Identification of a growth factor required for culturing specific fastidious oral bacteria.

Aims: The aim of this research was to isolate oral bacteria that are dependent for growth on adjacent bacteria producing a required growth factor and to identify the chemical structure of the growth factor. Methods: Porphyromonas pasteri strain KLE1280, could be cultivated with Staphylococcus hominis and Escherichia coli as helper strains. A deletion mutant library of E. coli was screened to determine genes involved in production of the growth factor. Compounds produced by the growth factor's pathway were screened to see if they would stimulate growth of strain P. pasteri KLE1280. The genomes of species related to P. pasteri KLE1280 were screened for presence of the factor's synthetic pathway. Results: Analysis of the E. coli deletion mutant library and growth studies identified 1,2-dihydroxy-2-naphthoic acid (DHNA) and menaquinone-4 (MK4) as the growth factors. Strain P. pasteri KLE1280 was shown to lack five genes in the menaquinone synthesis pathway but to possess the two genes necessary to convert DHNA to menaquinone. Genome analysis found that 8 species in genera Porphyromonas and Tannerella lack five genes in the menaquinone synthesis pathway. Conclusions: Addition of DHNA to culture media allows isolation of strains of several oral species that are not recovered using standard media.

An elusive electron shuttle from a facultative anaerobe.

Some anaerobic bacteria use insoluble minerals as terminal electron acceptors and discovering the ways in which electrons move through the membrane barrier to the exterior acceptor forms an active field of research with implications for both bacterial physiology and bioenergy. A previous study suggested that Shewanella oneidensis MR-1 utilizes a small, polar, redox active molecule that serves as an electron shuttle between the bacteria and insoluble acceptors, but the shuttle itself has never been identified. Through isolation and synthesis, we identify it as ACNQ (2-amino-3-carboxy-1,4-naphthoquinone), a soluble analog of menaquinone. ACNQ is derived from DHNA (1,4-dihydroxy-2-naphthoic acid) in a non-enzymatic process that frustrated genetic approaches to identify the shuttle. Both ACNQ and DHNA restore reduction of AQDS under anaerobic growth in menaquinone-deficient mutants. Bioelectrochemistry analyses reveal that ACNQ (-0.32 VAg/AgCl) contributes to the extracellular electron transfer (EET) as an electron shuttle, without altering menaquinone generation or EET related cytochrome c expression.

Quinones are growth factors for the human gut microbiota.

BACKGROUND: The human gut microbiome has been linked to numerous components of health and disease. However, approximately 25% of the bacterial species in the gut remain uncultured, which limits our ability to properly understand, and exploit, the human microbiome. Previously, we found that growing environmental bacteria in situ in a diffusion chamber enables growth of uncultured species, suggesting the existence of growth factors in the natural environment not found in traditional cultivation media. One source of growth factors proved to be neighboring bacteria, and by using co-culture, we isolated previously uncultured organisms from the marine environment and identified siderophores as a major class of bacterial growth factors. Here, we employ similar co-culture techniques to grow bacteria from the human gut microbiome and identify novel growth factors. RESULTS: By testing dependence of slow-growing colonies on faster-growing neighboring bacteria in a co-culture assay, eight taxonomically diverse pairs of bacteria were identified, in which an "induced" isolate formed a gradient of growth around a cultivatable "helper." This set included two novel species Faecalibacterium sp. KLE1255-belonging to the anti-inflammatory Faecalibacterium genus-and Sutterella sp. KLE1607. While multiple helper strains were identified, Escherichia coli was also capable of promoting growth of all induced isolates. Screening a knockout library of E. coli showed that a menaquinone biosynthesis pathway was required for growth induction of Faecalibacterium sp. KLE1255 and other induced isolates. Purified menaquinones induced growth of 7/8 of the isolated strains, quinone specificity profiles for individual bacteria were identified, and genome analysis suggests an incomplete menaquinone biosynthetic capability yet the presence of anaerobic terminal reductases in the induced strains, indicating an ability to respire anaerobically. CONCLUSIONS: Our data show that menaquinones are a major class of growth factors for bacteria from the human gut microbiome. These organisms are taxonomically diverse, including members of the genus Faecalibacterium, Bacteroides, Bilophila, Gordonibacter, and Sutterella. This suggests that loss of quinone biosynthesis happened independently in many lineages of the human microbiota. Quinones can be used to improve existing bacterial growth media or modulate the human gut microbiota by encouraging the growth of important symbionts, such as Faecalibacterium species.

Synthesis and structure confirmation of fuscachelins A and B, structurally unique natural product siderophores from Thermobifida fusca.

The fuscachelin siderophores have been prepared synthetically as have their metal chelation complexes. The heterodimeric nature of the fuscachelin decamer lends itself to a convergent synthetic strategy. Synthetic access to the natural products and intermediates will provide readily adaptable tools in future studies examining iron-sequestration and the biosynthetic machinery.

Structure elucidation and biosynthesis of fuscachelins, peptide siderophores from the moderate thermophile Thermobifida fusca.

Bacteria belonging to the order Actinomycetales have proven to be an important source of biologically active and often therapeutically useful natural products. The characterization of orphan biosynthetic gene clusters is an emerging and valuable approach to the discovery of novel small molecules. Analysis of the recently sequenced genome of the thermophilic actinomycete Thermobifida fusca revealed an orphan nonribosomal peptide biosynthetic gene cluster coding for an unknown siderophore natural product. T. fusca is a model organism for the study of thermostable cellulases and is a major degrader of plant cell walls. Here, we report the isolation and structure elucidation of the fuscachelins, siderophore natural products produced by T. fusca. In addition, we report the purification and biochemical characterization of the termination module of the nonribosomal peptide synthetase. Biochemical analysis of adenylation domain specificity supports the assignment of this gene cluster as the producer of the fuscachelin siderophores. The proposed nonribosomal peptide biosynthetic pathway exhibits several atypical features, including a macrocyclizing thioesterase that produces a 10-membered cyclic depsipeptide and a nonlinear assembly line, resulting in the unique heterodimeric architecture of the siderophore natural product.