Roles of Three Transporters, CbcXWV, BetT1, and BetT3, in Pseudomonas aeruginosa Choline Uptake for Catabolism

Abstract

Pseudomonas aeruginosa uses the quaternary amine choline as a carbon source, osmoprotectant, and macromolecular precursor. The importance of choline in P. aeruginosa physiology is highlighted by the presence of multiple known and putative choline transporters encoded within its genome. This report describes the relative roles of three choline transporters, the ABC transporter CbcXWV and two symporters, BetT1 and BetT3, in P. aeruginosa growth on choline under osmotic conditions that are physiologically relevant to eukaryotic hosts. The increased lag phases exhibited by the ⌬betT1 and ⌬betT1 ⌬betT3 mutants relative to the wild type upon transfer to medium with choline as a sole carbon source suggested roles for BetT1 and BetT3 in cells newly exposed to choline. BetT3 and CbcXWV, but not BetT1, were sufficient to support growth on choline. betT1 and betT3 expression was regulated by the repressor BetI and choline, whereas cbcXWV expression was induced by the activator GbdR and glycine betaine. The data support a model in which, upon transfer to a choline-based medium, the glycine betaine derived from choline taken up by BetT1 and BetT3 promotes subsequent GbdRmediated cbcXWV induction. Furthermore, growth data indicated that the relative contributions of each transporter varied under different conditions, as BetT1 and CbcXWV were the primary choline transporters under hypo-osmolar conditions whereas BetT3 was the major choline transporter under hyperosmolar conditions. This work represents the first systematic approach to unravel the mechanisms of choline uptake in P. aeruginosa, which has the most complex bacterial choline uptake systems characterized to date.

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

“…1). These data also support a model where initial uptake by BetT1 or BetT3 primes the induction of CbcXWV, creating a series of transport steps that can be regulated by choline and GB-sensitive transcription factors, as discussed below (48).…”

“…This section of the review focuses only on transport regulation by the osmolytes themselves, which occurs after the initial osmostress response and under nonstress conditions, and I refrain from review of sensing osmolarity and cell envelope stress, which has been well reviewed elsewhere (18,49,50). Choline and GB transporters in P. aeruginosa can be regulated at the transcriptional level (48). In most bacteria, one of the BCCT-family transporters is close to, and generally divergently transcribed from, the choline oxidase genes and its associated choline-responsive transcription factor (51-53).…”

“…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).…”

“…There are strongly supported GbdR orthologues and orthologues of the entire P. aeruginosa choline degradation pathway in all other sequenced pseudomonads, suggesting similar capabilities across the genus. GbdR controls transcription of the cbcXWV operon in P. aeruginosa, thus stimulating increased transport as part of a positive-feedback loop (48). CbcW is the transmembrane-spanning subunit that assembles to form the transport pore, and CbcX is the In P. aeruginosa, choline and GB import are mediated by the BCCT-family transporters BetT1, BetT2, and BetT3 and the ABC-family transporter CbcWV, but the importance of each changes based on the external osmolarity.…”

“…The P. aeruginosa genome encodes three members of the BCCT (betaine-choline-carnitine transport) family and a single specific ABC (ATP binding cassette) family transporter predicted to be involved in quaternary amine transport. The BCCT family was largely thought to transport compatible solutes for the purpose of osmoprotection, while the ABC transporter has been shown to be important for bulk transport of choline as a primary carbon source (27,47,48). Recent work has elaborated the model of choline and GB uptake by P. aeruginosa, where it was shown that the four active transporters (BetT1, BetT2, BetT3, and CbcXWV) function to span the range of physiologic osmolarities to provide optimal uptake from the environment (48,117) (Fig.…”