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. Electronic supplementary material The online version of this article (10.1186/s40168-017-0380-5) contains supplementary material, which is available to authorized users

Additional file 5: Table S3. of Quinones are growth factors for the human gut microbiota

Quinone-induced bacteria have a disrupted menaquinone biosynthesis pathway, while related organisms not induced by quinones have a complete pathway. The genomes of the nearest type strains of all E. coli- or quinone-induced cultured bacteria were surveyed manually for the presence of a functional menaquinone biosynthesis pathway using a published dataset [24]. All organisms induced by E. coli or quinones in earlier co-culture experiments were missing large components of the menaquinone biosynthesis pathway, while Bacteroides species not induced by E. coli or quinones were predicted to have complete menaquinone biosynthetic capabilities. No strains were found to have predicted copies of genes in the futalosine pathway, an alternative means to generate menaquinone. ubiE/menG: 2-methoxy-6-polyprenyl-1,4-benzoquinol methylase; menF = Menaquinone-specific isochorismate synthase; menD = 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic-acid synthase; menH = 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase; menY = 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate dehydrogenase; menC = o-succinylbenzoate synthase; menE = o-succinylbenzoic acid--CoA ligase; menB = Naphthoate synthase. menI = 1,4-dihydroxy-2-naphthoyl-CoA hydrolase; menJ = 1,4-dihydroxy-2-naphthoyl-CoA hydrolasein (putative); menA = 1,4-dihydroxy-2-naphthoate polyprenyltransferase; mqnA = Chorismate dehydratase; mqnE = Aminodeoxyfutalosine synthase; mqnC = Cyclic dehypoxanthine futalosine synthase; mqnD = 1,4-dihydroxy-6-naphthoate synthase; mqnZ = 1,4-dihydroxy-6-naphthoate synthase (alternative); mqnX = Aminodeoxyfutalosine deaminase; mqnB = Futalosine hydrolase (EC 3.2.2.26); mtnN = Aminodeoxyfutalosine nucleosidase; mqnL = 1,4-dihydroxy-6-naphthoate carboxy-lyase, UbiD-like; mqnM = 2-heptaprenyl-1,4-naphthoquinone methyltransferase; mqnP = 1,4-naphthoquinone polyprenyltransferase. Data was taken and modified from Racheev, 2016. (XLSX 10 kb

Additional file 6: Table S4. of Quinones are growth factors for the human gut microbiota

Quinone-dependent and control strains have predicted anaerobic reductases. The genomes of nearest type strains of all E. coli- or quinone-induced cultured bacteria were surveyed manually for the presence of individual annotated anaerobic reductases using a published dataset [35]. All analyzed organisms have the genetic capability to utilize anaerobic reductases for anaerobic respiration. Arx = Arsenate reductase; Cyd = Cytochrome bd reductase; Dms = Dimethyl sulfoxide reductase; Dsr = Sulfite reductase; Frd = Fumarate reductase; Nap = Nitrate reductase; Nar = Nitrate reductase; Nrf = Nitrite reductase; Phs = Thiosulfate reductase; Psr = Polysulfite reductase; Tor = Trimethylamine N-oxide reductase; Ttr = Tetrathionate reductase; Ynf = Selenate reductase. Data was taken and modified from Racheev, 2014 [35] and Ravcheev, 2016 [24]. (XLSX 8 kb

Additional file 4: Figure S2. of Quinones are growth factors for the human gut microbiota

Single deletions in the E. coli menaquinone-8 pathway, but not ubiquinone-8 pathway, prevented growth induction of KLE1255. Single deletion mutants for all genes involved in ubiquinone-8 and menaquinone-8 biosynthesis were tested for induction capabilities of KLE1255. Red boxes indicate E. coli mutants with impaired growth induction capabilities for KLE1255. (PNG 364 kb

Additional file 1: Table S1. of Quinones are growth factors for the human gut microbiota

Entire chromosome E. coli deletion library reformatted. 283 strains were first compiled from E. coli small-, medium-, and large-scale deletion libraries to cover all non-essential genes of the E. coli genome. Strains were taken from the Keio collection [19] and two larger deletion libraries [20, 21]. (XLS 36 kb

Additional file 2: Table S2. of Quinones are growth factors for the human gut microbiota

Strains identified in the E. coli knockout screen unable to induce the growth of KLE1255. Includes information on which genes are absent for each clone. (XLSX 18 kb

Additional file 3: Figure S1. of Quinones are growth factors for the human gut microbiota

Single deletions in the E. coli chorismate biosynthesis pathway prevented growth induction of KLE1255. Single deletion mutants for all genes involved in chorismate biosynthesis were tested for induction capabilities of KLE1255. Red boxes indicate E. coli mutants with impaired growth induction capabilities for KLE1255. (PNG 449 kb