GcsR, a TyrR-Like Enhancer-Binding Protein, Regulates Expression of the Glycine Cleavage System in Pseudomonas aeruginosa PAO1

Abstract

Glycine serves as a major source of single carbon units for biochemical reactions within bacterial cells. Utilization of glycine is tightly regulated and revolves around a key group of proteins known as the glycine cleavage system (GCS). Our lab previously identified the transcriptional regulator GcsR (PA2449) as being required for catabolism of glycine in the opportunistic pathogen Pseudomonas aeruginosa PAO1. In an effort to clarify and have an overall better understanding of the role of GcsR in glycine metabolism, a combination of transcriptome sequencing and electrophoretic mobility shift assays was used to identify target genes of this transcriptional regulator. It was found that GcsR binds to an 18-bp consensus sequence (TGTAACG-N4-CGTTCCG) upstream of the gcs2 operon, consisting of the gcvH2, gcvP2, glyA2, sdaA, and gcvT2 genes. The proteins encoded by these genes, namely, the GCS (GcvH2-GcvP2-GcvT2), serine hydroxymethyltransferase (GlyA2), and serine dehydratase (SdaA), form a metabolic pathway for the conversion of glycine into pyruvate, which can enter the central metabolism. GcsR activates transcription of the gcs2 operon in response to glycine. Interestingly, GcsR belongs to a family of transcriptional regulators known as TyrR-like enhancer-binding proteins (EBPs). Until this study, TyrR-like EBPs were only known to function in regulating aromatic amino acid metabolism. GcsR is the founding member of a new class of TyrR-like EBPs that function in the regulation of glycine metabolism. Indeed, homologs of GcsR and its target genes are present in almost all sequenced genomes of the Pseudomonadales order, suggesting that this genetic regulatory mechanism is a common theme for pseudomonads. IMPORTANCE Glycine is required for various cellular functions, including cell wall synthesis, protein synthesis, and the biosynthesis of several important metabolites. Regulating levels of glycine metabolism allows P. aeruginosa to maintain the metabolic flux of glycine through several pathways, including the metabolism of glycine to produce other amino acids, entry into the trichloroacetic acid cycle, and the production of virulence factors such as hydrogen cyanide. In this study, we characterized GcsR, a transcriptional regulator that activates the expression of genes involved in P. aeruginosa PAO1 glycine metabolism. Our work reveals that GcsR is the founding member of a novel class of TyrR-like EBPs that likely regulate glycine metabolism in Pseudomonadales.

Keywords: Glycine metabolism; Pseudomonas aeruginosa PAO1; TyrR; enhancer-binding proteins; transcription factors.

FIG 1

ΔgcsR PAO1 is unable to grow in glycine as a sole carbon source. P. aeruginosa PAO1 and ΔgcsR PAO1 were grown with glycine as the sole carbon source for 48 h at 37°C. Data points represent mean values ± the standard deviations (n = 3). Student’s t test was performed to identify significant differences (P < 0.0001; marked with an asterisk).

FIG 2

The five gcs2 genes are transcribed as an operon. (A) At the top is a schematic of the gcs2 gene cluster. Intergenic regions that were amplified are designated A to D. At the bottom is a schematic of the rplU gene, encoding the 50S ribosomal protein (L21), which was was used as a control for the RT-PCR analysis (amplified region designated E). (B) RT-PCR analysis of the gcs2 gene cluster. The regions designated A to E were amplified by PCR with cDNA, RNA, or genomic DNA obtained from P. aeruginosa PAO1 as the template.

FIG 3

Expression of P_gcvH2::lacZ is induced by glycine. (A) Wild-type or ΔgcvA or ΔgcvR E. coli cells harboring the P_gcvH2::lacZ reporter construct and the pBRL620-5 plasmid for expression of GcsR were grown in LB to an OD₆₀₀ of ~0.3 and then challenged with 10 mM glycine, 10 mM glutamate, 10 mM serine, or no substrate. (B) E. coli ΔgcvA mutant cells harboring empty plasmids pTrc99a and ΔP_lac-pBBR1MCS-5, plasmids pBRL456 containing the P_gcvH2::lacZ reporter and pBRL620-5 for the expression of GcsR, plasmid pBRL456 and plasmid pBRL620-3 harboring gcsR without a promoter, plasmids ΔP_lac-pBBR1MCS-5 and pBRL620-5, or plasmids pBRL456 and pTrc99a were grown in LB to an OD₆₀₀ of ~0.3 and then challenged with 10 mM glycine or no substrate. Data points represent mean values ± the standard deviations (n = 3). Analysis of variance was performed by using Dunnett’s post hoc test (α value of 0.05) to identify significant differences (P < 0.0001; marked with an asterisk).

FIG 4

Expression of P_gcvH2::lacZ is induced with glycine in P. aeruginosa PAO1. P. aeruginosa wild-type (WT) PAO1, ΔgcsR PAO1, or ΔrpoN PAO1 cells harboring the P_gcvH2::lacZ reporter construct was grown in M9 minimal medium to an OD₆₀₀ of ~0.3 and then challenged with 10 mM glycine, 10 mM glutamate, 10 mM serine, or no substrate. Data points represent mean values ± the standard deviations (n = 3). Analysis of variance was performed by using Dunnett’s post hoc test (α value of 0.05) to identify significant differences (P < 0.0001; marked with an asterisk).

FIG 5

GcsR binds the gcvH2 promoter region. EMSAs were performed with His₆-GcsR and 2 nM Cy5-labeled probe DNA unless specified otherwise. (A) P_PA5530 (nonspecific) or P_gcvH2 (specific) was incubated in the absence (lanes 1 and 3, respectively) or presence (lanes 2 and 4, respectively) of 200 nM His₆-GcsR. (B) His₆-GcsR (0 to 200 nM) was incubated with P_gcvH2. (C) A 200 nM concentration of His₆-GcsR was incubated with P_gcvH2 and increasing concentrations of unlabeled specific competitor *P_gcvH2 (lane 3 to 5) or 100 nM unlabeled nonspecific competitor *P_PA5530 (lane 6).

FIG 6

GcsR binds to three 18-bp tandem repeats in the gcvH2 promoter region. EMSAs were performed with His₆-GcsR and 2 nM Cy5-labeled probe DNA. (A) Sequence of the 217-bp region upstream of the gcvH2 gene. The three 18-bp GcsR binding sites are in bold type. The underlined nucleotides in the GcsR binding sites were mutated to G in this study. The RpoN binding site is in bold italic type. (B) A 200 nM concentration of His₆-GcsR was incubated with P_gcvH2 (lane 2) or P_gcvH2-mut1 (lane 4). (C) P_gcvH2-2, P_gcvH2-2-mut, P_gcvH2-3, and P_gcvH2-3-mut were incubated in the absence (lanes 1, 3, 5, and 7, respectively) or presence (lanes 2, 3, 6, and 8, respectively) of 200 nM His₆-GcsR. (D) Consensus sequence of the GcsR binding site derived from the three 18-bp tandem repeats in the P_gcvH2 sequence constructed with WebLogo 3.4.

FIG 7

GcsR has divalent-cation-dependent phosphatase activity. A 5 µM sample of His₆-GcsR was incubated with the substrate p-nitrophenylphosphate in the absence of any cofactor or in the presence of 2 mM glycine, 2 mM Mg²⁺, 2 mM Zn²⁺, or 2 mM Co²⁺. Data points represent mean values ± the standard deviations (n = 3). Analysis of variance was performed by using Dunnett’s post hoc test (α value of 0.05) to identify significant differences (P < 0.0001; marked with an asterisk).

FIG 8

GcsR affects pyocyanin production through the MexEF-OprN efflux pump. (A) Overexpression of rhlI or deletion of the mexF gene inactivating the MexEF-OprN pump in the gcsR::Tn mutant strain PW5126 restores pyocyanin production in PW5126. The ΔgcsR PAO1 strain produces pyocyanin levels similar to those of PAO1. Data points represent mean values ± the standard deviations (n = 3). Analysis of variance was performed by using Dunnett’s post hoc test (α value of 0.05) to identify significant differences (P < 0.0001; marked with an asterisk). (B) The P_mexE probe was incubated in the absence (lane 1) or presence (lane 2) of 200 nM His₆-GcsR.

FIG 9

P. aeruginosa ΔgcsR PAO1 is lethal to C. elegans in a paralytic-killing model. (A) Course of C. elegans survival in a paralytic-killing assay with E. coli OP50 (closed circles; n = 195), P. aeruginosa lecA::luxΔlasR PAO1 (closed squares; n = 197), P. aeruginosa PAO1 (closed triangles; n = 273), and P. aeruginosa ΔgcsR PAO1 (open triangles; n = 351). C. elegans survival was significantly reduced when it was exposed to P. aeruginosa ΔgcsR PAO1. The log rank test was performed to identify significant differences (P < 0.0001; marked with asterisks). (B, C) Appearance of C. elegans after a 2-h exposure to P. aeruginosa PAO1. Scale bars are 100 µm. (D, E) Appearance of C. elegans after a 2-h exposure to P. aeruginosa ΔgcsR PAO1. Scale bars are 100 µm.

FIG 10

The P. fluorescens Pf0-1, P. putida KT2440, and P. syringae pv. tomato DC3000 genomes harbor gcsR orthologs. (A) Organization of the gcsR gene and the gcs gene cluster in P. fluorescens Pf0-1, P. putida KT2440, and P. syringae pv. tomato DC3000. (B) Consensus sequence of putative binding sites of GcsR orthologs from P. fluorescens Pf0-1, P. putida KT2440, and P. syringae pv. tomato DC3000 constructed with WebLogo 3.4.