Identification of Two Gene Clusters and a Transcriptional Regulator Required for Pseudomonas aeruginosa Glycine Betaine Catabolism

Abstract

Glycine betaine (GB), which occurs freely in the environment and is an intermediate in the catabolism of choline and carnitine, can serve as a sole source of carbon or nitrogen in Pseudomonas aeruginosa. Twelve mutants defective in growth on GB as the sole carbon source were identified through a genetic screen of a nonredundant PA14 transposon mutant library. Further growth experiments showed that strains with mutations in two genes, gbcA (PA5410) and gbcB (PA5411), were capable of growth on dimethylglycine (DMG), a catabolic product of GB, but not on GB itself. Subsequent nuclear magnetic resonance (NMR) experiments with 1,2-13 C-labeled choline indicated that these genes are necessary for conversion of GB to DMG. Similar experiments showed that strains with mutations in the dgcAB (PA5398-PA5399) genes, which exhibit homology to genes that encode other enzymes with demethylase activity, are required for the conversion of DMG to sarcosine. Mutant analyses and 13 C NMR studies also confirmed that the soxBDAG genes, predicted to encode a sarcosine oxidase, are required for sarcosine catabolism. Our screen also identified a predicted AraC family transcriptional regulator, encoded by gbdR (PA5380), that is required for growth on GB and DMG and for the induction of gbcA, gbcB, and dgcAB in response to GB or DMG. Mutants defective in the previously described gbt gene (PA3082) grew on GB with kinetics similar to those of the wild type in both the PAO1 and PA14 strain backgrounds. These studies provided important insight into both the mechanism and the regulation of the catabolism of GB in P. aeruginosa.A number of microbes, including Pseudomonas aeruginosa, can utilize glycine betaine (GB) as a sole carbon, nitrogen, and energy source (17,35,41). GB, an important osmoprotectant for many bacteria (6), is available to organisms in a variety of environments (5,14,34,41). Free GB can be released by roots (9), microbes (14, 15), or decaying animal (20) and plant (10) matter. Alternatively, GB can be derived from choline or carnitine (4,5,12,15,20,36). Choline and carnitine can be found in many eukaryote-associated environments, and bacteria, including P. aeruginosa, can use phospholipases and choline phosphatases to release choline from phosphatidylcholine (30, 38). In P. aeruginosa, choline is oxidized to GB by a two-step process catalyzed by BetA and BetB (29,36), while carnitine is predicted to be reduced and deacetylated by uncharacterized enzymes, ultimately yielding GB (16).The aerobic catabolism of GB in bacteria is best understood in Sinohizobium (35), Corynebacterium (8, 37), and Arthrobacter species (24). The data from these studies suggest that GB catabolism occurs via serial demethylation that forms dimethylglycine (DMG), then sarcosine (also called monomethylglycine), and finally glycine (Fig. 1). Thin-layer chromatographic analyses indicated that in P. aeruginosa DMG and sarcosine are also intermediates formed during GB catabolism (11). Furthermore, in the same study, a proteomics analysis of P. aeruginosa cul...

Homeostasis and Catabolism of Choline and Glycine Betaine: Lessons from Pseudomonas aeruginosa

“…Sarcosine demethylation is conducted by a heterotetrameric enzyme, SoxBDAG, which is homologous to the proteins encoded by sarcosine oxidase genes in Corynebacterium and Stenotrophomonas (formerly Pseudomonas) maltophilia (78,79). The genes in this catabolic pathway in P. aeruginosa have been established on the basis of genetic analysis (56), and those findings are supported by protein induction data (80) (Fig. 2).…”

“…In P. aeruginosa, GB-responsive transcripts are induced via the AraC-family transcriptional activator GbdR (56) (Fig. 2).…”

“…The AraC family members canonically regulate genes involved in metabolism (e.g., AraC and XylS for regulation of arabinose and xylene/benzoate metabolism, respectively [59,60]) and/or virulence (61) (e.g., ExsA regulation of type III secretion in P. aeruginosa [62] and ToxT regulation of cholera toxin and the toxincoregulated pilus in Vibrio cholerae [63]). Evidence suggests that GbdR senses GB and dimethylglycine levels in the cell and induces transcription of genes involved in virulence, GB transport, GB catabolism, and detoxification of the catabolic byproducts, hydrogen peroxide and formaldehyde (48,56,(64)(65)(66)(67)(68) (Fig. 2).…”

“…Pseudomonads oxidize choline to GB using a pair of enzymes encoded in the same operon, choline oxidase (BetA) and betaine aldehyde dehydrogenase (BetB). The GB resulting from choline oxidation is then demethylated, a reaction that is predicted to use the oxygenase activity of GbcAB to demethylate GB to dimethylglycine and formaldehyde (56). The demethylation of dimethylglycine is carried out by a heterodimeric flavin-linked oxidoreductase comprised of DgcA and DgcB (56,64), which are homologous to the proteins encoded by dimethylglycine catabolic genes of Arthrobacter spp.…”

“…BetI is a member of the TetR family of transcriptional repressors that bind palindromic sequences as homodimers and are generally involved in resistance to antimicrobials and stress, including osmotic stress (88). Detection of GB by GbdR mediates the transcriptional response involved in GB catabolism (56,67) (Fig. 2).…”