Tn5 Mutagenesis Research Articles

Tracking and characterization of a novel conjugative transposon identified by shotgun transposon mutagenesis.

The horizontal transfer of mobile genetic elements (MGEs) is an essential process determining the functional and genomic diversity of bacterial populations. MGEs facilitate the exchange of fitness determinant genes like antibiotic resistance and virulence factors. Various computational methods exist to identify potential MGEs, but confirming their ability to transfer requires additional experimental approaches. Here, we apply a transposon (Tn) mutagenesis technique for confirming mobilization without the need for targeted mutations. Using this method, we identified two MGEs, including a previously known conjugative transposon (CTn) called BoCTn found in Bacteroides ovatus and a novel CTn, PvCTn, identified in Phocaeicola vulgatus. In addition, Tn mutagenesis and subsequent genetic deletion enabled our characterization of a helix-turn-helix motif gene, BVU3433 which negatively regulates the conjugation efficiency of PvCTn in vitro. Furthermore, our transcriptomics data revealed that BVU3433 plays a crucial role in the repression of PvCTn genes, including genes involved in forming complete conjugation machinery [Type IV Secretion System (T4SS)]. Finally, analysis of individual strain genomes and community metagenomes identified the widespread prevalence of PvCTn-like elements with putative BVU3433 homologs among human gut-associated bacteria. In summary, this Tn mutagenesis mobilization method (TMMM) enables observation of transfer events in vitro and can ultimately be applied in vivo to identify a broader diversity of functional MGEs that may underly the transfer of important fitness determinants.

Identification and characterization of Acinetobacter nosocomialis BfmRS, two-component regulatory system, essential for biofilm development.

Biofilm development by bacteria is considered to be an essential stage in the bacterial infection. Acinetobacter nosocomialis is an important nosocomial pathogen causing a variety of human infections. However, characteristics and specific determinants of biofilm development have been poorly characterized in A. nosocomialis. The aim of this study was to investigate the factors involved in the biofilm development by A. nosocomialis. Library of random transposon mutants was constructed using the Tn5 mutagenesis. The mutant strains, in which the ability of biofilm formation was significantly impaired, were screened by gentian violet staining. The roles of BfmR and BfmS were determined by constructing a bfmR and bfmS deletion mutant and analyzing the effects of bfmR and bfmS mutation on the biofilm development and motility of A. nosocomialis. We identified a biofilm-defective mutant in which a transposon insertion inactivated an open reading frame encoding the BfmR in a two-component regulatory system consisting of BfmR and BfmS. The bfmR mutant revealed a significant reduction in biofilm formation and motility compared to wild-type strain. Deficiency in the biofilm formation and motility of the bfmR mutant was restored by single copy bfmR complementation. In contrast, the bfmS mutant had no effect on biofilm formation. A. nosocomialis has a two-component regulatory system, BfmRS. BfmR is a response regulator required for the initial attachment and maturation of biofilm during the biofilm development as well as the bacterial growth. BfmR could be a potential drug target for A. nosocomialis infection.

A system for transposon mutagenesis of Bartonella bacilliformis

Bartonella bacilliformis is the etiologic agent of Carrión's disease in South America. Lack of a system for random mutagenesis has significantly hampered research on the pathogen's molecular biology. Here, we describe a transposon (Tn)-based mutagenesis strategy for B. bacilliformis using pSAM_Rl; a Tn-mariner delivery vector originally constructed for members of the Rhizobiaceae family. Following electroporation of the vector, five candidate mutant strains were selected based on aberrant colony morphologies, and four mutations confirmed and identified using arbitrarily-primed PCR coupled with Sanger sequencing. One mutant strain, 4B2, was found to have a disrupted flgI gene, encoding the P-ring component of the flagellar motor. We therefore investigated the flgI strain's motility phenotype in a novel motility medium and found that insertional mutagenesis produced a non-motile mutant. Taken as a whole, the results show that: 1) pSAM_R1 is a practical Tn delivery vector for B. bacilliformis, 2) the plasmid can be used to create random Tn mariner mutants, 3) arbitrarily-primed PCR coupled with Sanger sequencing is a rapid and simple method for identifying and locating mutations generated by this Tn, and 4) in silico-predicted mutant phenotypes can be verified in vitro following mutagenesis. This system of Tn mutagenesis and mutation identification provides a novel and straightforward approach to investigate the molecular biology of B. bacilliformis.

Proline confers acid stress tolerance to Bacillus megaterium G18

Proline plays a multifunctional role in several organisms including bacteria in conferring protection under stress conditions. In this paper we report the role of proline in conferring acid tolerance to Bacillus megaterium G18. An acid susceptible mutant of B. megaterium G18 which required proline for its growth under acid stress condition was generated through Tn5 mutagenesis. Further, targeted inactivation of proC involved in osmo-adaptive proline synthesis in B. megaterium G18 resulted in the loss of ability of the bacterium to grow at low pH (pH 4.5). Exogenous supply of proline (1 mM) to the growth medium restored the ability of the mutant cells to grow at pH 4.5 which was not the same in case of other osmoprotectants tested. Proline was produced and secreted to extracellular medium by B. megaterium G18 when growing in low pH condition as evidenced by the use of Escherichia coli proline auxotrophs and HPLC analysis. Further, pHT01 vector based expression of full length proC gene in the ∆proC mutant cells restored the survival capacity of the mutant cells in acidic pH, suggesting that proline production is an important strategy employed by B. megaterium G18 to survive under acid stress induced osmotic stress.

A black-pigmented pseudomonad isolate with antibacterial activity against phyllospheric pathogens

Bacteria of the Pseudomonas genus have been widely studied due to their antagonistic potential against a diverse group of fungal and bacterial phytopathogens, and their competence to colonize different plant tissues. We have isolated a rhizospheric pseudomonad that produced a black pigment, which is not a widespread trait within this genus. We confirmed that the isolate belonged to the P. putida complex through a MLSA analysis. We observed that the pigment synthesis was enhanced under high C:N ratios (25:1) and it was dependent of the carbon source, being maximized when we added glucose to M9. Besides, the supplementation of M9 with tryptophan inhibited the pigment production under C:N ratios of 4:1, and the addition of kojic acid reduced notably the pigment under favorable conditions. Ps. black presented several traits associated with plant-growth promoting potential with classical in vitro assays. Through a Tn5 mutagenesis approach, we found 2 representative clones, PB1 and PB5, that were consistently unable to produce the pigment under several growth conditions and were not altered in their in vitro probiotic traits. When comparing with PB1 and PB5 performances, we observed that the pigment gives Ps. black a higher tolerance to oxidative stress and UV radiation exposure. When confronting Ps. black with different bacterial phytopathogens, we demonstrated that Ps. black could inhibit in vitro the growth of Xanthomonas vesicatoria Bv5-4a, Pseudomonas syringae pv. tomato DC3000, P. syringae pv. syringae B728a, P. savastanoi pv. glycinea B076 and Clavibacter michiganensis subsp. michiganensis Cm9. Except for Psg B076, this antagonism was lost for PB1 and PB5 and when performing the test for Ps. black with tryptophan supplementation. Thus, we suggest that the pigment should be involved in the bacterial antagonisms, and that Ps black contains more than one antibacterial mechanism.

Escherichia coli O157:H7 Curli Fimbriae Promotes Biofilm Formation, Epithelial Cell Invasion, and Persistence in Cattle.

Escherichia coli O157:H7 (O157) is noninvasive and a weak biofilm producer; however, a subset of O157 are exceptions. O157 ATCC 43895 forms biofilms and invades epithelial cells. Tn5 mutagenesis identified a mutation responsible for both phenotypes. The insertion mapped within the curli csgB fimbriae locus. Screening of O157 strains for biofilm formation and cell invasion identified a bovine and a clinical isolate with those characteristics. A single base pair A to T transversion, intergenic to the curli divergent operons csgDEFG and csgBAC, was present only in biofilm-producing and invasive strains. Using site-directed mutagenesis, this single base change was introduced into two curli-negative/noninvasive O157 strains and modified strains to form biofilms, produce curli, and gain invasive capability. Transmission electron microscopy (EM) and immuno-EM confirmed curli fibers. EM of bovine epithelial cells (MAC-T) co-cultured with curli-expressing O157 showed intracellular bacteria. The role of curli in O157 persistence in cattle was examined by challenging cattle with curli-positive and -negative O157 and comparing carriage. The duration of bovine colonization with the O157 curli-negative mutant was shorter than its curli-positive isogenic parent. These findings definitively demonstrate that a single base pair stably confers biofilm formation, epithelial cell invasion, and persistence in cattle.

Transposon mutagenesis in Mycobacterium kansasii links a small RNA gene to colony morphology and biofilm formation and identifies 9,885 intragenic insertions that do not compromise colony outgrowth.

Mycobacterium kansasii (Mk) is a resilient opportunistic human pathogen that causes tuberculosis‐like chronic pulmonary disease and mortality stemming from comorbidities and treatment failure. The standard treatment of Mk infections requires costly, long‐term, multidrug courses with adverse side effects. The emergence of drug‐resistant isolates further complicates the already challenging drug therapy regimens and threatens to compromise the future control of Mk infections. Despite the increasingly recognized global burden of Mk infections, the biology of this opportunistic pathogen remains essentially unexplored. In particular, studies reporting gene function or generation of defined mutants are scarce. Moreover, no transposon (Tn) mutagenesis tool has been validated for use in Mk, a situation limiting the repertoire of genetic approaches available to accelerate the dissection of gene function and the generation of gene knockout mutants in this poorly characterized pathogen. In this study, we validated the functionality of a powerful Tn mutagenesis tool in Mk and used this tool in conjunction with a forward genetic screen to establish a previously unrecognized role of a conserved mycobacterial small RNA gene of unknown function in colony morphology features and biofilm formation. We also combined Tn mutagenesis with next‐generation sequencing to identify 12,071 Tn insertions that do not compromise viability in vitro. Finally, we demonstrated the susceptibility of the Galleria mellonella larva to Mk, setting the stage for further exploration of this simple and economical infection model system to the study of this pathogen.

7-hydroxytropolone is the main metabolite responsible for the fungal antagonism of Pseudomonas donghuensis strain SVBP6.

Pseudomonas donghuensis strain SVBP6, an isolate from an agricultural plot in Argentina, displays a broad-spectrum and diffusible antifungal activity, which requires a functional gacS gene but could not be ascribed yet to known secondary metabolites typical of Pseudomonas biocontrol species. Here, we report that Tn5 mutagenesis allowed the identification of a gene cluster involved in both the fungal antagonism and the production of a soluble tropolonoid compound. The ethyl acetate extract from culture supernatant showed a dose-dependent inhibitory effect against the phytopathogenic fungus Macrophomina phaseolina. The main compound present in the organic extract was identified by spectroscopic and X-ray analyses as 7-hydroxytropolone (7HT). Its structure and tautomerism was confirmed by preparing the two key derivatives 2,3-dimethoxy- and 2,7-dimethoxy-tropone. 7HT, but not 2,3- or 2,7-dimethoxy-tropone, mimicked the fungal inhibitory activity of the ethyl acetate extract from culture supernatant. The activity of 7HT, as well as its production, was barely affected by the presence of up to 50 μM added iron (Fe+2 ). To summarize, P. donghuensis SVBP6 produces 7HT under the positive control of the Gac-Rsm cascade and is the main active metabolite responsible for the broad-spectrum inhibition of different phytopathogenic fungi.

High-throughput analysis of genes involved in biocontrol performance of Pseudomonas fluorescens NBC275 against Gray mold.

Many physiological and microbial characteristics influence the biocontrol performance of the biological control agents (BCAs) in agricultural fields. To implement effective biocontrol, the contribution of specific genes, mechanisms and traits to the biocontrol performance of BCAs need to be characterized and explored in greater detail. In this study, a transposon (Tn) mutant library using the BCA Pseudomonas fluorescens NBC275 (Pf275) was generated to explore genes and bacterial characteristics involved in antifungal activity and biocontrol performance. Among the Tn mutants, 205 strains showing variations in antifungal activity compared to wild-type (WT) were selected and further analysed for biocontrol efficacy against gray mold in pepper fruits. The genes involved in pyoverdine biosynthesis (pvdI and pvdD) and chitin-binding protein (gbpA) played essential roles in the antifungal activity and biocontrol capacity of Pf275. In addition, a mutation in phlD completely abolished the antifungal activity and significantly suppressed the biocontrol ability of the strain. Genes affecting antifungal activity of Pf275 significantly influenced swimming motility, which was identified as an important trait for the biocontrol ability of the bacterial strain. Overall, our results suggest that antifungal compound production, siderophore biosynthesis and swimming motility synergistically contribute to Pf275 biocontrol performance. The utility of this library was demonstrated by identifying genes for antagonism and biocontrol ability in this BCA strain. The functional roles of many genes identified as contributing to antagonism and in vivo biocontrol activity require further study. Genes contributing to antifungal activity and biocontrol performance of P. fluorescens were identified and highlighted by Tn mutagenesis, which will give insight to improve the biocontrol performance of this BCA.

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.

Isolation, characterization, genomic sequencing, and GFP-marked insertional mutagenesis of a high-performance nitrogen-fixing bacterium, Kosakonia radicincitans GXGL-4A and visualization of bacterial colonization on cucumber roots.

A gram-negative bacterium GXGL-4A was originally isolated from maize roots. It displayed nitrogen-fixing (NF) ability under nitrogen-free culture condition, and had a significant promotion effect on cucumber growth in the pot inoculation test. The preliminary physiological and biochemical traits of GXGL-4A were characterized. Furthermore, a phylogenetic tree was constructed based on 16S ribosomal DNA (rDNA) sequences of genetically related species. To determine the taxonomic status of GXGL-4A and further utilize its nitrogen-fixing potential, genome sequence was obtained using PacBio RS II technology. The analyses of average nucleotide identity based on BLAST+ (ANIb) and correlation indexes of tetra-nucleotide signatures (Tetra) showed that the NF isolate GXGL-4A is closely related to the Kosakonia radicincitans type strain DSM 16656. Therefore, the isolate GXGL-4A was eventually classified into the species of Kosakonia radicincitans and designated K. radicincitans GXGL-4A. A high consistency in composition and gene arrangement of nitrogen-fixing gene cluster I (nif cluster I) was found between K. radicincitans GXGL-4A and other Kosakonia NF strains. The mutants tagged with green fluorescence protein (GFP) were obtained by transposon Tn5 mutagenesis, and then, the colonization of gfp-marked K. radicincitans GXGL-4A cells on cucumber seedling root were observed under fluorescence microscopy. The preferential sites of the labeled GXGL-4A cell population were the lateral root junctions, the differentiation zone, and the elongation zone. All these results should benefit for the deep exploration of nitrogen fixation mechanism of K. radicincitans GXGL-4A and will definitely facilitate the genetic modification process of this NF bacterium in sustainable agriculture.

Genomic insights into the broad antifungal activity, plant-probiotic properties, and their regulation, in Pseudomonas donghuensis strain SVBP6.

Plant-growth promotion has been linked to the Pseudomonas genus since the beginning of this research field. In this work, we mined the genome of an Argentinean isolate of the recently described species P. donghuensis. Strain SVBP6, isolated from bulk soil of an agricultural plot, showed a broad antifungal activity and several other plant-probiotic activities. As this species has been recently described, and it seems like some plant-growth promoting (PGP) traits do not belong to the classical pseudomonads toolbox, we decide to explore the SVBP6 genome via an bioinformatic approach. Genome inspection confirmed our previous in vitro results about genes involved in several probiotic activities. Other genetic traits possibly involved in survival of SVBP6 in highly competitive environments, such as rhizospheres, were found. Tn5 mutagenesis revealed that the antifungal activity against the soil pathogen Macrophomina phaseolina was dependent on a functional gacS gene, from the regulatory cascade Gac-Rsm, but it was not due to volatile compounds. Altogether, our genomic analyses and in vitro tests allowed the phylogenetic assignment and provided the first insights into probiotic properties of the first P. donghuensis isolate from the Americas.

Novel genes associated with enhanced motility of Escherichia coli ST131.

Uropathogenic Escherichia coli (UPEC) is the cause of ~75% of all urinary tract infections (UTIs) and is increasingly associated with multidrug resistance. This includes UPEC strains from the recently emerged and globally disseminated sequence type 131 (ST131), which is now the dominant fluoroquinolone-resistant UPEC clone worldwide. Most ST131 strains are motile and produce H4-type flagella. Here, we applied a combination of saturated Tn5 mutagenesis and transposon directed insertion site sequencing (TraDIS) as a high throughput genetic screen and identified 30 genes associated with enhanced motility of the reference ST131 strain EC958. This included 12 genes that repress motility of E. coli K-12, four of which (lrhA, ihfA, ydiV, lrp) were confirmed in EC958. Other genes represented novel factors that impact motility, and we focused our investigation on characterisation of the mprA, hemK and yjeA genes. Mutation of each of these genes in EC958 led to increased transcription of flagellar genes (flhD and fliC), increased expression of the FliC flagellin, enhanced flagella synthesis and a hyper-motile phenotype. Complementation restored all of these properties to wild-type level. We also identified Tn5 insertions in several intergenic regions (IGRs) on the EC958 chromosome that were associated with enhanced motility; this included flhDC and EC958_1546. In both of these cases, the Tn5 insertions were associated with increased transcription of the downstream gene(s), which resulted in enhanced motility. The EC958_1546 gene encodes a phage protein with similarity to esterase/deacetylase enzymes involved in the hydrolysis of sialic acid derivatives found in human mucus. We showed that over-expression of EC958_1546 led to enhanced motility of EC958 as well as the UPEC strains CFT073 and UTI89, demonstrating its activity affects the motility of different UPEC strains. Overall, this study has identified and characterised a number of novel factors associated with enhanced UPEC motility.

Characterization of the Pathogenicity of Streptococcus intermedius TYG1620 Isolated from a Human Brain Abscess Based on the Complete Genome Sequence with Transcriptome Analysis and Transposon Mutagenesis in a Murine Subcutaneous Abscess Model.

Streptococcus intermedius is known to cause periodontitis and pyogenic infections in the brain and liver. Here we report the complete genome sequence of strain TYG1620 (genome size, 2,006,877 bp; GC content, 37.6%; 2,020 predicted open reading frames [ORFs]) isolated from a brain abscess in an infant. Comparative analysis of S. intermedius genome sequences suggested that TYG1620 carries a notable type VII secretion system (T7SS), two long repeat regions, and 19 ORFs for cell wall-anchored proteins (CWAPs). To elucidate the genes responsible for the pathogenicity of TYG1620, transcriptome analysis was performed in a murine subcutaneous abscess model. The results suggest that the levels of expression of small hypothetical proteins similar to phenol-soluble modulin β1 (PSMβ1), a staphylococcal virulence factor, significantly increased in the abscess model. In addition, an experiment in a murine subcutaneous abscess model with random transposon (Tn) mutant attenuation suggested that Tn mutants with mutations in 212 ORFs in the Tn mutant library were attenuated in the murine abscess model (629 ORFs were disrupted in total); the 212 ORFs are putatively essential for abscess formation. Transcriptome analysis identified 37 ORFs, including paralogs of the T7SS and a putative glucan-binding CWAP in long repeat regions, to be upregulated and attenuated in vivo This study provides a comprehensive characterization of S. intermedius pathogenicity based on the complete genome sequence and a murine subcutaneous abscess model with transcriptome and Tn mutagenesis, leading to the identification of pivotal targets for vaccines or antimicrobial agents for the control of S. intermedius infections.

Essential Genes for In Vitro Growth of the Endophyte Herbaspirillum seropedicae SmR1 as Revealed by Transposon Insertion Site Sequencing.

ABSTRACTThe interior of plants contains microorganisms (referred to as endophytes) that are distinct from those present at the root surface or in the surrounding soil. Herbaspirillum seropedicae strain SmR1, belonging to the betaproteobacteria, is an endophyte that colonizes crops, including rice, maize, sugarcane, and sorghum. Different approaches have revealed genes and pathways regulated during the interactions of H. seropedicae with its plant hosts. However, functional genomic analysis of transposon (Tn) mutants has been hampered by the lack of genetic tools. Here we successfully employed a combination of in vivo high-density mariner Tn mutagenesis and targeted Tn insertion site sequencing (Tn-seq) in H. seropedicae SmR1. The analysis of multiple gene-saturating Tn libraries revealed that 395 genes are essential for the growth of H. seropedicae SmR1 in tryptone-yeast extract medium. A comparative analysis with the Database of Essential Genes (DEG) showed that 25 genes are uniquely essential in H. seropedicae SmR1. The Tn mutagenesis protocol developed and the gene-saturating Tn libraries generated will facilitate elucidation of the genetic mechanisms of the H. seropedicae endophytic lifestyle.IMPORTANCE A focal point in the study of endophytes is the development of effective biofertilizers that could help to reduce the input of agrochemicals in croplands. Besides the ability to promote plant growth, a good biofertilizer should be successful in colonizing its host and competing against the native microbiota. By using a systematic Tn-based gene-inactivation strategy and massively parallel sequencing of Tn insertion sites (Tn-seq), it is possible to study the fitness of thousands of Tn mutants in a single experiment. We have applied the combination of these techniques to the plant-growth-promoting endophyte Herbaspirillum seropedicae SmR1. The Tn mutant libraries generated will enable studies into the genetic mechanisms of H. seropedicae-plant interactions. The approach that we have taken is applicable to other plant-interacting bacteria.

Involvement of type VI secretion system in secretion of iron chelator pyoverdine in Pseudomonas taiwanensis.

Rice bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive rice diseases worldwide. Therefore, in addition to breeding disease-resistant rice cultivars, it is desirable to develop effective biocontrol agents against Xoo. Here, we report that a soil bacterium Pseudomonas taiwanensis displayed strong antagonistic activity against Xoo. Using matrix-assisted laser desorption/ionization imaging mass spectrometry, we identified an iron chelator, pyoverdine, secreted by P. taiwanensis that could inhibit the growth of Xoo. Through Tn5 mutagenesis of P. taiwanensis, we showed that mutations in genes that encode components of the type VI secretion system (T6SS) as well as biosynthesis and maturation of pyoverdine resulted in reduced toxicity against Xoo. Our results indicated that T6SS is involved in the secretion of endogenous pyoverdine. Mutations in T6SS component genes affected the secretion of mature pyoverdine from the periplasmic space into the extracellular medium after pyoverdine precursor is transferred to the periplasm by the inner membrane transporter PvdE. In addition, we also showed that other export systems, i.e., the PvdRT-OpmQ and MexAB-OprM efflux systems (for which there have been previous suggestions of involvement) and the type II secretion system (T2SS), are not involved in pyoverdine secretion.

Isolation of salt-tolerant mutants of Mesorhizobium ciceri strain Rch125 and identification of genes involved in salt sensitivity

Mesorhizobium ciceri Rch125 is a salt-sensitive strain isolated from root nodules of chickpea (Cicer arietinum L.). The aim of this work was to investigate the genes responsible for the sensitivity to salinity. Twelve Rch125 salt-tolerant mutants were isolated after random Tn5 mutagenesis and selected using a medium containing 300 mM NaCl, where growth of the wild-type is totally inhibited. In addition to this NaCl tolerance, the mutants also displayed higher tolerance to LiCl, CaCl2 and sucrose. Genes that were disrupted in the salt-tolerant mutants were in one of three functional categories: membrane transporters, outer membrane proteins, and genes of unknown function. Genetic complementation experiments demonstrated that the genes identified were involved in the salt sensitivity of the Rch125 strain. In most cases, disruption of the salt-sensitivity genes did not negatively affect the free-living or the symbiotic capabilities of Rch125 under non-saline conditions.

Important contribution of the novel locus comEB to extracellular DNA-dependent Staphylococcus lugdunensis biofilm formation.

The coagulase-negative species Staphylococcus lugdunensis is an emerging cause of serious and potentially life-threatening infections, such as infective endocarditis. The pathogenesis of these infections is characterized by the ability of S. lugdunensis to form biofilms on either biotic or abiotic surfaces. To elucidate the genetic basis of biofilm formation in S. lugdunensis, we performed transposon (Tn917) mutagenesis. One mutant had a significantly reduced biofilm-forming capacity and carried a Tn917 insertion within the competence gene comEB. Site-directed mutagenesis and subsequent complementation with a functional copy of comEB verified the importance of comEB in biofilm formation. In several bacterial species, natural competence stimulates DNA release via lysis-dependent or -independent mechanisms. Extracellular DNA (eDNA) has been demonstrated to be an important structural component of many bacterial biofilms. Therefore, we quantified the eDNA in the biofilms and found diminished eDNA amounts in the comEB mutant biofilm. High-resolution images and three-dimensional data obtained via confocal laser scanning microscopy (CSLM) visualized the impact of the comEB mutation on biofilm integrity. The comEB mutant did not show reduced expression of autolysin genes, decreased autolytic activities, or increased cell viability, suggesting a cell lysis-independent mechanism of DNA release. Furthermore, reduced amounts of eDNA in the comEB mutant biofilms did not result from elevated levels or activity of the S. lugdunensis thermonuclease NucI. In conclusion, we defined here, for the first time, a role for the competence gene comEB in staphylococcal biofilm formation. Our findings indicate that comEB stimulates biofilm formation via a lysis-independent mechanism of DNA release.

Merging genome mining with ancient three-way species co-evolution may open a novel gate for informative natural products discovery

Finger millet is an ancient African cereal crop, resistant to many pathogens. It was domesticated 7000 years ago in Ethiopia, reaching India by 3000 BC. Unlike other cereals, finger millet is resistant to the toxigenic fungal pathogen Fusarium graminearum. As this fungus is also ancient to Africa, we hypothesized that the crop may host microbes (endophytes) that co-evolved to combat Fusarium. Here we describe the first ever discovery of endophytes from finger millet. We report a diverse array of anti-Fusarium secondary metabolites isolated from the fungal endophytes. A bacterial endophyte (strain M6) showed potent broad-spectrum antifungal activity including against F.graminearum, and was subsequently shown to be a novel Enterobacter species. Confocal microscopy showed that GFP-tagged M6 colonized different cereal tissues. Mechanistically, M6 caused cleavage of F.graminearum hyphae at septa. At the molecular level, Tn5 mutagenesis aided by whole genome sequencing and gene annotation uncovered 10 operons responsible for the anti-fungal trait, including at least two phenazine biosynthetic ORFs. Real time PCR revealed that most of the candidate genes are inducible by F.graminearum. Knockouts of the genes caused loss of cereal resistance to F.graminearum. Biochemically, three new phenazine derivatives were discovered for the first time. We conclude, informative search for bioactive compounds opens a new revolutionary era for natural products discovery.

Rhizobium Mutant Deficient in Mineral Phosphate Solubilization Activity Shows Reduced Nodulation and Plant Growth in Green Gram

The aim of the present study was to understand the mineral phosphate solubilization (MPS) in Rhizobium sp. MR-54 (MPS+) by using forward genetic approach and its effect on nodulation as well as nitrogen accumulation on green gram. Tn5 mutants (MPS−) were generated from the MPS+ strain by Tn5 mutagenesis. Screening 1,603 colonies of MR-54::Tn5 resulted in identification of those mutants which failed to show any solubilization zone up to 72 h after incubation on Tri-calcium phosphate medium (TCP). pH drops and Pi release were highly correlated up to 15 days after incubation in TCP broth. Gluconic acid was detected in the culture supernatants of MPS+ isolates through thin layer chromatography indicating the presence of direct oxidation pathway in MR-54 strain. Detailed characterization of mutants was carried out for related properties like colony morphology, Pi release and pH change in TCP broth, organic acid production, surface characteristics and plant growth promoting ability etc. Mutants deficient in MPS showed reduced nodulation and nitrogen accumulation in green gram under green house conditions in leonard jar apparatus over the wild strain indicating the MPS by Rhizobium being involved in nodulation, nitrogen accumulation and plant growth.