Pseudomonas fortuita sp. nov., isolated from the endosphere of a wild yam.

A Gram-negative, strictly aerobic bacterial strain was isolated from asymptomatic leaf tissue of a wild yam plant. Optimal growth was observed at 28 °C and pH 7, and catalase and oxidase activities were detected. Polyphasic taxonomic and comparative genomics revealed that strain LMG 33091T represents a novel species of Pseudomonas. The nearest phylogenetic neighbours of strain LMG 33091T were Pseudomonas putida NBRC 14164T (with 99.79 % 16S rRNA sequence identity), Pseudomonas alkylphenolica KL28T (99.28 %) and Pseudomonas asplenii (99.07 %) ATCC 23835T. MALDI-TOF MS analysis yielded distinct profiles for strain LMG 33091T and the nearest phylogenetic neighbours. Average nucleotide identity analyses between the whole genome sequence of strain LMG 33091T and of the type strains of its nearest-neighbour taxa yielded values below the species delineation threshold and thus confirmed that the strain represented a novel Pseudomonas species, for which we propose the name Pseudomonas fortuita sp. nov., with strain LMG 33091T (=GMI12077T= CFBP 9143T) as the type strain.

Cyclitol metabolism is a central feature of Burkholderia leaf symbionts.

The symbioses between plants of the Rubiaceae and Primulaceae families with Burkholderia bacteria represent unique and intimate plant-bacterial relationships. Many of these interactions have been identified through PCR-dependent typing methods, but there is little information available about their functional and ecological roles. We assembled 17 new endophyte genomes representing endophytes from 13 plant species, including those of two previously unknown associations. Genomes of leaf endophytes belonging to Burkholderia s.l. show extensive signs of genome reduction, albeit to varying degrees. Except for one endophyte, none of the bacterial symbionts could be isolated on standard microbiological media. Despite their taxonomic diversity, all endophyte genomes contained gene clusters linked to the production of specialized metabolites, including genes linked to cyclitol sugar analog metabolism and in one instance non-ribosomal peptide synthesis. These genes and gene clusters are unique within Burkholderia s.l. and are likely horizontally acquired. We propose that the acquisition of secondary metabolite gene clusters through horizontal gene transfer is a prerequisite for the evolution of a stable association between these endophytes and their hosts.

Rhizobium leguminosarum symbiovar viciae strains are natural wheat endophytes that can stimulate root development.

Although rhizobia that establish a nitrogen-fixing symbiosis with legumes are also known to promote growth in non-legumes, studies on rhizobial associations with wheat roots are scarce. We searched for Rhizobium leguminosarum symbiovar viciae (Rlv) strains naturally competent to endophytically colonize wheat roots. We isolated 20 strains from surface-sterilized wheat roots and found a low diversity of Rlv compared to that observed in the Rlv species complex. We tested the ability of a subset of these Rlv for wheat root colonization when co-inoculated with other Rlv. Only a few strains, including those isolated from wheat roots, and one strain isolated from pea nodules, were efficient in colonizing roots in co-inoculation conditions, while all the strains tested in single strain inoculation conditions were found to colonize the surface and interior of roots. Furthermore, Rlv strains isolated from wheat roots were able to stimulate root development and early arbuscular mycorrhizal fungi colonization. These responses were strain and host genotype dependent. Our results suggest that wheat can be an alternative host for Rlv; nevertheless, there is a strong competition between Rlv strains for wheat root colonization. In addition, we showed that Rlv are endophytic wheat root bacteria with potential ability to modify wheat development.

Induction of antibiotic specialized metabolism by co-culturing in a collection of phyllosphere bacteria.

A diverse set of bacteria live on the above-ground parts of plants, composing the phyllosphere, and play important roles for plant health. Phyllosphere microbial communities assemble in a predictable manner and diverge from communities colonizing other plant organs or the soil. However, how these communities differ functionally remains obscure. We assembled a collection of 258 bacterial isolates representative of the most abundant taxa of the phyllosphere of Arabidopsis and a shared soil inoculum. We screened the collection for the production of metabolites that inhibit the growth of Gram-positive and Gram-negative bacteria either in isolation or in co-culture. We found that isolates capable of constitutive antibiotic production in monoculture were significantly enriched in the soil fraction. In contrast, the proportion of binary cultures resulting in the production of growth inhibitory compounds differed only marginally between the phyllosphere and soil fractions. This shows that the phyllosphere may be a rich resource for potentially novel molecules with antibiotic activity, but that production or activity is dependent upon induction by external signals or cues. Finally, we describe the isolation of antimicrobial acyloin metabolites from a binary culture of Arabidopsis phyllosphere isolates, which inhibit the growth of clinically relevant Acinetobacter baumannii.

PaSiT: a novel approach based on short-oligonucleotide frequencies for efficient bacterial identification and typing.

MOTIVATION: One of the most widespread methods used in taxonomy studies to distinguish between strains or taxa is the calculation of average nucleotide identity. It requires a computationally expensive alignment step and is therefore not suitable for large-scale comparisons. Short oligonucleotide-based methods do offer a faster alternative but at the expense of accuracy. Here, we aim to address this shortcoming by providing a software that implements a novel method based on short-oligonucleotide frequencies to compute inter-genomic distances. RESULTS: Our tetranucleotide and hexanucleotide implementations, which were optimized based on a taxonomically well-defined set of over 200 newly sequenced bacterial genomes, are as accurate as the short oligonucleotide-based method TETRA and average nucleotide identity, for identifying bacterial species and strains, respectively. Moreover, the lightweight nature of this method makes it applicable for large-scale analyses. AVAILABILITY AND IMPLEMENTATION: The method introduced here was implemented, together with other existing methods, in a dependency-free software written in C, GenDisCal, available as source code from https://github.com/LM-UGent/GenDisCal. The software supports multithreading and has been tested on Windows and Linux (CentOS). In addition, a Java-based graphical user interface that acts as a wrapper for the software is also available. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Paenibacillus foliorum sp. nov., Paenibacillus phytohabitans sp. nov., Paenibacillus plantarum sp. nov., Paenibacillus planticolens sp. nov., Paenibacillus phytorum sp. nov. and Paenibacillus germinis sp. nov., isolated from the Arabidopsis thaliana phyllosphere.

Six endospore-forming, Gram-stain-positive or variable, motile, rod-shaped, aerobic or facultatively anaerobic bacteria with different MALDI-TOF mass spectra (MS) were isolated from the phyllosphere of Arabidopsis thaliana plants grown in plant chambers after inoculation of surface sterilized seeds with a top soil microbial cell suspension. They were identified as members of the genus Paenibacillus through comparison with a commercial MALDI-TOF MS database and comparative 16S rRNA gene sequencing. Their genome sequences comprised multiple biosynthetic gene clusters and suggested they have unexplored biotechnological potential. Analyses of average nucleotide identity values between these strains and the type strains of their nearest neighbour species demonstrated that they represented a novel Paenibacillus species each. A detailed phenotypic comparison yielded distinctive biochemical characteristics for each of these novel species. We therefore propose to classify that these isolates into six novel species within genus Paenibacillus, for which we propose the names Paenibacillus foliorum sp. nov., Paenibacillus phytohabitans sp. nov., Paenibacillus plantarum sp. nov., Paenibacillus planticolens sp. nov., Paenibacillus phytorum sp. nov. and Paenibacillus germinis sp. nov., with strains LMG 31456T (=R-74617T=CECT 30138T), LMG 31459T (=R-74621T=CECT 30135T), LMG 31461T (=R-74618T=CECT 30133T), LMG 31457T (=R-74619T=CECT 30137T), LMG 31458T (=R-74620T=CECT 30136T) and LMG 31460T (=R-74622T=CECT 30134T) as the type strains, respectively.

Orrella amnicola sp. nov., isolated from a freshwater river, reclassification of Algicoccus marinus as Orrella marina comb. nov., and emended description of the genus Orrella.

A novel Gram-negative, aerobic, non-motile, ovoid to rod-shaped bacterium, designated NBD-18T, was isolated from a freshwater river in Taiwan. Optimal growth occurred at 30 °C, at pH 6 and in the absence of NaCl. The predominant fatty acids of strain NBD-18T were C16 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C17 : 0 cyclo and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and phosphatidyldimethylethanolamine. The major polyamine was putrescine. The major isoprenoid quinone was Q-8. The genomic DNA G+C content of strain NBD-18T was 50.9 %. Strain NBD-18T was most closely related to Orrella dioscoreae LMG 29303T and Algicoccus marinus HZ20T at a 16S rRNA gene sequence similarity of 97.7 %. 16S rRNA gene sequence similarity between O. dioscoreae LMG 29303T and A. marinus HZ20T was 97.7 %. Phylogenetic analyses based on 16S rRNA gene sequences and an up-to-date bacterial core gene set indicated that strain NBD-18T, O. dioscoreae LMG 29303T and A. marinus HZ20T are affiliated with the same genus. Digital DNA-DNA hybridization, average nucleotide identity and average amino acid identity values among these three strains supported that they belong to the same genus and that strain NBD-18T represents a novel species. Thus, A. marinus HZ20T should be reclassified as Orrella marina comb. nov. based on the rules for priority of publication and validation. On the basis of the genotypic, chemotaxonomic and phenotypic data, strain NBD-18T represents a novel species in the genus Orrella, for which the name Orrella amnicola sp. nov. is proposed. The type strain is NBD-18T (=BCRC 81197T=LMG 31338T).

Synthesis and Biological Evaluation of the Novel Growth Inhibitor Streptol Glucoside, Isolated from an Obligate Plant Symbiont.

The plant Psychotria kirkii hosts an obligatory bacterial symbiont, Candidatus Burkholderia kirkii, in nodules on their leaves. Recently, a glucosylated derivative of (+)-streptol, (+)-streptol glucoside, was isolated from the nodulated leaves and was found to possess a plant growth inhibitory activity. To establish a structure-activity relationship study, a convergent strategy was developed to obtain several pseudosugars from a single synthetic precursor. Furthermore, the glucosylation of streptol was investigated in detail and conditions affording specifically the α or ß glucosidic anomer were identified. Although (+)-streptol was the most active compound, its concentration in P. kirkii plant leaves extract was approximately ten-fold lower than that of (+)-streptol glucoside. These results provide compelling evidence that the glucosylation of (+)-streptol protects the plant host against the growth inhibitory effect of the compound, which might constitute a molecular cornerstone for this successful plant-bacteria symbiosis.

Pedobacter gandavensis sp. nov., Pedobacter foliorum sp. nov. and Pedobacter planticolens sp. nov., isolated from leaves of Arabidopsis thaliana.

Three rod-shaped, non-spore-forming, yellow or pale-yellow pigmented bacteria with distinct MALDI-TOF mass spectra were isolated from the phyllosphere of Arabidopsis thaliana seedlings. Their 16S rRNA gene sequences demonstrated that these isolates belong to the genus Pedobacter. The nearest phylogenetic neighbours of strain LMG 31462T were Pedobacter steynii DSM 19110T (98.3 % 16S rRNA sequence similarity) and Pedobacter caeni LMG 22862T (98.3 %); the nearest phylogenetic neighbours of strain LMG 31463T were Pedobacter panaciterrae Gsoil 042T (98.3 %) and Pedobacter nutrimenti DSM 27372T (98.1 %); and the nearest phylogenetic neighbours of strain LMG 31464T were Pedobacter boryungensis BR-9T (99.0 %) and Pedobacter daejeonensis THG-DN3.18T (98.7 %). Average nucleotide identity analyses between the whole genome sequences of the three strains and of the type strains of their respective nearest-neighbour taxa yielded values well below the species delineation threshold and thus confirmed that the three strains represented a novel Pedobacter species each. An extensive phenotypic comparison and an analysis of whole-cell fatty acid components yielded distinctive phenotypic characteristics for each of these strains. We therefore propose to classify these isolates as three novel species, for which we propose the names Pedobacter gandavensis with LMG 31462T (=R-74704T=CECT 30149T) as the type strain, Pedobacter foliorum with LMG 31463T (=R-74623T=CECT 30150T) as the type strain and Pedobacter planticolens with LMG 31464T (=R-74626T=CECT 30151T) as the type strain.

Heterologous Expression, Biosynthetic Studies, and Ecological Function of the Selective Gq-Signaling Inhibitor FR900359.

The cyclic depsipeptide FR900359 (FR), isolated from the tropical plant Ardisia crenata, is a strong and selective inhibitor of Gq proteins, making it an indispensable pharmacological tool to study Gq-related processes, as well as a promising drug candidate. Gq inhibition is a novel mode of action for defense chemicals and crucial for the ecological function of FR, as shown by in vivo experiments in mice, its affinity to insect Gq proteins, and insect toxicity studies. The uncultured endosymbiont of A. crenata was sequenced, revealing the FR nonribosomal peptide synthetase (frs) gene cluster. We here provide a detailed model of FR biosynthesis, supported by in vitro enzymatic and bioinformatic studies, and the novel analogue AC-1, which demonstrates the flexibility of the FR starter condensation domains. Finally, expression of the frs genes in E. coli led to heterologous FR production in a cultivable, bacterial host for the first time.

The Essential Genome of Burkholderia cenocepacia H111.

The study of the minimum set of genes required to sustain life is a fundamental question in biological research. Recent studies on bacterial essential genes suggested that between 350 and 700 genes are essential to support autonomous bacterial cell growth. Essential genes are of interest as potential new antimicrobial drug targets; hence, our aim was to identify the essential genome of the cystic fibrosis (CF) isolate Burkholderia cenocepacia H111. Using a transposon sequencing (Tn-Seq) approach, we identified essential genes required for growth in rich medium under aerobic and microoxic conditions as well as in a defined minimal medium with citrate as a sole carbon source. Our analysis suggests that 398 genes are required for autonomous growth in rich medium, a number that represents only around 5% of the predicted genes of this bacterium. Five hundred twenty-six genes were required to support growth in minimal medium, and 434 genes were essential under microoxic conditions (0.5% O2). A comparison of these data sets identified 339 genes that represent the minimal set of essential genes required for growth under all conditions tested and can be considered the core essential genome of B. cenocepacia H111. The majority of essential genes were found to be located on chromosome 1, and few such genes were located on chromosome 2, where most of them were clustered in one region. This gene cluster is fully conserved in all Burkholderia species but is present on chromosome 1 in members of the closely related genus Ralstonia, suggesting that the transfer of these essential genes to chromosome 2 in a common ancestor contributed toward the separation of the two genera.IMPORTANCE Transposon sequencing (Tn-Seq) is a powerful method used to identify genes that are essential for autonomous growth under various conditions. In this study, we have identified a set of "core essential genes" that are required for growth under multiple conditions, and these genes represent potential antimicrobial targets. We also identified genes specifically required for growth under low-oxygen and nutrient-limited environments. We generated conditional mutants to verify the results of our Tn-Seq analysis and demonstrate that one of the identified genes was not essential per se but was an artifact of the construction of the mutant library. We also present verified examples of genes that were not truly essential but, when inactivated, showed a growth defect. These examples have identified so-far-underestimated shortcomings of this powerful method.

The genome analysis of Candidatus†Burkholderia crenata reveals that secondary metabolism may be a key function of the Ardisia crenata leaf nodule symbiosis.

A majority of Ardisia species harbour Burkholderia sp. bacteria within specialized leaf nodules. The bacteria are transmitted hereditarily and have not yet been cultured outside of their host. Because the plants cannot develop beyond the seedling stage without their symbionts, the symbiosis is considered obligatory. We sequenced for the first time the genome of Candidatus Burkholderia crenata (Ca. B. crenata), the leaf nodule symbiont of Ardisia crenata. The genome of Ca. B. crenata is the smallest Burkholderia genome to date. It contains a large amount of insertion sequences and pseudogenes and displays features consistent with reductive genome evolution. The genome does not encode functions commonly associated with plant symbioses such as nitrogen fixation and plant hormone metabolism. However, we identified unique genes with a predicted role in secondary metabolism in the genome of Ca. B. crenata. Specifically, we provide evidence that the bacterial symbionts are responsible for the synthesis of compound FR900359, a cyclic depsipeptide with biomedical properties previously isolated from leaves of A. crenata.

A novel siderophore-independent strategy of iron uptake in the genus Burkholderia.

Like many other bacteria, Burkholderia sp. take up iron in its ferric form via siderophore-dependent transporters. We observed that mutant strains of B. cenocepacia H111 unable to synthesize siderophores did not exhibit any growth defect under iron limited conditions. This finding suggested that this opportunistic pathogen can adopt an alternative iron uptake strategy to compensate for the loss of siderophores. We identified a putative iron uptake locus, ftrBcc ABCD, in the genome of B. cenocepacia H111, which is also conserved in other members of the genus Burkholderia. Mutants deficient in both siderophore-dependent and FtrBcc ABCD systems failed to grow under iron-limited conditions and radiolabelled iron transport assays showed that these mutants were impaired in iron uptake. In addition, expression of ftrBcc ABCD can restore growth of an E. coli strain lacking all known high-affinity iron transport systems under iron-limited conditions. We show that all four proteins encoded by ftrBcc ABCD are essential for iron uptake. Furthermore, our results indicate that the expression of ftrBcc ABCD is regulated at the transcriptional level by iron concentration. This study provides evidence of an alternative, siderophore-independent, iron uptake system in Burkholderia species.

Isolation and Total Synthesis of Kirkamide, an Aminocyclitol from an Obligate Leaf Nodule Symbiont.

The new C7N aminocyclitol kirkamide (1) was isolated from leaf nodules of the plant Psychotria kirkii by using a genome-driven (1)H NMR-guided fractionation approach. The structure and absolute configuration were elucidated by HRMS, NMR, and single-crystal X-ray crystallography. An enantioselective total synthesis was developed, which delivered kirkamide (1) on a gram scale in 11 steps and features a Ferrier carbocyclization and a Pd-mediated hydroxymethylation. We propose that kirkamide is synthesized by Candidatus Burkholderia kirkii, the obligate leaf symbiont of Psychotria kirkii. Kirkamide (1) was shown to be toxic to aquatic arthropods and insects, thus suggesting that bacterial secondary metabolites play a protective role in the Psychotria/Burkholderia leaf nodule symbiosis.

The third replicon of members of the Burkholderia cepacia Complex, plasmid pC3, plays a role in stress tolerance.

The metabolically versatile Burkholderia cepacia complex (Bcc) occupies a variety of niches, including the plant rhizosphere and the cystic fibrosis lung (where it is often fatal to the patient). Bcc members have multipartite genomes, of which the third replicon, pC3 (previously chromosome 3), has been shown to be a nonessential megaplasmid which confers virulence and both antifungal and proteolytic activity on several strains. In this study, pC3 curing was extended to cover strains of 16 of the 17 members of the Bcc, and the phenotypes conferred by pC3 were determined. B. cenocepacia strains H111, MCO-3, and HI2424 were previously cured of pC3; however, this had not proved possible in the epidemic strain K56-2. Here, we investigated the mechanism of this unexpected stability and found that efficient toxin-antitoxin systems are responsible for maintaining pC3 of strain K56-2. Identification of these systems allowed neutralization of the toxins and the subsequent deletion of K56-2pC3. The cured strain was found to exhibit reduced antifungal activity and was attenuated in both the zebrafish and the Caenorhabditis elegans model of infection. We used a PCR screening method to examine the prevalence of pC3 within 110 Bcc isolates and found that this replicon was absent in only four cases, suggesting evolutionary fixation. It is shown that plasmid pC3 increases the resistance of B. cenocepacia H111 to various stresses (oxidative, osmotic, high-temperature, and chlorhexidine-induced stresses), explaining the prevalence of this replicon within the Bcc.

Structures and roles of BcsD and partner scaffold proteins in proteobacterial cellulose secretion.

Cellulose is the world's most abundant biopolymer, and similar to its role as a cell wall component in plants, it is a prevalent constituent of the extracellular matrix in bacterial biofilms. Although bacterial cellulose (BC) was first described in the 19th century, it was only recently revealed that it is produced by several distinct types of Bcs secretion systems that feature multiple accessory subunits in addition to a catalytic BcsAB synthase tandem. We recently showed that crystalline cellulose secretion in the Gluconacetobacter genus (α-Proteobacteria) is driven by a supramolecular BcsH-BcsD scaffold-the "cortical belt"-which stabilizes the synthase nanoarrays through an unexpected inside-out mechanism for secretion system assembly. Interestingly, while bcsH is specific for Gluconacetobacter, bcsD homologs are widespread in Proteobacteria. Here, we examine BcsD homologs and their gene neighborhoods from several plant-colonizing ß- and γ-Proteobacteria proposed to secrete a variety of non-crystalline and/or chemically modified cellulosic polymers. We provide structural and mechanistic evidence that through different quaternary structure assemblies BcsD acts with proline-rich BcsH, BcsP, or BcsO partners across the proteobacterial clade to form synthase-interacting intracellular scaffolds that, in turn, determine the biofilm strength and architecture in species with strikingly different physiology and secreted biopolymers.

The association between Dioscorea sansibarensis and Orrella dioscoreae as a model for hereditary leaf symbiosis.

Hereditary, or vertically-transmitted, symbioses affect a large number of animal species and some plants. The precise mechanisms underlying transmission of functions of these associations are often difficult to describe, due to the difficulty in separating the symbiotic partners. This is especially the case for plant-bacteria hereditary symbioses, which lack experimentally tractable model systems. Here, we demonstrate the potential of the leaf symbiosis between the wild yam Dioscorea sansibarensis and the bacterium Orrella dioscoreae (O. dioscoreae) as a model system for hereditary symbiosis. O. dioscoreae is easy to grow and genetically manipulate, which is unusual for hereditary symbionts. These properties allowed us to design an effective antimicrobial treatment to rid plants of bacteria and generate whole aposymbiotic plants, which can later be re-inoculated with bacterial cultures. Aposymbiotic plants did not differ morphologically from symbiotic plants and the leaf forerunner tip containing the symbiotic glands formed normally even in the absence of bacteria, but microscopic differences between symbiotic and aposymbiotic glands highlight the influence of bacteria on the development of trichomes and secretion of mucilage. This is to our knowledge the first leaf symbiosis where both host and symbiont can be grown separately and where the symbiont can be genetically altered and reintroduced to the host.

Identification of Burkholderia cenocepacia strain H111 virulence factors using nonmammalian infection hosts.

Burkholderia cenocepacia H111, a strain isolated from a cystic fibrosis patient, has been shown to effectively kill the nematode Caenorhabditis elegans. We used the C. elegans model of infection to screen a mini-Tn5 mutant library of B. cenocepacia H111 for attenuated virulence. Of the approximately 5,500 B. cenocepacia H111 random mini-Tn5 insertion mutants that were screened, 22 showed attenuated virulence in C. elegans. Except for the quorum-sensing regulator cepR, none of the mutated genes coded for the biosynthesis of classical virulence factors such as extracellular proteases or siderophores. Instead, the mutants contained insertions in metabolic and regulatory genes. Mutants attenuated in virulence in the C. elegans infection model were also tested in the Drosophila melanogaster pricking model, and those also attenuated in this model were further tested in Galleria mellonella. Six of the 22 mutants were attenuated in D. melanogaster, and five of these were less pathogenic in the G. mellonella model. We show that genes encoding enzymes of the purine, pyrimidine, and shikimate biosynthesis pathways are critical for virulence in multiple host models of infection.

Proteomics analysis of Psychotria leaf nodule symbiosis: improved genome annotation and metabolic predictions.

Several plant species of the genus Psychotria (Rubiaceae) harbor Burkholderia sp. bacteria within specialized leaf nodules. The bacteria are transmitted vertically between plant generations and have not yet been cultured outside of their host. This symbiosis is considered to be obligatory because plants devoid of symbionts fail to develop into mature individuals. The genome of 'Candidatus Burkholderia kirkii' has been sequenced recently and has revealed evidence of reductive genome evolution, as shown by the proliferation of insertion sequences and the presence of numerous pseudogenes. We employed shotgun proteomics to investigate the expression of 'Ca. B. kirkii' proteins in the leaf nodule. Drawing from this dataset and refined comparative genomics analyses, we designed a new pseudogene prediction algorithm and improved the genome annotation. We also found conclusive evidence that nodule bacteria allocate vast resources to synthesis of secondary metabolites, possibly of the C7N aminocyclitol family. Expression of a putative 2-epi-5-valiolone synthase, a key enzyme of the C7N aminocyclitol synthesis, is high in the nodule population but downregulated in bacteria residing in the shoot apex, suggesting that production of secondary metabolites is particularly important in the leaf nodule.