Biosynthesis of antifungal and antibacterial polyketides by Burkholderia gladioli in coculture with Rhizopus microsporus.

Fungi-bacteria interactions can impact the course of fungal infection and biotechnological use. The mucoralean fungus Rhizopus microsporus, traditionally used in food fermentations (tempe and sufu), is frequently accompanied by Burkholderia gladioli pv. cocovenenans. When producing tempe bongkrek, the bacterial contamination can lead to lethal food-related intoxications caused by the respiratory toxin bongkrekic acid. To unveil the metabolic potential of the fungus-associated bacterium, we sequenced its genome, assigned secondary metabolite biosynthesis gene clusters and monitored the metabolic profile under various growth conditions. In addition to the bongkrekic acid biosynthesis gene cluster we found gene clusters coding for the biosynthesis of toxoflavin and a complex polyketide. The orphan polyketide synthase gene cluster was activated under conditions that emulate tempe production, which enabled isolation and structure elucidation of four members of the enacyloxin family of antibiotics, out of which one is new. Moreover, we found that the fungus positively influences the growth of the bacteria and dramatically increases bongkrekic acid production in stationary culture, which inhibits the growth of the fungus. These results showcase the context-dependent formation of antifungal and antibacterial agents at the fungal-bacterial interface, which may also serve as a model for scenarios observed in mixed infections.

Biosynthesis of the respiratory toxin bongkrekic acid in the pathogenic bacterium Burkholderia gladioli.

Bongkrekic acid (BA), an infamous respiratory toxin of the pathogenic bacterium Burkholderia gladioli, causes lethal intoxications when tempe bongkrek is produced with contaminated Rhizopus oligosporus cultures. Genome sequencing of B. gladioli pathovar cocovenenans unveiled the genetic basis for BA biosynthesis, and pointed to a homologous bon gene cluster in a B. gladioli strain from an infected rice plant. For functional genetics in B. gladioli λ Red recombination was established. Dissection of the modular type I polyketide synthase (a trans-AT PKS) provided insights into complex polyketide assembly. Isoprenoid-like ß-branching events and a six-electron oxidation of a methyl group to a carboxylic acid give rise to the unique branched tricarboxylic fatty acid. The role of the cytochrome P450 monooxygenase, BonL, was proven by structural elucidation of deoxybongkrekic acid from a mutant.

Need to differentiate lethal toxin-producing strains of Burkholderia gladioli, which cause severe food poisoning: description of B. gladioli pathovar cocovenenans and an emended description of B. gladioli.

Burkholderia cocovenenans produces a lethal toxin (Bongkrekic acid) that leads to high fatality in food poisoning cases. However, B. cocovenenans was combined in Burkholderia gladioli in 1999. B. gladioli was originally described as a phytopathogenic bacteria that sometimes causes pneumonia in humans and that acts as an opportunistic pathogen. We thought that it was clinically dangerous to describe these two species without considering their pathogenicities. From our data of 16S rRNA sequence analysis, DNA-DNA hybridization, and fatty acid analysis, we could confirm that B. cocovenenans and B. gladioli should be categorized as a single species. However the species really weaved lethal toxin-producing strains with non-lethal strains. To emphasize that B. gladioli contains two different pathogens, we describe a new pathovar, B. gladioli pathovar cocovenenans, for the lethal toxin-producing strains. We provide characteristics that differentiate this lethal toxin-producing pathovar from other phytopathogenic pathovars within B. gladioli, together with an emended description of B. gladioli.

The effect of lipids on bongkrekic (Bongkrek) acid toxin production by Burkholderia cocovenenans in coconut media.

Tempe bongkrek is an Indonesian food made by fermentation of coconut presscake or coconut milk residue Rhizopus oligosporus. Consumption of tempe bongkrek is associated with a food-borne human intoxication and significant numbers of deaths annually. The bacterium Burkholderia cocovenenans, which is the causative organism, produces two toxins, toxoflavin and bongkrekic acid (also commonly referred to as bongkrek acid). The reasons why these poisonings occur only in a very limited number of foods and only in isolated regions of the world are unclear. Our preliminary experiments in defined media and coconut investigated several compositional and environmental factors and suggested that lipid type and/or concentration were important. The effect of lipid concentration and fatty acid type on the production of bongkrekic acid by B. cocovenenans was examined by adding different amounts of coconut fat or individual free fatty acids to defatted and sterilized Rich Coconut Media (dRCM). The dRCM with added lipid was inoculated with B. cocovenenans, incubated at 30 degrees C for 5 days and the amount of bongkrekic acid formed quantified by HPLC. Coconut fat concentrations of 10% (dry basis) or less did not result in detectable amounts of bongkrekic acid even though the B. cocovenenans grew to high levels. Forty and 50% coconut fat resulted in as much as 1.4 mg/g bonkrekic acid (dry weight) at the same level of growth. Of eight saturated fatty acids tested, only lauric (12:0), myristic (14:0), and palmitic (16:0) acids stimulated the production of detectable amounts of toxin. When four 18-carbon free fatty acids with different degrees of saturation were compared, significant amounts of bongkrekic acid (2.62 mg/g dry weight) were produced only with oleic acid (18:1). These data indicate that the concentration and type of lipid in the substrate is critical for bongkrekic acid formation. This may explain why bongkrekic acid intoxication is limited to certain foods. Outbreaks associated with foods containing less than 20% fat may be a result of toxoflavin formation and not bongkrekic acid formation.

[Study on mutagenicity of Toxoflavin from Pseudomonas farinofermentans].

Fermented corn flour containing toxoflavin and bongkretic acid produced by P. farinofermentans can cause acute food poisoning, but study on mutagenicity of toxoflavin has not been reported. We studied the mutagenicity of toxoflavin by Tradescantia Paludosa and animal micronucleus tests. The results showed that toxoflavin possesses distinct mutagenic action in both plant and animal cells. Mutagenic action of toxoflavin in cells may have important bearing on food hygiene examination.