Quinones are growth factors for the human gut microbiota

Abstract

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.

Keywords: Cultivation; Faecalibacterium; Growth factors; Menaquinones; Microbiome; Uncultured bacteria.

Fig. 1

Isolating uncultured bacteria using co-culture methods. Two approaches are used to identify bacterial isolates from stool exhibiting dependency phenotypes. a In an untargeted approach, early-forming colonies are picked and spotted on plates where late-forming neighboring colonies are spread. b In a targeted approach, a known helper bacterium, in this case Escherichia coli K12, can be spotted at the time of inoculation of stool sample. Neighboring colonies around the E. coli spot are then picked, spread on fresh agar, and an E. coli inoculum is then spotted on the plate. In both assays, induction of the candidate-dependent bacteria around the spotted helper indicates a positive hit. The plates in this figure are used as a representative example to explain the isolation technique

Fig. 2

Escherichia coli is a universal helper for the cultured helper-dependent and helper-induced strains. All strains identified in the co-culture screen were tested for induction by E. coli K12 BW25113. Identified induced strains were spread on brain heart infusion agar with 5.0 g/L yeast extract, 0.1% cysteine, and 15 mg/mL hemin (BHIych), and 5 μL of a 24-h culture of E. coli K12 BW25113 was then spotted on the same plate. a Taxonomic information on the induced isolates and their helper organisms. Different growth induction profiles were identified, including complete dependence, as observed with b Bilophila wadsworthia KLE 1795, and substantially increased colony size, as seen with c Bacteroides clarus KLE1792, d Gordonibacter pamelaeae KLE1812, e Faecalibacterium sp. KLE1255, and all other isolates (not pictured). Images are displayed in black and white to improve contrast

Fig. 3

Screening for deletion mutants unable to induce the growth of Faecalibacterium sp. KLE1255. a A selection of 283 strains that were compiled from E. coli small-, medium-, and large-scale deletion libraries to cover all non-essential genes of the E. coli genome were screened for induction of KLE1255 via co-culture assay. b Eight strains were identified as being unable to induce growth of KLE1255, harboring deletions in various regions of the genome. c One strain, E. coli OCL67, had a single large deletion consisting of 16 genes, including 6 in menaquinone biosynthesis. menE = 2-succinylbenzoate--CoA ligase; menC = o-succinylbenzoate synthase; menB = 1,4-dihydroxy-2-naphthoyl-CoA synthase; menH = 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase; menD = 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase; menF = isochorismate synthase; elaB = putative membrane-anchored DUF883 family ribosome-binding protein; elaA = putative N-acetyltransferase; rbn = ribonuclease BN; elaD = deubiquitinase; yfbK = putative lipoprotein; yfbL = putative membrane-associated peptidase; yfbM = DUF1877 family protein; yfbN = uncharacterized protein; yfbO = uncharacterized protein; yfbP = uncharacterized protein. d Wild type induced strong growth in KLE1255, while e OCL67 did not. Similarly, f WT E. coli also induces Faecalibacterium prausnitzii ATCC 27766

Fig. 4

Quinones are growth factors for E. coli-induced bacteria. Bacteria were plated on rich BHIych agar and spotted with 5 μL of 1 mM of a MK-4, b DHNA, or c menadione, or the vehicle of the quinones, ethanol. The exception was KLE1255, which was spotted with 10 μL of 10 mM stocks all quinones. d E. coli and quinone induction capabilities of dependent strains and several cultivable Bacteroides species. Bolded strains represent induced organisms. Examples of induction with purified quinones here include e Faecalibacterium sp. KLE1255, f Bilophila wadsworthia KLE1795, g Bacteroides clarus KLE1792, and h Gordonibacter pamelaeae KLE1812

Fig. 5

Model for menaquinone-dependence in Faecalibacterium sp. Genome analysis suggests that Faecalibacterium sp. possess a truncated electron transport chain that is directed towards fumarate respiration. The required menaquinone is provided by an external source (indicated by the dashed arrow). MK menaquinone, MKH₂ menaquinol