A chemically-defined growth medium to support Lactobacillus-Acetobacter sp. community analysis.

Lactobacilli and Acetobacter sp. are commercially important bacteria that often form communities in natural fermentations, including food preparations, spoilage, and in the digestive tract of the fruit fly Drosophila melanogaster. Communities of these bacteria are widespread and prolific, despite numerous strain-specific auxotrophies, suggesting they have evolved nutrient interdependencies that regulate their growth. The use of a chemically-defined medium (CDM) supporting the growth of both groups of bacteria would facilitate the identification of the molecular mechanisms for the metabolic interactions between them. While numerous CDMs have been developed that support specific strains of lactobacilli or Acetobacter, there has not been a medium formulated to support both genera. We developed such a medium, based on a previous CDM designed for growth of lactobacilli, by modifying the nutrient abundances to improve growth yield. We further simplified the medium by substituting casamino acids in place of individual amino acids and the standard Wolfe's vitamins and mineral stocks in place of individual vitamins and minerals, resulting in a reduction from 40 to 8 stock solutions. These stock solutions can be used to prepare several CDM formulations that support robust growth of numerous lactobacilli and Acetobacters. Here, we provide the composition and several examples of its use, which is important for tractability in dissecting the genetic and metabolic basis of natural bacterial species interactions

A chemically-defined growth medium to support Lactobacillus-Acetobacter sp. community analysis

Lactobacilli and Acetobacter sp. are commercially important bacteria that often form communities in natural fermentations, including food preparations, spoilage, and in the digestive tract of the fruit fly Drosophila melanogaster. Communities of these bacteria are widespread and prolific, despite numerous strain-specific auxotrophies, suggesting they have evolved nutrient interdependencies that regulate their growth. The use of a chemically-defined medium (CDM) supporting the growth of both groups of bacteria would facilitate the identification of the molecular mechanisms for the metabolic interactions between them. While numerous CDMs have been developed that support specific strains of lactobacilli or Acetobacter, there has not been a medium formulated to support both genera. We developed such a medium, based on a previous CDM designed for growth of lactobacilli, by modifying the nutrient abundances to improve growth yield. We further simplified the medium by substituting casamino acids in place of individual amino acids and the standard Wolfe's vitamins and mineral stocks in place of individual vitamins and minerals, resulting in a reduction from 40 to 8 stock solutions. These stock solutions can be used to prepare several CDM formulations that support robust growth of numerous lactobacilli and Acetobacters. Here, we provide the composition and several examples of its use, which is important for tractability in dissecting the genetic and metabolic basis of natural bacterial species interactions

Composition of CDM_<i>L</i>.

Lactobacilli and Acetobacter sp. are commercially important bacteria that often form communities in natural fermentations, including food preparations, spoilage, and in the digestive tract of the fruit fly Drosophila melanogaster. Communities of these bacteria are widespread and prolific, despite numerous strain-specific auxotrophies, suggesting they have evolved nutrient interdependencies that regulate their growth. The use of a chemically-defined medium (CDM) supporting the growth of both groups of bacteria would facilitate the identification of the molecular mechanisms for the metabolic interactions between them. While numerous CDMs have been developed that support specific strains of lactobacilli or Acetobacter, there has not been a medium formulated to support both genera. We developed such a medium, based on a previous CDM designed for growth of lactobacilli, by modifying the nutrient abundances to improve growth yield. We further simplified the medium by substituting casamino acids in place of individual amino acids and the standard Wolfe’s vitamins and mineral stocks in place of individual vitamins and minerals, resulting in a reduction from 40 to 8 stock solutions. These stock solutions can be used to prepare several CDM formulations that support robust growth of numerous lactobacilli and Acetobacters. Here, we provide the composition and several examples of its use, which is important for tractability in dissecting the genetic and metabolic basis of natural bacterial species interactions.</div

Growth of <i>Acetobacter sp.</i> isolates.

Acetobacter sp. from several different sources (S2 Table in S1 File) were grown with CDMA under aerobic conditions with continuous shaking. Solid lines represent means of 12 technical replicates. Shaded areas represent standard error on a 5-point rolling average. Time point OD600 readings were taken every 5 minutes.</p

Composition of CDM_<i>A</i>.

Lactobacilli and Acetobacter sp. are commercially important bacteria that often form communities in natural fermentations, including food preparations, spoilage, and in the digestive tract of the fruit fly Drosophila melanogaster. Communities of these bacteria are widespread and prolific, despite numerous strain-specific auxotrophies, suggesting they have evolved nutrient interdependencies that regulate their growth. The use of a chemically-defined medium (CDM) supporting the growth of both groups of bacteria would facilitate the identification of the molecular mechanisms for the metabolic interactions between them. While numerous CDMs have been developed that support specific strains of lactobacilli or Acetobacter, there has not been a medium formulated to support both genera. We developed such a medium, based on a previous CDM designed for growth of lactobacilli, by modifying the nutrient abundances to improve growth yield. We further simplified the medium by substituting casamino acids in place of individual amino acids and the standard Wolfe’s vitamins and mineral stocks in place of individual vitamins and minerals, resulting in a reduction from 40 to 8 stock solutions. These stock solutions can be used to prepare several CDM formulations that support robust growth of numerous lactobacilli and Acetobacters. Here, we provide the composition and several examples of its use, which is important for tractability in dissecting the genetic and metabolic basis of natural bacterial species interactions.</div

Co-culture growth of <i>Lp. plantarum</i> and <i>A. pasteurianus</i>.

Lp. plantarum and A. pasteurianus were grown in co-culture in CDMA with glucose and potassium acetate added. (A) Growth curves (solid lines) display means of 12 experimental replicates. Shaded areas represent standard error on a 5-point rolling average. Time point OD600 readings were taken every 5 minutes. (B) OD measurements were taken at the end of the growth curve after resuspension of the culture supernatant for each of the 12 replicates. Mean and standard deviation are shown. We note that the apparently 4-fold lower OD of Lp. plantarum in the resupsended culture versus the time course in A is likely due to settling of the cells and adherence to the plate during the time course. The CFU/mL counts are in C. (C) CFU/ml was counted by plating the resuspended cultures on MRS and MYPL for each of the 12 replicates. Mean and standard deviation shown.</p

S1 File -

Lactobacilli and Acetobacter sp. are commercially important bacteria that often form communities in natural fermentations, including food preparations, spoilage, and in the digestive tract of the fruit fly Drosophila melanogaster. Communities of these bacteria are widespread and prolific, despite numerous strain-specific auxotrophies, suggesting they have evolved nutrient interdependencies that regulate their growth. The use of a chemically-defined medium (CDM) supporting the growth of both groups of bacteria would facilitate the identification of the molecular mechanisms for the metabolic interactions between them. While numerous CDMs have been developed that support specific strains of lactobacilli or Acetobacter, there has not been a medium formulated to support both genera. We developed such a medium, based on a previous CDM designed for growth of lactobacilli, by modifying the nutrient abundances to improve growth yield. We further simplified the medium by substituting casamino acids in place of individual amino acids and the standard Wolfe’s vitamins and mineral stocks in place of individual vitamins and minerals, resulting in a reduction from 40 to 8 stock solutions. These stock solutions can be used to prepare several CDM formulations that support robust growth of numerous lactobacilli and Acetobacters. Here, we provide the composition and several examples of its use, which is important for tractability in dissecting the genetic and metabolic basis of natural bacterial species interactions.</div

A symbiotic physical niche in Drosophila melanogaster regulates stable association of a multi-species gut microbiota.

The gut is continuously invaded by diverse bacteria from the diet and the environment, yet microbiome composition is relatively stable over time for host species ranging from mammals to insects, suggesting host-specific factors may selectively maintain key species of bacteria. To investigate host specificity, we used gnotobiotic Drosophila, microbial pulse-chase protocols, and microscopy to investigate the stability of different strains of bacteria in the fly gut. We show that a host-constructed physical niche in the foregut selectively binds bacteria with strain-level specificity, stabilizing their colonization. Primary colonizers saturate the niche and exclude secondary colonizers of the same strain, but initial colonization by Lactobacillus species physically remodels the niche through production of a glycan-rich secretion to favor secondary colonization by unrelated commensals in the Acetobacter genus. Our results provide a mechanistic framework for understanding the establishment and stability of a multi-species intestinal microbiome

Composition of the chemically-defined growth medium.

Composition of the chemically-defined growth medium.</p

Growth of <i>Lactiplantibacillus plantarum</i> isolates in CDM_<i>L</i>.

Growth of Lactiplantibacillus plantarum isolates from different sources grown with variations of CDML in different laboratories (S2 Table in S1 File). (A) 10 isolates predominantly from Drosophila guts grown aerobically with continuous shaking in the CDML with a starting inoculum of 0.01 OD600. Note Lp WCFS1 is a human mouth isolate. (B) Seven isolates of L. plantarum from food fermentations and mammals grown anaerobically in CDML, shaken intermittently before each OD reading. Note Lp WCFS1 in panel A is a single colony derivative of Lp NCIMB8826 in panel B. Both are from the same human mouth isolate. (C) Seven isolates of L. plantarum from food fermenatations grown anaerobically in a rich formulation of CDML with 10x L-cysteine. Solid lines represent means of 12 technical replicates. In panel A, Time point OD600 readings were taken every 5 minutes, and shaded areas represent standard error on a 5-point rolling average. for panels B and C, time point OD600 readings were taken every every 1 hour for panels B and C, and shaded areas represent standard error at each time point (note the error in B and C is very small except at early time points).</p