Bacillus amyloliquefaciens FZB42 is a Gram-positive, plant-associated bacterium, which stimulates plant growth and produces secondary metabolites that suppress soil-borne plant pathogens. Its 3,918-kb genome, containing an estimated 3,693 protein-coding sequences, lacks extended phage insertions, which occur ubiquitously in the closely related Bacillus subtilis 168 genome. The B. amyloliquefaciens FZB42 genome reveals an unexpected potential to produce secondary metabolites, including the polyketides bacillaene and difficidin. More than 8.5% of the genome is devoted to synthesizing antibiotics and siderophores by pathways not involving ribosomes. Besides five gene clusters, known from B. subtilis to mediate nonribosomal synthesis of secondary metabolites, we identified four giant gene clusters absent in B. subtilis 168. The pks2 gene cluster encodes the components to synthesize the macrolactin core skeleton.

Although bacterial polyketides are of considerable biomedical interest, the molecular biology of polyketide biosynthesis in Bacillus spp., one of the richest bacterial sources of bioactive natural products, remains largely unexplored. Here we assign for the first time complete polyketide synthase (PKS) gene clusters to Bacillus antibiotics. Three giant modular PKS systems of the trans-acyltransferase type were identified in Bacillus amyloliquefaciens FZB 42. One of them, pks1, is an ortholog of the pksX operon with a previously unknown function in the sequenced model strain Bacillus subtilis 168, while the pks2 and pks3 clusters are novel gene clusters. Cassette mutagenesis combined with advanced mass spectrometric techniques such as matrixassisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-electrospray ionization mass spectrometry revealed that the pks1 (bae) and pks3 (dif) gene clusters encode the biosynthesis of the polyene antibiotics bacillaene and difficidin or oxydifficidin, respectively. In addition, B. subtilis OKB105 (pheA sfp 0 ), a transformant of the B. subtilis 168 derivative JH642, was shown to produce bacillaene, demonstrating that the pksX gene cluster directs the synthesis of that polyketide.Environmental Bacillus amyloliquefaciens strain FZB 42 is distinguished from the domesticated model organism Bacillus subtilis 168 (23) by several features important for rhizosphere competence particularly by its abilities to suppress competitive organisms present in the plant rhizosphere (17, 21) and to promote plant growth (16). In a previous contribution (20), we have reported that B. amyloliquefaciens FZB 42 is a producer of three families of lipopeptides, surfactins, bacillomycins D, and fengycins, which are well-known secondary metabolites with mainly antifungal activity. They are also produced by numerous B. subtilis strains (48). Furthermore, three giant gene clusters containing genes with homology to polyketide synthase (PKS) genes of modular organization were identified but not assigned functional roles. Mutants of FZB 42 deficient in the synthesis of cyclic lipopeptides were unable to suppress phytopathogenic fungi but still retained their antibacterial potency.Polyketides belong to a large family of secondary metabolites that include many bioactive compounds with antibacterial, immunosuppressive, antitumor, or other physiologically relevant bioactivities. Their biosynthesis is accomplished by stepwise decarboxylative Claisen condensations between the extender unit and the growing polyketide chain, generating enzyme-bound ␤-ketoacyl intermediates. Before a subsequent round of chain extension, a variable set of modifying enzymes can locally introduce structural variety. Similar to the nonribosomal synthesis of peptides, the PKS multienzyme system uses acyl carrier proteins (ACPs) that are posttranslationally modified with the 4Ј-phosphopantetheine prosthetic group to channel the growing polyketide intermediate during elongation processes (3). Type I PKSs...

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Salmochelins represent novel carbohydrate containing catecholate siderophores, which are excreted by Salmonella enterica and uropathogenic Escherichia coli strains under low-iron stress. While previous analytical data showed salmochelins to contain 2,3-dihydroxybenzoyl-L-serine and glucose, the molecular structure remained elusive. Structure elucidation with electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry (ESI-FTICR-MS), GC-MS and 2D-NMR now revealed that salmochelins are enterobactin-related compounds, which are beta-C-glucosylated at the 5-position of a 2,3-dihydroxybenzoyl residue. The key compound salmochelin S4 is a twofold beta-C-glucosylated enterobactin analogue. Comparison of partial structures of salmochelin with a C-glycosylated compound previously characterized by another group strongly suggest that salmochelins represent the long sought compounds termed Salmonella resistance factors (SRF) or pacifarins. Transformation of iro-genes into enterobactin-producing E. coli K12 confers the ability to produce salmochelins. A detailed analysis proved iroB to be the sole gene with glycosyltransferase activity necessary for salmochelin production. Salmochelins compared to enterobactin are the better siderophores in the presence of serum albumin. This may indicate for salmochelins a considerably more important role for pathogenic processes in certain Escherichia coli and Salmonella infections than formerly assigned to enterobactin. This conclusion is supported by the location of the iro genes on pathogenicity islands of uropathogenic E. coli strains.

BackgroundIn standard reference sources, the incidence of coronary artery disease (CAD) in patients with atrial fibrillation (AF) ranged between 24 and 46.5%. Since then, the incidence of cardiovascular risk factors (CRF) has increased and modern treatment strategies (“pill in the pocket”) are only applicable to patients without structural heart disease. The aim of this study was to investigate the incidence and severity of CAD in patients with AF.MethodsFrom January 2005 until December 2009, we included 261 consecutive patients admitted to hospital with paroxysmal, persistent or permanent AF in this prospective study. All patients underwent coronary angiography and the Framingham risk score (FRS) was calculated. Patients with previously diagnosed or previously excluded CAD were excluded.ResultsThe overall incidence of CAD in patients presenting with AF was 34%; in patients >70 years, the incidence of CAD was 41%. The incidence of patients undergoing a percutaneous coronary intervention (PCI) or coronary artery bypass graft (CABG) was 21%. Patients with CAD were older (73±8 years vs 68±10 years, p = 0.001), had significantly more frequent hypercholesterolemia (60% vs 30%, p<0.001), were more frequent smokers (26% vs 13%, p = 0.017) and suffered from angina more often (37% vs 2%, p<0.001). There was a significant linear trend among the FRS categories in percentage and the prevalence of CAD and PCI/CABG (p<0.0001).ConclusionsThe overall incidence of CAD in patients presenting with AF was relatively high at 34%; the incidence of PCI/CABG was 21%. Based upon increasing CRF in the western world, we recommend a careful investigation respecting the FRS to either definitely exclude or establish an early diagnosis of CAD – which could contribute to an early and safe therapeutic strategy considering type Ic antiarrhythmics and oral anticoagulation.

In the genome of Bacillus amyloliquefaciens FZB42, three operons pks1, pks2, and pks3 were identified which encode the biosynthesis of polyketides. pks1 and pks3 have been attributed to the production of bacillaene and difficidin/oxydifficidin, respectively, while the pks2 product remained hitherto unknown. Mass spectrometric analysis of the culture filtrates of the wild-type B. amyloliquefaciens FZB42 and mutants revealed pks2-specific metabolites. By combination of the mass spectrometric and UV/vis data with a database search, these compounds were attributed to four members of the macrolactin family, macrolactin A and D as well as 7-O-malonyl- and 7-O-succinyl-macrolactin. This conclusion was verified by the isolation and structure elucidation of macrolactin A using mass spectrometric and 2D-NMR studies. Macrolactin biosynthesis was investigated using feeding experiments with (13)C-acetate. (13)C-labelled macrolactin A revealed an alternating labelling of its carbon skeleton with (13)C, indicating that acetate/malonate was used as the sole precursor. The macrolactin structure is compatible with the domain organization of the pks2-operon. Similarly to pks1 and pks3, pks2 is a modular polyketide synthase system of type I which exhibits a trans-acyltransferase architecture using a discrete acyltransferase enzyme iteratively in the assembly of macrolactin. Finally, the potential for macrolactin production on a genetic and metabolic basis was found to be widely distributed among Bacillus amyloliquefaciens strains.

BackgroundMethylotrophic microorganisms are playing a key role in biogeochemical processes - especially the global carbon cycle - and have gained interest for biotechnological purposes. Significant progress was made in the recent years in the biochemistry, genetics, genomics, and physiology of methylotrophic bacteria, showing that methylotrophy is much more widespread and versatile than initially assumed. Despite such progress, system-level description of the methylotrophic metabolism is currently lacking, and much remains to understand regarding the network-scale organization and properties of methylotrophy, and how the methylotrophic capacity emerges from this organization, especially in facultative organisms.ResultsIn this work, we report on the integrated, system-level investigation of the metabolic network of the facultative methylotroph Methylobacterium extorquens AM1, a valuable model of methylotrophic bacteria. The genome-scale metabolic network of the bacterium was reconstructed and contains 1139 reactions and 977 metabolites. The sub-network operating upon methylotrophic growth was identified from both in silico and experimental investigations, and 13C-fluxomics was applied to measure the distribution of metabolic fluxes under such conditions. The core metabolism has a highly unusual topology, in which the unique enzymes that catalyse the key steps of C1 assimilation are tightly connected by several, large metabolic cycles (serine cycle, ethylmalonyl-CoA pathway, TCA cycle, anaplerotic processes). The entire set of reactions must operate as a unique process to achieve C1 assimilation, but was shown to be structurally fragile based on network analysis. This observation suggests that in nature a strong pressure of selection must exist to maintain the methylotrophic capability. Nevertheless, substantial substrate cycling could be measured within C2/C3/C4 inter-conversions, indicating that the metabolic network is highly versatile around a flexible backbone of central reactions that allows rapid switching to multi-carbon sources.ConclusionsThis work emphasizes that the metabolism of M. extorquens AM1 is adapted to its lifestyle not only in terms of enzymatic equipment, but also in terms of network-level structure and regulation. It suggests that the metabolism of the bacterium has evolved both structurally and functionally to an efficient but transitory utilization of methanol. Besides, this work provides a basis for metabolic engineering to convert methanol into value-added products.