later all groups inoculated with mutated bacteria showed clinical signs of tick-borne fever and bacteremia while the group initially inoculated with the Ap.Norvar1, showed protection against clinical disease. The current study shows a weak, but partial protection against infection in animals inoculated with mutated bacteria, while animals that received Ap.Norvar1 both for inoculation and challenge, responded with homologues protection.

Creating a Library of Random Transposon Mutants in Leptospira.

Generation of a random transposon mutant library is advantageous in Leptospira as site-directed mutagenesis remains a challenge, especially in pathogenic species. This procedure is typically completed by transformation of Leptospira with a Himar1 containing plasmid via conjugation with Escherichia coli as a donor cell. Here we describe the methodology to generate random transposon mutants in the saprophyte Leptospira biflexa via conjugation of plasmid pSW29T-TKS2 harbored in E. coli ß2163. Determination of transposon insertion site by semi-random nested PCR will also be described. A similar methodology may be employed to generate Tn mutants of pathogenic Leptospira species.

Development of a Counterselectable Transposon To Create Markerless Knockouts from an 18,432-Clone Ordered Mycobacterium bovis Bacillus Calmette-Guérin Mutant Resource.

Mutant resources are essential to improve our understanding of the biology of slow-growing mycobacteria, which include the causative agents of tuberculosis in various species, including humans. The generation of deletion mutants in slow-growing mycobacteria in a gene-by-gene approach in order to make genome-wide ordered mutant resources is still a laborious and costly approach, despite the recent development of improved methods. On the other hand, transposon mutagenesis in combination with Cartesian pooling-coordinate sequencing (CP-CSeq) allows the creation of large archived Mycobacterium transposon insertion libraries. However, such mutants contain selection marker genes with a risk of polar gene effects, which are undesired both for research and for use of these mutants as live attenuated vaccines. In this paper, a derivative of the Himar1 transposon is described which allows the generation of clean, markerless knockouts from archived transposon libraries. By incorporating FRT sites for FlpE/FRT-mediated recombination and I-SceI sites for ISceIM-based transposon removal, we enable two thoroughly experimentally validated possibilities to create unmarked mutants from such marked transposon mutants. The FRT approach is highly efficient but leaves an FRT scar in the genome, whereas the I-SceI-mediated approach can create mutants without any heterologous DNA in the genome. The combined use of CP-CSeq and this optimized transposon was applied in the BCG Danish 1331 vaccine strain (WHO reference 07/270), creating the largest ordered, characterized resource of mutants in a member of the Mycobacterium tuberculosis complex (18,432 clones, mutating 83% of the nonessential M. tuberculosis homologues), from which markerless knockouts can be easily generated.IMPORTANCE While speeding up research for many fields of biology (e.g., yeast, plant, and Caenorhabditis elegans), genome-wide ordered mutant collections are still elusive in mycobacterial research. We developed methods to generate such resources in a time- and cost-effective manner and developed a newly engineered transposon from which unmarked mutants can be efficiently generated. Our library in the WHO reference vaccine strain of Mycobacterium bovis BCG Danish targets 83% of all nonessential genes and was made publicly available via the BCCM/ITM Mycobacteria Collection. This resource will speed up Mycobacterium research (e.g., drug resistance research and vaccine development) and paves the way to similar genome-wide mutant collections in other strains of the Mycobacterium tuberculosis complex. The stretch to a full collection of mutants in all nonessential genes is now much shorter, with just 17% remaining genes to be targeted using gene-by-gene approaches, for which highly effective methods have recently also been described.

Discovery of in vivo Virulence Genes of Obligatory Intracellular Bacteria by Random Mutagenesis.

Ehrlichia spp. are emerging tick-borne obligatory intracellular bacteria that cause febrile and sometimes fatal diseases with abnormal blood cell counts and signs of hepatitis. Ehrlichia HF strain provides an excellent mouse disease model of fatal human ehrlichiosis. We recently obtained and established stable culture of Ehrlichia HF strain in DH82 canine macrophage cell line, and obtained its whole genome sequence and annotation. To identify genes required for in vivo virulence of Ehrlichia, we constructed random insertional HF strain mutants by using Himar1 transposon-based mutagenesis procedure. Of total 158 insertional mutants isolated via antibiotic selection in DH82 cells, 74 insertions were in the coding regions of 55 distinct protein-coding genes, including TRP120 and multi-copy genes, such as p28/omp-1, virB2, and virB6. Among 84 insertions mapped within the non-coding regions, seven are located in the putative promoter region since they were within 50 bp upstream of the seven distinct genes. Using limited dilution methods, nine stable clonal mutants that had no apparent defect for multiplication in DH82 cells, were obtained. Mouse virulence of seven mutant clones was similar to that of wild-type HF strain, whereas two mutant clones showed significantly retarded growth in blood, livers, and spleens, and the mice inoculated with them lived longer than mice inoculated with wild-type. The two clones contained mutations in genes encoding a conserved hypothetical protein and a staphylococcal superantigen-like domain protein, respectively, and both genes are conserved among Ehrlichia spp., but lack homology to other bacterial genes. Inflammatory cytokine mRNA levels in the liver of mice infected with the two mutants were significantly diminished than those infected with HF strain wild-type, except IL-1ß and IL-12 p40 in one clone. Thus, we identified two Ehrlichia virulence genes responsible for in vivo infection, but not for infection and growth in macrophages.

Loci Identification of a N-acyl Homoserine Lactone Type Quorum Sensing System and a New LysR-type Transcriptional Regulator Associated with Antimicrobial Activity and Swarming in Burkholderia Gladioli UAPS07070.

A random transposition mutant library of B. gladioli UAPS07070 was analyzed for searching mutants with impaired microbial antagonism. Three derivates showed diminished antimicrobial activity against a sensitive strain. The mutated loci showed high similarity to the quorum sensing genes of the AHL-synthase and its regulator. Another mutant was affected in a gene coding for a LysrR-type transcriptional regulator. The production of toxoflavin, the most well known antimicrobial-molecule and a major virulence factor of plant-pathogenic B. glumae and B. gladioli was explored. The absence of a yellowish pigment related to toxoflavin and the undetectable transcription of toxA in the mutants indicated the participation of the QS system and of the LysR-type transcriptional regulator in the regulation of toxoflavin. Additionally, those genes were found to be related to the swarming phenotype. Lettuce inoculated with the AHL synthase and the lysR mutants showed less severe symptoms. We present evidence of the participation of both, the quorum sensing and for the first time, of a LysR-type transcriptional regulator in antibiosis and swarming phenotype in a strain of B. gladioli.

MarTrack: A versatile toolbox of mariner transposon derivatives used for functional genetic analysis of bacterial genomes.

The mariner transposon family of Himar1 has been widely used for the random mutagenesis of bacteria to generate single insertions into the chromosome. Here, a versatile toolbox of mariner transposon derivatives was generated and applied to the functional genomics investigation of fish pathogen Edwardsiella piscicida. In this study, we combined the merits of the random mutagenesis of mariner transposon and common efficient reporter marker genes or regulatory elements, mcherry, gfp, luxAB, lacZ, sacBR, and PBAD and antibiotic resistance cassettes to construct a series of derivative transposon vectors, pMmch, pMKGR, pMCGR, pMXKGR, pMLKGR, pMSGR, and pMPR, based on the initial transposon pMar2xT7. The function and effectiveness of the modified transposons were verified by introducing them into E. piscicida EIB202. Based on the toolbox, a transposon insertion mutant library containing approximately 3.0 × 105 separated mutants was constructed to explore the upstream regulators of esrB, the master regulator of the type III and type VI secretion systems (T3/T6SS) in E. piscicida. Following analysis by Con-ARTIST, ETAE_2184 (renamed as EsrR) was screened out and identified as a novel regulator mediating T3SS expression. In addition, the esrR mutants displayed critical virulence attenuation. Due to the broad-range host compatibility of mariner transposons, the newly built transposon toolbox can be broadly applied for functional genomics studies in various bacteria.

MarTrack: A versatile toolbox of mariner transposon derivatives used for functional genetic analysis of bacterial genomes.

The mariner transposon family of Himar1 has been widely used for the random mutagenesis of bacteria to generate single insertions into the chromosome. Here, a versatile toolbox of mariner transposon derivatives was generated and applied to the functional genomics investigation of fish pathogen Edwardsiella piscicida. In this study, we combined the merits of the random mutagenesis of mariner transposon and common efficient reporter marker genes or regulatory elements, mCherry, gfp, luxAB, lacZ, sacBR, and PBAD and antibiotic resistance cassettes to construct a series of derivative transposon vectors, pMmch, pMKGR, pMCGR, pMXKGR, pMLKGR, pMSGR, and pMPR, based on the initial transposon pMar2xT7. The function and effectiveness of the modified transposons were verified by introducing them into E. piscicida EIB202. Based on the toolbox, a transposon insertion mutant library containing approximately 3.0 × 105 distinct mutants was constructed to explore the upstream regulators of esrB, the master regulator of the type III and type VI secretion systems (T3/T6SS) in E. piscicida. Following analysis by Con-ARTIST, ETAE_3474, annotated as fabR and involved in fatty acid metabolism, was screened out and identified as a novel regulator mediating T3SS and T6SS expression. In addition, the fabR mutants displayed critical virulence attenuation in turbot. Due to the broad-range host compatibility of mariner transposons, the newly built transposon toolbox can be applied for functional genomics studies in various bacteria.

Development of a metabolic pathway transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii.

BACKGROUND: Clostridium spp. can synthesize valuable chemicals and fuels by utilizing diverse waste-stream substrates, including starchy biomass, lignocellulose, and industrial waste gases. However, metabolic engineering in Clostridium spp. is challenging due to the low efficiency of gene transfer and genomic integration of entire biosynthetic pathways. RESULTS: We have developed a reliable gene transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii based on the conjugal transfer of donor plasmids containing large transgene cassettes (> 5 kb) followed by the inducible activation of Himar1 transposase to promote integration. We established a conjugation protocol for the efficient generation of transconjugants using the Gram-positive origins of replication repL and repH. We also investigated the impact of DNA methylation on conjugation efficiency by testing donor constructs with all possible combinations of Dam and Dcm methylation patterns, and used bisulfite conversion and PacBio sequencing to determine the DNA methylation profile of the C. ljungdahlii genome, resulting in the detection of four sequence motifs with N6-methyladenosine. As proof of concept, we demonstrated the transfer and genomic integration of a heterologous acetone biosynthesis pathway using a Himar1 transposase system regulated by a xylose-inducible promoter. The functionality of the integrated pathway was confirmed by detecting enzyme proteotypic peptides and the formation of acetone and isopropanol by C. ljungdahlii cultures utilizing syngas as a carbon and energy source. CONCLUSIONS: The developed multi-gene delivery system offers a versatile tool to integrate and stably express large biosynthetic pathways in the industrial promising syngas-fermenting microorganism C. ljungdahlii. The simple transfer and stable integration of large gene clusters (like entire biosynthetic pathways) is expanding the range of possible fermentation products of heterologously expressing recombinant strains. We also believe that the developed gene delivery system can be adapted to other clostridial strains as well.

Fluorescent Protein Expressing Rickettsia buchneri and Rickettsia peacockii for Tracking Symbiont-Tick Cell Interactions.

Rickettsiae of indeterminate pathogenicity are widely associated with ticks. The presence of these endosymbionts can confound a One Health approach to combatting tick-borne diseases. Genomic analyses of symbiotic rickettsiae have revealed that they harbor mutations in gene coding for proteins involved in rickettsial pathogenicity and motility. We have isolated and characterized two rickettsial symbionts-Rickettsia peacockii and R. buchneri-both from ticks using tick cell cultures. To better track these enigmatic rickettsiae in ticks and at the tick-mammal interface we transformed the rickettsiae to express fluorescent proteins using shuttle vectors based on rickettsial plasmids or a transposition system driving insertional mutagenesis. Fluorescent protein expressing R. buchneri and R. peacockii will enable us to elucidate their interactions with tick and mammalian cells, and track their location and movement within individual cells, vector ticks, and host animals.