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

genotype_file_MER_Geraldesetal2012.csv

The file genotype_file_MER_Geraldesetal2012.csv contains the genotyping data used in Geraldes et al. 2012 "A 34K SNP genotyping array for Populus trichocarpa: Design, application to the study of natural populations and transferability to other Populus species". The file contains genotypes for 32,683 loci in 72 accessions. These loci were genotyped with the 34K Populus SNP array described in this manuscript. Details regarding each locus and the genotyping methods can be found in the manuscript. The file contains 32,684 lines and 73 fields per line separated with commas (.csv). The first line is a header line. The first field reads "SNP" and indicates that for each line, the first field contains the name of the Single Nucleotide Polymorphism (SNP). This name has three parts separated with underscores, where the first part is always "scaffold", the second part is the linkage group to which the locus is mapped and the last part is the location (in base pairs) in that linkage group. All names refer to version 2 of the Populus trichocarpa genome available at http://www.phytozome.net/. Each following field in line one, is the name of the accession genotyped. Accession details are provided in Geraldes et al 2012. Each subsequent line has the genotypes for each individual/locus. Each genotype is followed by a "|" and a number ranging from 0 to 1. This number is the GenCall Score, a measure of the confidence in the genotytpe call. Details are in Geraldes et al. 2012. If the Gencall Score is <0.15, the genotype is considered missing and is represented by "--". Other missing genotypes are indicated with #NA

Data from: A 34K SNP genotyping array for Populus trichocarpa: Design, application to the study of natural populations and transferability to other Populus species

Genetic mapping of quantitative traits requires genotypic data for large numbers of markers in many individuals. For such studies, the use of large single nucleotide polymorphism (SNP) genotyping arrays still offers the most cost-effective solution. Herein we report on the design and performance of a SNP genotyping array for Populus trichocarpa (black cottonwood). This genotyping array was designed with SNPs pre-ascertained in 34 wild accessions covering most of the species latitudinal range. We adopted a candidate gene approach to the array design that resulted in the selection of 34 131 SNPs, the majority of which are located in, or within 2 kb of, 3543 candidate genes. A subset of the SNPs on the array (539) was selected based on patterns of variation among the SNP discovery accessions. We show that more than 95% of the loci produce high quality genotypes and that the genotyping error rate for these is likely below 2%. We demonstrate that even among small numbers of samples (n = 10) from local populations over 84% of loci are polymorphic. We also tested the applicability of the array to other species in the genus and found that the number of polymorphic loci decreases rapidly with genetic distance, with the largest numbers detected in other species in section Tacamahaca. Finally, we provide evidence for the utility of the array to address evolutionary questions such as intraspecific studies of genetic differentiation, species assignment and the detection of natural hybrids