Tn5 Insertion Research Articles

High-efficiency nuclear transformation of the microalgae Nannochloropsis oceanica using Tn5 Transposome for the generation of altered lipid accumulation phenotypes

BackgroundOne of the major problems in the production of lipids for biotechnological purposes using microalgae is maintaining a high productivity of these molecules without reducing cellular biomass. High production rates are usually obtained by cultivating microalgae under different stress conditions. However, many of these changes usually result in lower biomass productivity. Therefore, the optimization of the culture conditions and genetic modification techniques in these organisms is needed to generate robust new strains for profitable economic use.ResultsIn this work, we describe a new strategy for random mutation of genomic DNA in the microalgae Nannochloropsis oceanica by insertion of a Transposome complex Tn5. This complex contains an antibiotic-resistance cassette commanded by a CMV viral promoter that allows high efficiency of transformation and the generation of mutants. This strategy, complemented with a large-scale identification and selection system for mutants, such as flow cytometry with cell selection, allowed us to obtain clonal cultures of mutants with altered phenotypes in the accumulation of intracellular lipids. The characterization of some of these mutants uncovered new genes that are likely to be involved in the regulation of lipid synthesis, revealing possible cellular responses that influence the intracellular homeostasis of lipids.ConclusionThe strategies proposed here are easy to implement in different types of microalgae and provide a promising scenario for improving biotechnological applications.

Rhizobium tropici CIAT 899 copA gene plays a fundamental role in copper tolerance in both free life and symbiosis with Phaseolus vulgaris.

Rhizobium tropici CIAT 899 is a facultative symbiotic diazotroph able to deal with stressful concentrations of metals. Nevertheless the molecular mechanisms involved in metal tolerance have not been elucidated. Copper (Cu2+) is a metal component essential for the heme-copper respiratory oxidases and enzymes that catalyse redox reactions, however, it is highly toxic when intracellular trace concentrations are surpassed. In this study, we report that R. tropici CIAT 899 is more tolerant to Cu2+ than other Rhizobium and Sinorhizobium species. Through Tn5 random mutagenesis we identify a R. tropici mutant strain with a severe reduction in Cu2+ tolerance. The Tn5 insertion disrupted the gene RTCIAT899_CH17575, encoding a putative heavy metal efflux P1B-1-type ATPase designated as copA. Phaseolus vulgaris plants inoculated with the copA::Tn5 mutant in the presence of toxic Cu2+ concentrations showed a drastic reduction in plant and nodule dry weight, as well as nitrogenase activity. Nodules induced by the copA::Tn5 mutant present an increase in H2O2 concentration, lipoperoxidation and accumulate 40-fold more Cu2+ than nodules formed by the wild-type strain. The copA::Tn5 mutant complemented with the copA gene recovered the wild-type symbiotic phenotypes. Therefore, the copA gene is essential for R. tropici CIAT 899 to survive in copper-rich environments in both free life and symbiosis with P. vulgaris plants.

A mutation in the glta gene from a native isolate of the Pseudomonas chlororaphis subgroup induces a phenotypic change associated with phenazine production

Members of the Pseudomonas genus colonise the rhizosphere of different plant species and display plant-probiotic traits. Among our collection of 19 isolates of native pseudomonads, isolate SPAN5 was obtained from a bulk soil sample of grasslands. This isolate, related with the Pseudomonas chlororaphis subgroup, showed several plant-growth promoting activities in vitro. We carried out Tn5 mutagenesis for identifying genes related to the biocontrol. We here report that the mutant clone SPAN5-135, which lost its orange pigmentation, suffered the insertion of Tn5 into the gltA gene encoding a type II citrate synthase (CS). The CS enzyme activity was reduced significantly in SPAN-135, as well as its phenazine production in several media. Also, siderophore and exoprotease secretion were affected. SPAN-135 could not recover the complete inhibition potential of the wild type version against the phytopathogenic fungi tested. This is the first report of the requirement of gltA for phenazine and siderophores production.

Analysis of traits for biocontrol performance of Pseudomonas parafulva JBCS1880 against bacterial pustule in soybean plants

Many biocontrol mechanisms including antimicrobial activity have been identified for control of plant diseases. However, the influence of various traits on the biocontrol performance of a specific biological control agent (BCA) remains to be revealed. In this study, to dissect the contribution of various traits to the biocontrol performance of a bacterial BCA, we performed genetic screening using a random transposon (Tn) mutant collection constructed from Pseudomonas parafulva JBCS1880 that shows strong antagonism against Xanthomonas axonopodis pv. glycines and Burkholderia glumae. Suppression of antibacterial activity by Tn insertion reduced the biocontrol performance of the strain. The disruption of viscosin-like lipopeptide, which is predicted to have D-Ser at the second position (called VILS2 hereafter), significantly suppressed not only antagonism, but also swarming motility and biofilm formation of P. parafulva JBCS1880. In addition, surface colonization with swarming motility and siderophore production also contributed to the biocontrol ability of the strain. The results indicate that the coordinated action of VILS2-mediated antibacterial activity and surface movement is critical for biocontrol activity of P. parafulva JBCS1880. Overall, our results suggest that antagonism, swarming motility, and siderophore production are important traits for the biocontrol performance of P. parafulva JBCS1880. This is the first report of the biocontrol of soybean bacterial pustules using a strain of P. parafulva, which is expected to produce a novel lipopeptide. The traits essential for biocontrol performance of the potential BCA were determined, but the functional roles of many genes in biocontrol remain a focus for further investigation.

A two-component regulatory system VfmIH modulates multiple virulence traits in Dickeya zeae.

Bacterial pathogen Dickeya zeae strain EC1 produces antibiotics-like phytotoxins called zeamines, which are major virulence determinants encoded by the zms gene cluster. In this study, we identified a zeamine-deficient mutant with a Tn5 insertion in a gene designated as vfmI encoding a two-component system (TCS) sensor histidine kinase (HK), which is accompanied by vfmH encoding a response regulator (RR) at the same genetic locus. Domain analysis shows this TCS is analogous to the VfmIH of D. dadantii, with typical characteristics of sensor HK and RR, respectively, and sharing the same operon. Deletion of either vfmI or vfmH resulted in decreased production of zeamines and cell wall degrading enzymes (CWDEs), and alleviated virulence on rice seeds and potato tubers. In D. dadantii 3937, VfmH was shown to bind to the promoters of vfmA and vfmE, while in D. zeae EC1, VfmH could bind to the promoters of vfmA, vfmE and vfmF. RNA-seq analysis of strain EC1 and its vfmH mutant also showed that the TCS positively regulated a range of virulence genes, including zms, T1SS, T2SS, T3SS, T6SS, flagellar and CWDE genes.

Colanic Acid Is a Novel Phage Receptor of Pectobacterium carotovorum subsp. carotovorum Phage POP72.

The emergence and widespread nature of pathogen resistance to antibiotics and chemicals has led to the re-consideration of bacteriophages as an alternative biocontrol agent in several fields, including agriculture. In this study, we isolated and characterized a novel bacteriophage, POP72, that specifically infects Pectobacterium carotovorum subsp. carotovorum (Pcc), which frequently macerates agricultural crops. POP72 contains a 44,760 bp double-stranded DNA genome and belongs to the family Podoviridae. To determine the phage receptor for POP72, a random mutant library of Pcc was constructed using a Tn5 transposon and screened for resistance against POP72 infection. Most of the resistant clones had a Tn5 insertion in various genes associated with colanic acid (CA) biosynthesis. The phage adsorption rate and CA production decreased dramatically in the resistant clones. Complementation of the clones with the pUHE21-2 lacIq vector harboring genes associated with CA biosynthesis restored their sensitivity to POP72, as well as their ability to produce CA. These results suggest that CA functions as a novel phage receptor for POP72. The application of POP72 protected Chinese cabbage from Pcc infection, suggesting that phage POP72 would be an effective alternative antimicrobial agent to protect agricultural products from Pcc.

The histone-like protein HupB influences biofilm formation and virulence in Xanthomonas citri ssp. citri through the regulation of flagellar biosynthesis.

SummaryCitrus canker is an important disease of citrus, whose causal agent is the bacterium Xanthomonas citri ssp. citri (Xcc). In previous studies, we found a group of Xcc mutants, generated by the insertion of the Tn5 transposon, which showed impaired ability to attach to an abiotic substrate. One of these mutants carries the Tn5 insertion in hupB, a gene encoding a bacterial histone‐like protein, homologue to the β‐subunit of the Heat‐Unstable (HU) nucleoid protein of Escherichia coli. These types of protein are necessary to maintain the bacterial nucleoid organization and the global regulation of gene expression. Here, we characterized the influence of the mutation in hupB regarding Xcc biofilm formation and virulence. The mutant strain hupB was incapable of swimming in soft agar, whereas its complemented strain partially recovered this phenotype. Electron microscope imaging revealed that impaired motility of hupB was a consequence of the absence of the flagellum. Comparison of the expression of flagellar genes between the wild‐type strain and hupB showed that the mutant exhibited decreased expression of fliC (encoding flagellin). The hupB mutant also displayed reduced virulence compared with the wild‐type strain when they were used to infect Citrus lemon plants using different infection methods. Our results therefore show that the histone‐like protein HupB plays an essential role in the pathogenesis of Xcc through the regulation of biofilm formation and biosynthesis of the flagellum.

PGN_0297 is an essential component of the type IX secretion system (T9SS) in Porphyromonas gingivalis: Tn-seq analysis for exhaustive identification of T9SS-related genes.

The type IX secretion system (T9SS) was originally discovered in Porphyromonas gingivalis, one of the pathogenic bacteria associated with periodontal disease and is now known to be present in many members of the phylum Bacteroidetes. The T9SS secretes a number of potent virulence factors, including the highly hydrolytic proteases called gingipains, across the outer membrane in P. gingivalis. To understand the entire machinery of T9SS, an exhaustive search for T9SS‐related genes in P. gingivalis using the mariner family transposon (Tn) and Tn‐seq analysis was performed. Seven hundred and two Tn insertion sites in Tn mutants with no colony pigmentation that is associated with Lys‐gingipain (Kgp) defectiveness were determined, and it was found that the Tn was inserted in the kgp gene and 54 T9SS‐related candidate genes. Thirty‐three out of the 54 genes were already known as T9SS‐related genes. Furthermore, deletion mutant analysis of the remaining 21 genes revealed that they were not related to the T9SS. The 33 T9SS‐related genes include a gene for PGN_0297, which was found to be associated with the T9SS components PorK and PorN. A PGN_0297 gene deletion mutant was constructed, and it was found that the mutant showed no colony pigmentation, hemagglutination or gingipain activities, indicating that PGN_0297 was an essential component of the T9SS. The 33 genes did not include the six genes (gppX, omp17, porY, rfa, sigP and wzx) that were also reported as T9SS‐related genes. gppX deletion and insertion mutants were constructed, and it was found that they did not show deficiency in the T9SS.

A practical random mutagenesis system for Ralstonia solanacearum strains causing bacterial wilt of Pogostemon cablin using Tn5 transposon.

A practical random mutagenesis system of Ralstonia solanacearum by electroporation with Tn5 transposon was established, which may be utilized to provide genetic approach to study virulence genes of R. solanacearum strains and create nonpathogenic mutants for biological control of bacterial wilt in Pogostemon cablin. R. solanacearum strain PRS-84 used in this study was isolated from P. cablin plants infected with bacterial wilt. The bacterial suspension of R. solanacearum strain PRS-84 was mixed with Tn5 transposome complex and the mixture was transformed by electroporation. The electroporated cells were then spread on the 2, 3, 5-triphenyltetrazolium chloride agar plates containing kanamycin to select the kanamycin-resistant colonies. Several factors which determined the bacterial transformation efficiency were optimized. The transformation process was shown to be optimal at the electric field strength of 12.5kVcm-1. Bacterial cells harvested at mid-exponential phase gave the highest transformation efficiency. 10µgmL-1 kanamycin was found to be the optimal concentration for transformant selection. Tn5 insertion mutants of R. solanacearum strain PRS-84 were identified by PCR amplification and Southern blot analysis. Mutants subcultured for 100 passages were also detected by PCR amplification and Southern blot analysis. Furthermore, pathogenicity screening test of mutants was performed by inoculating in vitro regenerated patchouli plants. Results revealed that mutants with a single Tn5 insertion in their genomes were obtained from R. solanacearum strain PRS-84, and the Tn5 insertion could be stably inherited in the mutants. Then, mutants with reduced pathogenicity were selected.

Gene ssfg_01967 (miaB) for tRNA modification influences morphogenesis and moenomycin biosynthesis in Streptomyces ghanaensis ATCC14672.

Streptomyces ghanaensis ATCC14672 is remarkable for its production of phosphoglycolipid compounds, moenomycins, which serve as a blueprint for the development of a novel class of antibiotics based on inhibition of peptidoglycan glycosyltransferases. Here we employed mariner transposon (Tn) mutagenesis to find new regulatory genes essential for moenomycin production. We generated a library of 3000 mutants which were screened for altered antibiotic activity. Our focus centred on a single mutant, HIM5, which accumulated lower amounts of moenomycin and was impaired in morphogenesis as compared to the parental strain. HIM5 carried the Tn insertion within gene ssfg_01967 for putative tRNA (N6-isopentenyl adenosine(37)-C2)-methylthiotransferase, or MiaB, and led to a reduced level of thiomethylation at position 37 in the anticodon of S. ghanaensis transfer ribonucleic acid (tRNA). It is likely that the mutant phenotype of HIM5 stems from the way in which ssfg_01967::Tn influences translation of the rare leucine codon UUA in several genes for moenomycin production and life cycle progression in S. ghanaensis. This is the first report showing that quantitative changes in tRNA modification status in Streptomyces have physiological consequences.

Characterization of the ModABC Molybdate Transport System of Pseudomonas putida in Nicotine Degradation.

Pseudomonas putida J5 is an efficient nicotine-degrading bacterial strain that catabolizes nicotine through the pyrrolidine pathway. In our previous study, we used Tn5 transposon mutagenesis to investigate nicotine metabolism-associated genes, and 18 nicotine degradation-deficient mutants were isolated from 16,324 Tn5-transformants. Three of the mutants were Tn5 inserts into the modABC gene cluster that encoded an ABC-type, high-affinity, molybdate transporter. In-frame deletion of the modABC genes abolished the nicotine-degrading ability of strain J5, and complementation with modABC either from P. putida or Arthrobacter oxidans restored the degrading activity of the mutant to wild-type level. Nicotine degradation of J5 was inhibited markedly by addition of tungstate, a specific antagonist of molybdate. Molybdate at a non-physiologically high concentration (100 μM) fully restored nicotine-degrading activity and recovered growth of the modABC mutant in a nicotine minimal medium. Transcriptional analysis revealed that the expression of modABC was up-regulated at low molybdate concentrations and down-regulated at high moybdate concentrations, which indicated that at least one other system was able to transport molybdate, but with lower affinity. These results suggested that the molybdate transport system was essential to nicotine metabolism in P. putida J5.

Heat shock transcription factor σ32 defective in membrane transport can be suppressed by transposon insertion into genes encoding a restriction enzyme subunit or a putative autotransporter in Escherichia coli.

Heat shock transcription factor σ32 of Escherichia coli plays a major role in protein homeostasis and requires membrane localization for regulation. We here report that a strongly deregulated I54N-σ32 mutant defective in association with the membrane can be phenotypically suppressed by Tn5 insertion into the mcrC or ydbA2 gene, encoding a restriction enzyme subunit or part of a putative autotransporter, respectively. The suppression is specific for mutant I54N-σ32 and reduces its activity but not its abundance or stability. Moreover, the deregulated phenotype of I54N-σ32 is effectively suppressed by a plasmid carrying the same mcrC::Tn5 mutation. In contrast, deletion of the mcrC or ydbA2 gene hardly affects I54N-σ32 activity. These results, taken together, suggest that the truncated form of McrC (and presumably also of YdbA2) protein produced by the Tn5 insertion interacts specifically with I54N-σ32 to reduce its activity without substantially affecting its amount or stability.

Efficient transposon mutagenesis mediated by an IPTG-controlled conditional suicide plasmid

BackgroundTransposon mutagenesis is highly valuable for bacterial genetic and genomic studies. The transposons are usually delivered into host cells through conjugation or electroporation of a suicide plasmid. However, many bacterial species cannot be efficiently conjugated or transformed for transposon saturation mutagenesis. For this reason, temperature-sensitive (ts) plasmids have also been developed for transposon mutagenesis, but prolonged incubation at high temperatures to induce ts plasmid loss can be harmful to the hosts and lead to enrichment of mutants with adaptive genetic changes. In addition, the ts phenotype of a plasmid is often strain- or species-specific, as it may become non-ts or suicidal in different bacterial species.ResultsWe have engineered several conditional suicide plasmids that have a broad host range and whose loss is IPTG-controlled. One construct, which has the highest stability in the absence of IPTG induction, was then used as a curable vector to deliver hyperactive miniTn5 transposons for insertional mutagenesis. Our analyses show that these new tools can be used for efficient and regulatable transposon mutagenesis in Escherichia coli, Acinetobacter baylyi and Pseudomonas aeruginosa. In P. aeruginosa PAO1, we have used this method to generate a Tn5 insertion library with an estimated diversity of ~ 108, which is ~ 2 logs larger than the best transposon insertional library of PAO1 and related Pseudomonas strains previously reported.ConclusionWe have developed a number of IPTG-controlled conditional suicide plasmids. By exploiting one of them for transposon delivery, a highly efficient and broadly useful mutagenesis system has been developed. As the assay condition is mild, we believe that our methodology will have broad applications in microbiology research.

The Catabolite Repressor/Activator Cra Is a Bridge Connecting Carbon Metabolism and Host Colonization in the Plant Drought Resistance-Promoting Bacterium Pantoea alhagi LTYR-11Z.

Efficient root colonization is a prerequisite for application of plant growth-promoting (PGP) bacteria in improving health and yield of agricultural crops. We have recently identified an endophytic bacterium, Pantoea alhagi LTYR-11Z, with multiple PGP properties that effectively colonizes the root system of wheat and improves its growth and drought tolerance. To identify novel regulatory genes required for wheat colonization, we screened an LTYR-11Z transposon (Tn) insertion library and found cra to be a colonization-related gene. By using transcriptome (RNA-seq) analysis, we found that transcriptional levels of an eps operon, the ydiV gene encoding an anti-FlhD4C2 factor, and the yedQ gene encoding an enzyme for synthesis of cyclic dimeric GMP (c-di-GMP) were significantly downregulated in the Δcra mutant. Further studies demonstrated that Cra directly binds to the promoters of the eps operon, ydiV, and yedQ and activates their expression, thus inhibiting motility and promoting exopolysaccharide (EPS) production and biofilm formation. Consistent with previous findings that Cra plays a role in transcriptional regulation in response to carbon source availability, the activating effects of Cra were much more pronounced when LTYR-11Z was grown within a gluconeogenic environment than when it was grown within a glycolytic environment. We further demonstrate that the ability of LTYR-11Z to colonize wheat roots is modulated by the availability of carbon sources. Altogether, these results uncover a novel strategy utilized by LTYR-11Z to achieve host colonization in response to carbon nutrition in the environment, in which Cra bridges a connection between carbon metabolism and colonization capacity of LTYR-11Z.IMPORTANCE Rapid and appropriate response to environmental signals is crucial for bacteria to adapt to competitive environments and to establish interactions with their hosts. Efficient colonization and persistence within the host are controlled by various regulatory factors that respond to specific environmental cues. The most common is nutrient availability. In this work, we unraveled the pivotal role of Cra in regulation of colonization ability of Pantoea alhagi LTYR-11Z in response to carbon source availability. Moreover, we identified three novel members of the Cra regulon involved in EPS synthesis, regulation of flagellar biosynthesis, and synthesis of c-di-GMP and propose a working model to explain the Cra-mediated regulatory mechanism that links carbon metabolism to host colonization. This study elucidates the regulatory role of Cra in bacterial attachment and colonization of plants, which raises the possibility of extending our studies to other bacteria associated with plant and human health.

Generation and In Vivo Characterization of Tn5-Induced Biofilm Mutants of Vibrio cholerae O139.

The Gram-negative bacterium Vibrio cholerae is a unique pathogen with an ability to colonize human intestine as well as outside environments. The biofilm, an organized polymeric structure produced by this bacterium known to be a significant factor for the survival and persistence in hostile conditions. However, the direct role of biofilm formation by this bacterium in environmental persistence, in vivo colonization, and pathogenesis remains unexplored. In this study, we have generated biofilm-altered Tn5 mutants of V. cholerae O139 and evaluated their in vivo colonization ability on mouse model. These Tn5 mutants were found to harbor an independent, single Tn5 insertion in their genome. TheDNA sequence analysis revealed that genomic region wherein Tn5 insertion occurred is identified to be involved in functions like LPS biosynthesis, efflux transporters, motility, purine metabolism, stringent response, VPS synthesis, and a hypothetical protein of unknown function. In single-strain infection with the planktonic culture, the biofilm-altered as well as the biofilm intermediate mutants were found to be more or less similar in their intestinal colonization ability, however infection with their biofilm form, a marked difference was observed between the biofilm deficient and other biofilm forming strains. Further, in the competition experiments, biofilm deficient and proficient mutants were found reduced in their colonization ability and outcompeted by their parent strain. In conclusion, biofilm formation in V. cholerae O139 is a genetically complex process and the controlled and regulated production of biofilm appeared to be necessary for its efficient colonization of mouse intestine.

Dot/Icm-Translocated Proteins Important for Biogenesis of the Coxiella burnetii-Containing Vacuole Identified by Screening of an Effector Mutant Sublibrary.

Coxiella burnetii is an intracellular pathogen that replicates in a lysosome-derived vacuole. A determinant necessary for C. burnetii virulence is the Dot/Icm type IVB secretion system (T4SS). The Dot/Icm system delivers more than 100 proteins, called type IV effectors (T4Es), across the vacuolar membrane into the host cell cytosol. Several T4Es have been shown to be important for vacuolar biogenesis. Here, transposon (Tn) insertion sequencing technology (INSeq) was used to identify C. burnetii Nine Mile phase II mutants in an arrayed library, which facilitated the identification and clonal isolation of mutants deficient in 70 different T4E proteins. These effector mutants were screened in HeLa cells for deficiencies in Coxiella-containing vacuole (CCV) biogenesis. This screen identified and validated seven new T4Es that were important for vacuole biogenesis. Loss-of-function mutations in cbu0414 (coxH1), cbu0513, cbu0978 (cem3), cbu1387 (cem6), cbu1524 (caeA), cbu1752, or cbu2028 resulted in a small-vacuole phenotype. These seven mutant strains produced small CCVs in all cells tested, which included macrophage-like cells. The cbu2028::Tn mutant, though unable to develop large CCVs, had intracellular replication rates similar to the rate of the parental strain of C. burnetii, whereas the other six effector mutants defective in CCV biogenesis displayed significant reductions in intracellular replication. Vacuoles created by the cbu0513::Tn mutant did not accumulate lipidated microtubule-associated protein 1A/1B light chain 3 (LC3-II), suggesting a failure in fusion of the CCV with autophagosomes. These seven T4E proteins add to the growing repertoire of C. burnetii factors that contribute to CCV biogenesis.

Transposon insertion upstream of a putative sodium/sulphate symporter is associated with hypervirulence in the soft rot bacterium, Pectobacterium carotovorum

AbstractPathogenesis by Pectobacterium carotovorum and the other soft rot bacteria results mostly from the synthesis and secretion of a plethora of exoproteins including plant cell wall‐degrading enzymes and type III effectors. A mini‐Tn5 transposon mutant of P. carotovorum (KD200) was isolated that is highly increased in activity and transcript levels of extracellular cell wall‐degradative enzymes, pectate lyase, polygalacturonase, cellulase and protease. Accordingly, the mutant was more virulent as it macerated more host tissue than its parent. KD200 mutant harbours a Tn5 insertion directly upstream of nssAEcc71, a putative sodium/sulphate symporter gene and member of a family of divalent anion sodium symporters (DASS). The findings reported here suggest that DASS proteins may be involved in soft rot pathogenesis.

Molecular diversity and conjugal transferability of class 2 integrons among Escherichia coli isolates from food, animal and human sources

Integrons are genetic platforms able to excise, integrate and express antibiotic resistance gene cassettes (GCs). Here we investigated the complete genetic organisation, genetic environment, location and conjugative transferability of a collection of class 2 integrons carried by Escherichia coli strains from different sources (poultry/pork meat, animals and humans). PCR cartography was conducted to determine the genetic arrangement of the integrons, their physical linkage to Tn7 and chromosomal insertion at the attTn7 site. Clonal relatedness of specific isolates was determined by MLST and DO-PCR. Transferability of class 2 integrons was tested by conjugation. The resulting transconjugants were characterised by antimicrobial resistance genotyping, S1-PFGE and replicon typing. Although a limited diversity of GCs was shown, a high percentage of novel structures was identified owing to the integration of insertion sequence (IS) elements at different sites (IS3/IS4/IS5/IS21 families). Insertion of IS10 in the attI2 site of a class 2 integron, between Pc2B and Pc2C promoters, was likely mediated by a site-specific transposition event. Chromosomal insertion of integrons at attTn7 was confirmed in 80% of the isolates. Conjugation experiments demonstrated that 29% of class 2 integrons could be mobilised to E. coli CHS26, demonstrating that they can be located in conjugative/mobilisable elements at a low frequency. Reported structures evidence how class 2 integrons have evolved by the activity of integron integrases and the invasion of ISs. Since most of them are chromosomally located, dispersion is predominantly vertical, although conjugation events also contribute to the spread of class 2 integrons among bacterial communities.

The unexpected function of a Flavin-dependent oxidoreductase from Variovorax paradoxus TBEA6.

3,3'-Thiodipropionic acid (TDP) is used as an additive in food and cosmetic industry and as precursor substrate for biotechnical polythioester production. Its catabolism was investigated in Variovorax paradoxus TBEA6 previous to this study. It was reported that the insertion of the transposon Tn5::mob into a gene showing high homology to flavin-dependent oxidoreductases (fox) resulted in impaired growth with TDP. Therefore, it was assumed that the initial cleavage of TDP is catalyzed by an FAD-dependent oxidoreductase (Fox, VPARA_05580). Accordingly, fox was heterologously expressed as a thioredoxin fusion protein. Analytical size exclusion chromatography revealed a homodimeric structure and the presence of the cofactor FAD. In vitro experiments showed that FoxTBEA6 is a D-2-hydroxy acid specific dehydrogenase and that its activity is enhanced in presence of either Ni2+, Co2+ or Zn2+. Cleavage of TDP by FoxTBEA6 was not observed. The findings are contrary to restricted growth with TDP of the transposon mutants and the previously published deletion mutant V. paradoxus TBEA6 Δfox. In this study, this contradiction was investigated by generation of additional deletion mutants, in which partial or complete deletion of fox did not affect utilization of TDP, and the mapping of single nucleotide polymorphisms (SNPs) in V. paradoxus TBEA6 Δfox.

Identification of genes required for Mycobacterium abscessus growth in vivo with a prominent role of the ESX-4 locus

Mycobacterium abscessus, a rapidly growing mycobacterium (RGM) and an opportunistic human pathogen, is responsible for a wide spectrum of clinical manifestations ranging from pulmonary to skin and soft tissue infections. This intracellular organism can resist the bactericidal defense mechanisms of amoebae and macrophages, an ability that has not been observed in other RGM. M. abscessus can up-regulate several virulence factors during transient infection of amoebae, thereby becoming more virulent in subsequent respiratory infections in mice. Here, we sought to identify the M. abscessus genes required for replication within amoebae. To this end, we constructed and screened a transposon (Tn) insertion library of an M. abscessus subspecies massiliense clinical isolate for attenuated clones. This approach identified five genes within the ESX-4 locus, which in M. abscessus encodes an ESX-4 type VII secretion system that exceptionally also includes the ESX conserved EccE component. To confirm the screening results and to get further insight into the contribution of ESX-4 to M. abscessus growth and survival in amoebae and macrophages, we generated a deletion mutant of eccB4 that encodes a core structural element of ESX-4. This mutant was less efficient at blocking phagosomal acidification than its parental strain. Importantly, and in contrast to the wild-type strain, it also failed to damage phagosomes and showed reduced signs of phagosome-to-cytosol contact, as demonstrated by a combination of cellular and immunological assays. This study attributes an unexpected and genuine biological role to the underexplored mycobacterial ESX-4 system and its substrates.