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.

FIG. 1.

Predicted pathway for choline catabolism in P. aeruginosa (5, 11).

FIG. 2.

Genomic arrangement of the genes involved in GB catabolism. Genes identified in the screen are indicated by filled arrows, and the transposon insertion sites are indicated by open triangles. (A) Genomic region surrounding PA5410 and PA5411. (B) Genomic region including the PA5396-PA5399 putative operon. (C) Genes in the vicinity of PA5380.

FIG. 3.

Semiquantitative RT-PCR analysis of P. aeruginosa cells after 2 h of induction with pyruvate (P), GB, DMG (D), or sarcosine (S). PA5410 (gbcA), PA5411 (gbcB), PA5398 (dgcA), PA5399 (dgcB), and PA5380 (gbdR) transcript levels are shown. rplU was used as a control transcript.

FIG. 4.

¹³C NMR spectra of P. aeruginosa cell extracts after incubation with [1,2-¹³C]choline as the sole carbon source. (A) Wild type at 3 h. (B) ΔgbcA-gbcB mutant at 9 h. (C) ΔdgcB::TnM mutant at 9 h. (D) soxA::TnM mutant at 9 h. (E) ΔPA5380 (ΔgbdR) mutant at 9 h. The peaks are labeled. cho, choline; S, sarcosine.

FIG. 5.

Semiquantitative RT-PCR analysis of P. aeruginosa RNA after 2 h of induction by different compounds. The primer sets used in the PCR are indicated on the left. (A) Wild-type (WT) and ΔgbdR (Δ) cells induced with different carbon sources, including choline (cho), GB, DMG, and sarcosine (sarc). (B) ΔgbcA-gbcB and dgcB::TnM cells induced with different carbon sources, including GB (G), DMG (D), and pyruvate (P). (C) Wild-type and betA::TnM cells induced with choline.

FIG. 6.

Model of P. aeruginosa GB catabolism modified from the model of Diab et al. (11), with additions based on the data presented in this paper. BetA is the choline oxidase. BetB is betaine aldehyde dehydrogenase. BetI is the transcriptional repressor of the betABI locus. GbcA and GbcB are the predicted GB demethylase. DgcAB are the predicted DMG demethylase. SoxBDAG are the sarcosine oxidase complex members. Question marks indicate steps that have not been thoroughly evaluated in P. aeruginosa.