Siderophores from neighboring organisms promote the growth of uncultured bacteria

Abstract

The majority of bacterial species do not grow on synthetic media. Many non-growers require growth factors from other bacteria, but the nature of these compounds is largely unknown. We show here that previously uncultured isolates from marine sediment biofilm grow on a Petri dish in the presence of cultured organisms from the same environment. The growth factors produced by one cultured helper strain were identified as new acyl-desferrioxamine siderophores. A panel of previously uncultured isolates exhibited a range of siderophore promiscuity for growth promotion. This siderophore-based approach has enabled the culturing of organisms only distantly related to previously cultured microbes. The lack of growth in the laboratory for many strains from this habitat stems from an inability to autonomously produce siderophores, and the resulting chemical dependence on other microorganisms regulates community establishment in the environment.

Fig. 1

Intertidal sand grains. (A) Photograph of a collection of washed sand grains in seawater, scale bar = 3 mm. (B) Overview scanning electron micrograph (SEM) of a single sand grain, scale bar = 50 µm. (C) Higher resolution SEM of the boxed region in image B. Scale bar = 10 µm. (D) SEM image of a biofilm that can be seen attached to the surface of the sand particle, which represents the source of the bacterial cells that were resuspended by vortexing for isolation in this study. Scale bar = 10 µm. (E) SEM image of individual bacteria attached to the surface of the sand particle. Scale bar = 3 µm.

Fig. 2

Growth of M. polysiphoniae KLE1104 is induced by M. luteus KLE1011. Bacterial cells were re-suspended from sand biofilms and plated to isolate species dependent on neighboring colonies. (A) Isolation spread plate of bacteria from sand grains. (B) Environmental helper M. luteus KLE1011 cross-streaked (right side of plate) with unculturable isolate M. polysiphoniae KLE1104 (left side of plate). Colonies of the unculturable strain are larger when in closer proximity to the helper. (C) Filtered spent supernatant of M. luteus KLE1011 induces growth of M. polysiphoniae KLE1104. In all cases tested, the uncultured phenotypes, including KLE1011, remained stable. See also Table S7.

Fig. 3

Growth induction of M. polysiphoniae KLE1104 by enterobactin from E. coli. Filtered spent supernatant of E. coli BW25113 induces growth of M. polysiphoniae KLE1104, but spent culture media from enterobactin mutant strains do not. (A) Supernatant from the parental E. coli BW25113 strain induces growth, as does supernatant from strains deficient in (B) ΔluxS and (C) ΔtnaA. There was more growth around the tnaA mutant in this experiment, but subsequent examination showed that it was due to variation in this qualitative test. Supernatant of strains deficient in enterobactin synthesis (D) ΔentB and (E) ΔentC do not induce growth of the unculturable. Growth was not observed even at high densities of KLE1104. (F) Purified enterobactin spotted on a plate evenly spread with M. polysiphoniae KLE1104 induced growth of the isolate. Colony size decreased with increasing distance from the enterobactin source and no growth of the unculturable was seen further away from the source.

Fig. 4

Active siderophores produced by helper strain M. luteus KLE1011. (A) Reversed-phase C18 HPLC-MS/UV-visible spectroscopic analysis. The ferric and desferric siderophores were separated over C18 HPLC, demonstrating a range of hydrophobic interactions with the nonpolar resin. The more polar ferric forms eluted faster than the desferric forms, showed masses indicative of iron complexation (MS), and exhibited characteristic iron-ligand charge transfer absorption (UV-visible). (B) Acyl-desferrioxamine siderophore structures. See also Figures S1–S2 and Tables S1–S6.

Fig. 5

Siderophores tested for growth induction ability. The structures of 21 representative siderophores are shown, which were each tested for growth induction of 8 uncultured species. The siderophores include cyclic and linear trihydroxamates, dihydroxamates, carboxylic acid-types, phenols, catechols, oxazolines, and thiazolines that provide a variety of iron-binding structural types that would target various siderophore receptors.

Fig. 6

Isolation of distantly related uncultured microorganisms. Three organisms only distantly related to typed strains were isolated on high [Fe(II)] media. (A) The cultivable Lacinutrix sp. KLE1211 (yellow spot) induces growth of the uncultured Verrucomicrobia isolate, KLE1210 (orange ring). (B,C) SEM images of Verrucomicrobia KLE1210, scale bars B,C = 1 µm, 5 µm. (D) Parvularculaceae KLE1250 (orange ring), growing near a filter disk (white spot) containing 10 µl of 1% iron sulfate. (E–F) SEM images of Parvularculaceae KLE1250, scale bars E,F = 1 µm, 10 µm. (G) Gammaproteobacterium KLE1212 (yellow ring) is induced by a cultivable species, Vibrio tasmaniensis KLE1213 (beige spot), isolated from the same sand biofilm. (H–I) SEM images of the uncultured isolate, Gammaproteobacterium KLE1212, scale bars H,I = 1 µm, 5 µm. See also Figures S3–S6.