Secretion of Flavins by Three Species of Methanotrophic Bacteria

Abstract

We detected flavins in the growth medium of the methanotrophic bacterium Methylocystis species strain M. Flavin secretion correlates with growth stage and increases under iron starvation conditions. Two other methanotrophs, Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), secrete flavins, suggesting that flavin secretion may be common to many methanotrophic bacteria.Methanotrophic bacteria utilize methane as their sole carbon source and play a key role in the global carbon cycle. The first step in methane metabolism, the oxidation of methane to methanol, is catalyzed by methane monooxygenase (MMO) metalloenzymes by either a copper-dependent membranebound particulate MMO (pMMO) (9) or an iron-dependent, soluble MMO (sMMO) (18). Therefore, metals are necessary for the growth of methanotrophic bacteria (12,13). Despite the importance of metals in methanotroph physiology, uptake of metal ions by methanotrophic bacteria is not well understood. To study metal acquisition, we purified and characterized components of the spent growth medium from Methylocystis species strain M. Unexpectedly, we detected extracellular flavins (riboflavin and flavin mononucleotide [FMN]).To perform these experiments, the type II methanotroph Methylocystis sp. strain M was grown using a procedure similar to that described for Methylosinus trichosporium OB3b (7, 8, 10) (see the supplemental material). The spent growth medium from Methylocystis sp. strain M was purified by fast protein liquid chromatography (FPLC) using a Discovery DSC-18 reverse-phase column. The eluted fractions exhibit UV-visible spectral features that suggest the presence of flavins (Fig. 1A). When the fractions were purified further using a Supelco C 18 column, the purified flavins exhibited absorption features that were identical to those of commercial riboflavin (see Fig. S1 in the supplemental material). Upon excitation at 441 nm, the fluorescence emission spectrum of the purified fractions exhibited a broad peak between 450 and 700 nm, with a maximum at 523 nm (Fig. 1B). This feature is characteristic of the isoalloxazine ring in flavins (11,21).To characterize the amount of extracellular flavins secreted throughout growth, the cells were grown under three metal availability conditions: default growth ( Fig. S2 of the supplemental material. The growth rates, from an average of biological triplicates, determined for default, iron-starved, and copper-starved growth conditions between 12 and 28 h of growth are 0.043 Ϯ 0.003 h Ϫ1 , 0.014 Ϯ 0.003 h Ϫ1 , and 0.052 Ϯ 0.002 h Ϫ1 , respectively. These values imply that the availability of iron affects the growth of Methylocystis sp. strain M.We then characterized the amount of extracellular flavins in the spent medium isolated from three different stages of growth. The samples used for quantitation were normalized based on optical density measured at 600 nm. The dry weight and cell length, normalized to the optical density, remained unaffected by growth under different metal concentrations (see Fig. S4). ...

Dual Pathways for Copper Uptake by Methanotrophic Bacteria

“…It would not be surprising if Methylocystis sp. strain M could internalize Cu-Mb purified from M. trichosporium OB3b because it appears to produce a Mb of identical mass (20). The chemical composition of M. capsulatus (Bath) Mb is not known (17), and it is not clear whether M. alcaliphilum 20Z produces Mb.…”

“…The peptidic nature of Mb is analogous to the structures of many iron siderophores (14 -16), suggesting that it is a chalkophore ("sidero" is Greek for iron; "chalko" is Greek for copper) (13). Other methanotrophs, including Methylococcus capsulatus (Bath), Methylomicrobium album BG8, Methylocystis species strain M, and Methylocystis strain SB2, have been reported to produce Mb (17)(18)(19)(20), but it remains unclear whether there are different structural classes of Mb. It is not known how Mb is biosynthesized, and both nonribosomal and ribosomal peptide synthesis pathways have been suggested (1,12,18,21).…”

Extracellular Electron Transport-Mediated Fe(III) Reduction by a Community of Alkaliphilic Bacteria That Use Flavins as Electron Shuttles

“…Given that even mesophilic bacteria can adopt a wide variety of mechanisms to perform similar physiological functions when interacting with their environment (74) and the stress of a challenging environment has led extremophilic bacteria to evolve distinctly different mechanisms in many cases (45,75), it is striking that the electronshuttling compound found in this study of alkaliphiles is indistinguishable from that used by mesophiles. Interestingly, flavins have also been found in the culture supernatants of several methanotrophic species (36), indicating that this method of extracellular electron transfer may be more widespread among anaerobic communities living on the brink of life than first thought.…”

“…To date it has been shown that Shewanella spp. and several methanotrophic bacteria can release flavins (i.e., flavin mononucleotide [FMN] and riboflavin [30,36]) as electron shuttles. As yet it is uncertain whether bacteria can also release quinone-like compounds as electron shuttles in response to a metabolic requirement (37) or whether this is an opportunistic use of substances found in the environment.…”

Description of a Riboflavin Biosynthetic Gene Variant Prevalent in the Phylum Proteobacteria

“…More recently, a wider role for flavins in the physiology of microorganisms is coming to light, as a number of bacteria have been found to use free, extracytoplasmic flavins to carry out vital processes beyond the borders of the cell. Flavins are important for assimilatory iron reduction in Campylobacter jejuni, Helicobacter pylori, and three species of methanotrophic bacteria (2)(3)(4). Shewanella oneidensis and Geothrix fermentans use secreted flavin electron shuttles to accelerate respiration of insoluble minerals and electrodes (5)(6)(7)(8).…”