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Fungus-associated Burkholderia gladioli bacteria use a unique ‘dual-priming’ nonribosomal peptide synthetase to assemble icosalide A1., Burkholderia is a multi-talented genus of Gram-negative bacteria, which in recent years has become increasingly recognised as a promising source of bioactive natural products. Metabolite profiling of Burkholderia gladioli BCC0238 showed that it produces the asymmetric lipopeptidiolide antibiotic icosalide A1, originally isolated from a fungus. Comparative bioinformatics analysis of several genome-sequenced B. gladioli isolates identified a gene encoding a nonribosomal peptide synthase (NRPS) with an unusual architecture that was predicted to be responsible for icosalide biosynthesis. Inactivation of this gene in B. gladioli BCC0238 abolished icosalide production. PCR analysis and sequencing of total DNA from the original fungal icosalide A1 producer revealed it has a B. gladioli strain associated with it that harbours an NRPS with an identical architecture to that responsible for icosalide A1 assembly in B. gladioli BCC0238. Sequence analysis of the icosalide NRPS indicated that it contains two chain-initiating condensation (CI) domains. One of these is appended to the N-terminus of module 1 – a common architecture for NRPSs involved in lipopeptide assembly. The other is embedded in module 3, immediately downstream of a putative chain-elongating condensation domain. Analysis of the reactions catalysed by a tridomain construct from module 3 of the NRPS using intact protein mass spectrometry showed that the embedded CI domain initiates assembly of a second lipopeptide chain, providing key insights into the mechanism for asymmetric diolide assembly.

Pseudomonas mesoacidophila ATCC 31433 is a Gram-negative bacterium, first isolated from Japanese soil samples, that produces the monobactam isosulfazecin and the β-lactam-potentiating bulgecins. To characterize the biosynthetic potential of P. mesoacidophila ATCC 31433, its complete genome was determined using single-molecule real-time DNA sequence analysis. The 7.8-Mb genome comprised four replicons, three chromosomal (each encoding rRNA) and one plasmid. Phylogenetic analysis demonstrated that P. mesoacidophila ATCC 31433 was misclassified at the time of its deposition and is a member of the Burkholderia cepacia complex, most closely related to Burkholderia ubonensis The sequenced genome shows considerable additional biosynthetic potential; known gene clusters for malleilactone, ornibactin, isosulfazecin, alkylhydroxyquinoline, and pyrrolnitrin biosynthesis and several uncharacterized biosynthetic gene clusters for polyketides, nonribosomal peptides, and other metabolites were identified. Furthermore, P. mesoacidophila ATCC 31433 harbors many genes associated with environmental resilience and antibiotic resistance and was resistant to a range of antibiotics and metal ions. In summary, this bioactive strain should be designated B. cepacia complex strain ATCC 31433, pending further detailed taxonomic characterization.IMPORTANCE This work reports the complete genome sequence of Pseudomonas mesoacidophila ATCC 31433, a known producer of bioactive compounds. Large numbers of both known and novel biosynthetic gene clusters were identified, indicating that P. mesoacidophila ATCC 31433 is an untapped resource for discovery of novel bioactive compounds. Phylogenetic analysis demonstrated that P. mesoacidophila ATCC 31433 is in fact a member of the Burkholderia cepacia complex, most closely related to the species Burkholderia ubonensis Further investigation of the classification and biosynthetic potential of P. mesoacidophila ATCC 31433 is warranted.

Invasion of nonphagocytic cells through rearrangement of the actin cytoskeleton is a common immune evasion mechanism used by most intracellular bacteria. However, some pathogens modulate host microtubules as well by a still poorly understood mechanism. In this study, we aim at deciphering the mechanisms by which the opportunistic bacterial pathogen Pseudomonas aeruginosa invades nonphagocytic cells, although it is considered mainly an extracellular bacterium. Using confocal microscopy and immunofluorescence, we show that the evolved VgrG2b effector of P. aeruginosa strain PAO1 is delivered into epithelial cells by a type VI secretion system, called H2-T6SS, involving the VgrG2a component. An in vivo interactome of VgrG2b in host cells allows the identification of microtubule components, including the γ-tubulin ring complex (γTuRC), a multiprotein complex catalyzing microtubule nucleation, as the major host target of VgrG2b. This interaction promotes a microtubule-dependent internalization of the bacterium since colchicine and nocodazole, two microtubule-destabilizing drugs, prevent VgrG2b-mediated P. aeruginosa entry even if the invasion still requires actin. We further validate our findings by demonstrating that the type VI injection step can be bypassed by ectopic production of VgrG2b inside target cells prior to infection. Moreover, such uncoupling between VgrG2b injection and bacterial internalization also reveals that they constitute two independent steps. With VgrG2b, we provide the first example of a bacterial protein interacting with the γTuRC. Our study offers key insight into the mechanism of self-promoting invasion of P. aeruginosa into human cells via a directed and specific effector-host protein interaction., IMPORTANCE Innate immunity and specifically professional phagocytic cells are key determinants in the ability of the host to control P. aeruginosa infection. However, among various virulence strategies, including attack, this opportunistic bacterial pathogen is able to avoid host clearance by triggering its own internalization in nonphagocytic cells. We previously showed that a protein secretion/injection machinery, called the H2 type VI secretion system (H2-T6SS), promotes P. aeruginosa uptake by epithelial cells. Here we investigate which H2-T6SS effector enables P. aeruginosa to enter nonphagocytic cells. We show that VgrG2b is delivered by the H2-T6SS machinery into epithelial cells, where it interacts with microtubules and, more particularly, with the γ-tubulin ring complex (γTuRC) known as the microtubule-nucleating center. This interaction precedes a microtubule- and actin-dependent internalization of P. aeruginosa. We thus discovered an unprecedented target for a bacterial virulence factor since VgrG2b constitutes, to our knowledge, the first example of a bacterial protein interacting with the γTuRC.

Pseudomonas aeruginosa is a versatile and prevalent opportunistic pathogen. It encodes a large arsenal of pathogenicity factors, and secrets a plethora of proteins using specialised protein secretion systems. The type VI secretion system (T6SS) delivers proteins directly into neighbouring bacteria or eukaryotic cells using a mechanism homologous to the T4 bacteriophage tail spike. Three T6SS are encoded on the P. aeruginosa genome. The study of the H1-T6SS has been facilitated by the fact it can be activated by the manipulation of the RetS/Gac/Rsm regulatory cascade by deletion of retS. However, the precise signals required for activation of this cascade, resulting in H1-T6SS activation, are unknown. This work investigates the role of subinhibitory concentrations of antibiotics in activating the system, and shows that kanamycin is able to induce production of core H1-T6SS components. This activation requires a functional Gac/Rsm cascade, but it is not known if this is due to direct signalling via the cascade, or due to a dominant effect of RsmA repression. The H2-T6SS is characterized in this work. We highlight key differences between the H2-T6SS cluster in PAO1 and PA14, including the presence of additional core T6SS components and putative secreted effectors. A strain is generated in which expression of the PA14 H2-T6SS cluster can be activated and tightly controlled by arabinose inducible promoters. The activity of the promoters is confirmed by the H2-T6SS dependent secretion of Hcp2 specifically upon arabinose induction. We further consider two putative H2-T6SS secreted substrates, VgrG14 and Rhs14. Production of these proteins is observed following arabinose induction, but their secretion is not detected. The Rhs14 protein is characterised, and its possible role as a H2-T6SS dependent effector is discussed. Finally, the H2-T6SS system in PA14 is shown to inhibit the internalisation of P. aeruginosa PA14, in contrast to the previously published observations of the H2-T6SS promoting internalisation of PAO1. Open Access

An antimicrobial activity screen of Burkholderia gladioli BCC0238, a clinical isolate from a cystic fibrosis patient, led to the discovery of gladiolin, a novel macrolide antibiotic with potent activity against Mycobacterium tuberculosis H37Rv. Gladiolin is structurally related to etnangien, a highly unstable antibiotic from Sorangium cellulosum that is also active against Mycobacteria. Like etnangien, gladiolin was found to inhibit RNA polymerase, a validated drug target in M. tuberculosis. However, gladiolin lacks the highly labile hexaene moiety of etnangien and was thus found to possess significantly increased chemical stability. Moreover, gladiolin displayed low mammalian cytotoxicity and good activity against several M. tuberculosis clinical isolates, including four that are resistant to isoniazid and one that is resistant to both isoniazid and rifampicin. Overall, these data suggest that gladiolin may represent a useful starting point for the development of novel drugs to tackle multidrug-resistant tuberculosis. The B. gladioli BCC0238 genome was sequenced using Single Molecule Real Time (SMRT) technology. This resulted in four contiguous sequences: two large circular chromosomes and two smaller putative plasmids. Analysis of the chromosome sequences identified 49 putative specialized metabolite biosynthetic gene clusters. One such gene cluster, located on the smaller of the two chromosomes, encodes a trans-acyltransferase (trans-AT) polyketide synthase (PKS) multienzyme that was hypothesized to assemble gladiolin. Insertional inactivation of a gene in this cluster encoding one of the PKS subunits abrogated gladiolin production, confirming that the gene cluster is responsible for biosynthesis of the antibiotic. Comparison of the PKSs responsible for the assembly of gladiolin and etnangien showed that they possess a remarkably similar architecture, obfuscating the biosynthetic mechanisms responsible for most of the structural differences between the two metabolites.

Beneficial microorganisms are widely used in agriculture for control of plant pathogens, but a lack of efficacy and safety information has limited the exploitation of multiple promising biopesticides. We applied phylogeny-led genome mining, metabolite analyses and biological control assays to define the efficacy of Burkholderia ambifaria, a naturally beneficial bacterium with proven biocontrol properties but potential pathogenic risk. A panel of 64 B. ambifaria strains demonstrated significant antimicrobial activity against priority plant pathogens. Genome sequencing, specialized metabolite biosynthetic gene cluster mining and metabolite analysis revealed an armoury of known and unknown pathways within B. ambifaria. The biosynthetic gene cluster responsible for the production of the metabolite cepacin was identified and directly shown to mediate protection of germinating crops against Pythium damping-off disease. B. ambifaria maintained biopesticidal protection and overall fitness in the soil after deletion of its third replicon, a non-essential plasmid associated with virulence in Burkholderia cepacia complex bacteria. Removal of the third replicon reduced B. ambifaria persistence in a murine respiratory infection model. Here, we show that by using interdisciplinary phylogenomic, metabolomic and functional approaches, the mode of action of natural biological control agents related to pathogens can be systematically established to facilitate their future exploitation.

The genomes of 450 members of Burkholderiaceae , isolated from clinical and environmental sources, were sequenced and assembled as a resource for genome mining. Genomic analysis of the collection has enabled the identification of multiple metabolites and their biosynthetic gene clusters, including the antibiotics gladiolin, icosalide A, enacyloxin, and cepacin A.

Polyketide synthases assemble diverse natural products with numerous important applications. The thioester intermediates in polyketide assembly are covalently tethered to acyl carrier protein domains of the synthase. Several mechanisms for polyketide chain release are known, contributing to natural product structural diversification. Here, we report a dual transacylation mechanism for chain release from the enacyloxin polyketide synthase, which assembles an antibiotic with promising activity against Acinetobacter baumannii. A non-elongating ketosynthase domain transfers the polyketide chain from the final acyl carrier protein domain of the synthase to a separate carrier protein, and a non-ribosomal peptide synthetase condensation domain condenses it with (1S,3R,4S)-3,4-dihydroxycyclohexane carboxylic acid. Molecular dissection of this process reveals that non-elongating ketosynthase domain-mediated transacylation circumvents the inability of the condensation domain to recognize the acyl carrier protein domain. Several 3,4-dihydroxycyclohexane carboxylic acid analogues can be employed for chain release, suggesting a promising strategy for producing enacyloxin analogues. ispartof: Nature Chemistry vol:11 issue:10 pages:1-7 ispartof: location:England status: Published online

Burkholderia is a multi-talented genus of Gram-negative bacteria, which in recent years has become increasingly recognised as a promising source of bioactive natural products. Metabolite profiling of Burkholderia gladioli BCC0238 showed that it produces the asymmetric lipopeptidiolide antibiotic icosalide A1, originally isolated from a fungus. Comparative bioinformatics analysis of several genome-sequenced B. gladioli isolates identified a gene encoding a nonribosomal peptide synthase (NRPS) with an unusual architecture that was predicted to be responsible for icosalide biosynthesis. Inactivation of this gene in B. gladioli BCC0238 abolished icosalide production. PCR analysis and sequencing of total DNA from the original fungal icosalide A1 producer revealed it has a B. gladioli strain associated with it that harbours an NRPS with an identical architecture to that responsible for icosalide A1 assembly in B. gladioli BCC0238. Sequence analysis of the icosalide NRPS indicated that it contains two chain-initiating condensation (CI) domains. One of these is appended to the N-terminus of module 1 - a common architecture for NRPSs involved in lipopeptide assembly. The other is embedded in module 3, immediately downstream of a putative chain-elongating condensation domain. Analysis of the reactions catalysed by a tridomain construct from module 3 of the NRPS using intact protein mass spectrometry showed that the embedded CI domain initiates assembly of a second lipopeptide chain, providing key insights into the mechanism for asymmetric diolide assembly. Open Access article ispartof: Chemical Science vol:10 issue:21 ispartof: location:England status: published