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Evidence That COG0325 Proteins are involved in PLP Homeostasis
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Laurence Prunettia, Basma El Yacoubia, Cara R. Schiavona, Ericka Kirkpatricka, Lili Huangb,
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Marc Baillya*, Mona ElBadawi-Sidhuc, Katherine Harrisona, Jesse F. Gregory 3rd b, Oliver
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Fiehnc, Andrew D. Hansond and Valérie de Crécy-Lagarda#
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Department of Microbiology and Cell Science, Institute for Food and Agricultural Sciences and
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Genetic Institute, University of Florida, Gainesville, Florida, USAa; Department of Food Science
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and Human Nutrition, University of Florida, Gainesville, Florida, USAb; Department of
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Molecular and Cellular Biology & Genome Center, University of California, Davis, California,
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USAc; Department of Horticultural Sciences, University of Florida, Gainesville, Florida, USAd
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* Current address: Merck, Palo Alto California, USA
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L. P. and B. E.-Y. contributed equally to this work
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#Address correspondence to Valérie de Crécy-Lagard, [email protected]
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Running title: Function of E.coli yggS
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Keywords
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yggS; COG0325; vitamin B6; PLP protein; PROSC; pyridoxine toxicity; protein of unknown
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function.
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Subject category: Physiology and metabolism
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Word count
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Abstract= 195
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Main text (excluding abstract, and references) =5695
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Abbreviations
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GSA: Glutamic 5-semialdehyde; P5C: Δ1-pyrroline-5 carboxylic acid;; THF: Tetrahydrofolate;
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CH2-THF: 5,10-methylene-tetrahydrofolate; Gcv: Glycine-cleavage complex; PLP: Pyridoxal 5’-
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phosphate; PL: pyridoxal; PN: pyridoxine; PM: pyridoxamine; PNP: Pyridoxine 5’-phosphate;
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PROSC: PROline Synthase Co-transcribed homolog; ALR: alanine racemase; ODC: ornithine
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decarboxylase; meth: methionine; dT: thymidine; Cm: chloramphenicol; Amp: ampicillin.; Kan:
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kanamycin; Tet: tetracycline; aTet: anhydrotetracycline.
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ABSTRACT
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Pyridoxal 5'-phosphate (PLP) is an essential cofactor for nearly 60 Escherichia coli enzymes but
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is a highly reactive molecule that is toxic in its free form. How PLP levels are regulated and how
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PLP is delivered to target enzymes are still open questions. The COG0325 protein family
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belongs to the fold-type III class of PLP enzymes and binds PLP but has no known biochemical
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activity although it occurs in all kingdoms of life. Various pleiotropic phenotypes of the
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Escherichia coli COG0325 (yggS) mutant have been reported, some of which were reproduced
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and extended in this study. Comparative genomic, genetic and metabolic analyses suggest that
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these phenotypes reflect an imbalance in pyridoxal 5'-phosphate (PLP) homeostasis. The E. coli
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yggS mutant accumulates the PLP precursor pyridoxine 5'-phosphate (PNP), and is sensitive to
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an excess of pyridoxine but not of pyridoxal. The pyridoxine toxicity phenotype is
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complemented by the expression of eukaryotic yggS orthologs. It is also suppressed by the
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presence of amino acids specifically isoleucine, threonine and leucine suggesting the PLP
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dependent enzyme transaminase B (IlvE) is affected. These genetic results lay a foundation for
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future biochemical studies of the role of COG0325 proteins in PLP homeostasis.
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Introduction
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Pyridoxal 5’-phosphate (PLP) is one of six interconvertible vitamin B6 species (pyridoxal or PL,
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pyridoxine or PN, pyridoxamine or PM and their 5'-phosphate forms). Enzymes utilizing PLP as
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cofactor are found in all organisms, catalyze diverse reactions including transamination,
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decarboxylation, racemization and β- and - elimination, and are mostly associated with amino
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acid metabolism (Percudani and Peracchi 2009). PLP-enzymes belong to seven structurally
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distinct families (fold types I-VII) that probably arose independently (Christen and Mehta 2001;
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Percudani and Peracchi 2003) and encompass 184 enzyme activities. PLP metabolism is
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particularly relevant to human health because several disorders have been linked to PLP
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deficiency (Clayton 2006; Halsted 2013; Paul et al. 2013).
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While the biosynthesis, interconversion and salvage pathways for vitamin B6 species are
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well characterized (Fitzpatrick et al. 2010; Mooney and Hellmann 2010; Herrero et al. 2011;
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Mukherjee et al. 2011; Sang et al. 2011; Rueschhoff et al. 2013; Szydlowski et al. 2013), little is
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known about the regulation of PLP synthesis or about the connection between PLP and general
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metabolism (Shi et al. 2002; Chen and Xiong 2005; Titiz et al. 2006; Rueschhoff et al. 2013;
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Vanderschuren et al. 2013). How PLP molecules are delivered to their target enzymes and how
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the free/bound PLP pool is regulated are also poorly understood (di Salvo et al. 2012). PLP
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reacts with apo-B6 enzymes by forming an aldimine linkage with the ε-amino group of the active
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site lysine residue to produce the catalytically active holo-B6 enzyme forms. Alternatively, its
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highly reactive 4′-aldehyde group can spontaneously form unwanted aldimines with the ε-amino
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group of lysine residues of non-B6 proteins and with many other amines, and thiazolidine
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adducts with sulfhydryl groups of molecules such as cysteine, potentially leading to enzyme
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inactivation (Ohsawa and Gualerzi 1981; Dong and Fromm 1990; Mizushina et al. 2003;
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Vermeersch et al. 2004) and accumulation of damaged metabolites. For example, PLP reacts
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through a Knoevenagel condensation with Δ1-pyrroline-5-carboxylate (P5C) (Fig. 1) and Δ1-
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piperidine-6-carboxylate (P6C) (intermediates in proline and lysine metabolism, respectively)
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that accumulate in patients deficient in P5C-dehydrogenase and in α-aminoadipic
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semialdehyde/P6C dehydrogenase, respectively, leading to PLP deficiency (Fig. 1) (Farrant et al.
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2001; Clayton 2006; Mills et al. 2006).
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The high chemical reactivity of PLP requires a tight control of the free PLP pool such
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that an appropriate supply is available to apo-B6 enzymes while undesirable interactions are
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minimized. It has been proposed that PLP availability is regulated through product feedback
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inhibition and tight binding to PL kinase (PdxK), PNP oxidase (PdxH) as well as PLP synthase
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(PdxJ) (Yang and Schirch 2000; Moccand et al. 2011; Ghatge et al. 2012). However, a precise
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model of PLP homeostasis and how PLP is delivered to target enzymes is still lacking in any
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organism and is required to understand and manage PLP-related diseases.
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A candidate for a missing player in PLP homeostasis is the YggS/PROSC/YBL036C
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family (COG0325). This family belongs to the fold-type III class, along with alanine racemases
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and certain decarboxylases (Percudani and Peracchi 2009), and has been shown to bind PLP
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(Eswaramoorthy et al. 2003). Crystal structures of the yeast and Escherichia coli COG0325
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proteins have been determined [(Eswaramoorthy et al. 2003) and PDB id 1W8G]. Like the N-
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terminal domain of alanine racemase (ALR) and ornithine decarboxylase (ODC), this protein
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folds as a TIM barrel with a characteristic long N-terminal helix, and binds PLP in a similar
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mode. Unlike the dimeric ALR, YBL036C was found to be monomeric, and it was recently
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reported that the E. coli member of the family, YggS, has no racemase activity towards any of
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the 20 protein amino acids or their D enantiomers (Ito et al. 2013).
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Both the broad phylogenetic distribution and the pleiotropic phenotypes linked to the
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mutations of members of the COG0325 family are suggestive of a core conserved function. In E.
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coli, the ΔyggS ΔglyA double mutant is not viable on LB medium (Nichols et al. 2011). GlyA
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(SHMT, serine hydroxymethyltransferase) converts serine to glycine and in the process transfers
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a hydroxymethyl group to tetrahydrofolate (THF) forming 5,10-methylene-tetrahydrofolate
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(CH2-THF), the major source of C1 units in the cell (Green et al. 1996) (Fig. 1). GlyA, and the
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glycine cleavage enzyme system (Gcv), another source of one carbon units, are key to the
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biosynthesis of purines, thymidine, methionine, and lipids (Fig. 1). Mutants in the THF pathway
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are sensitive to sulfonamides (targeting FolP) and trimethoprim (targeting FolA) (Nichols et al.
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2011). Like mutants in the Gcv system, the ΔyggS strain is sensitive to sulfonamides and not to
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trimethoprim while the serine hydroxymethyltransferase mutant (ΔglyA::KanR) is sensitive to
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trimethoprim and not to sulfonamides (Nichols et al. 2011). Recently, ΔyggS strains were shown
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to display altered intracellular amino acid and acetyl coenzyme A (CoA) pools, and to excrete L-
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valine in the culture medium while accumulating pyruvate, 2-ketobutyrate and 2-aminobutyrate
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(Ito et al. 2013). Another connection with amino acid metabolism comes from Pseudomonas
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aeruginosa, whose yggS homolog was proposed to be co-transcribed with proC, the gene
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encoding P5C reductase that catalyzes the last step of proline synthesis (Fig. 1). This observation
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gave the PROline Synthase Co-transcribed homolog (PROSC) name to the first studied members
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of the family including the human homolog, which is expressed in all organs (De Wergifosse et
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al. 1994). Finally, in yeast, the COG0325 protein YBL036C was induced three-fold in response
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to the DNA-damaging agent, methyl methanesulphonate (MMS), implying involvement in
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processes ensuring genetic integrity (Lee et al. 2007).
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In this work, genetic, biochemical and comparative genomics approaches were combined
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to show that COG0325 family proteins have a role in PLP homeostasis that could explain the
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pleiotropic phenotypes of the yggS strain.
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MATERIAL AND METHODS
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Bioinformatic analyses. The BLAST tools (Altschul et al. 1997) and resources at NCBI
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(http://www.ncbi.nlm.nih.gov/) were routinely used. Multiple sequence alignments were built
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using Clustal Omega (Li et al. 2015) or Multalin (Corpet 1988). Protein domain analysis was
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performed using the Pfam database tools (Finn et al. 2014). Analysis of phylogenetic distribution
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and physical clustering was performed in the SEED database (Overbeek et al. 2014). Results are
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available
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(http://pubseed.theseed.org/SubsysEditor.cgi). The representative genome sets ( set 1 of ~1000
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genomes and set 2 of ~1500 genomes) were chosen based on phylogenetic diversity
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previously described (Dailey et al. 2015; Niehaus et al. 2015).The Ortho-MCL database (Chen et
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al. 2006) was queried for the analysis of COG0325 orthologs in Eukarya and BLASTp (Altschul
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et al. 1997) searches were performed specifically against sequences of archaeal genomes.
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Physical clustering was analyzed with the SEED subsystem coloring tool or the SeedViewer
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Compare Regions tool (Overbeek et al. 2014). The protein association network analysis was
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performed on the STRING database (string-db.org/) (Szklarczyk et al. 2015). The Enzyme
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Function Initiative-Enzyme Similarity Tool (EFI-EST) (http://enzymefunction.org/) was used to
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extract a physical clustering network (Gerlt et al. 2015) as follows. The amino acid sequences of
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proteins of the IPR011078 InterPro family was extracted to generate a sequence similarity
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network with an original alignment score threshold of 30 and no restrictions for alignment
in
the
“YggS_2015_Minimal”
subsystem
on
the
public
SEED
server
as
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lengths. A 65% sequence identity representative node network for IPR011078 was also edited in
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Cytoscape (Shannon et al. 2003) to produce multiple networks with alignment score thresholds
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of 40, 50, 60, 70, 80, 90 and 100 by deleting all edges with –log10 (E) values below those
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thresholds. These seven networks were then used to generate genome neighborhood networks
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using the EFI Genome Neighborhood Tool with default parameters. The top 11 Pfam protein
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families in the genome neighborhood networks with alignment scores of 70 and 80 were
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identified by restricting the network to Pfam protein families with ≥3,000 neighbors. These
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specific networks were chosen because they are the ones that separate into the most clusters
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before the network begins to disintegrate into mostly single nodes. The Interactive Tree of Life
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v2 (ITOL) platform was used to build the gene distribution trees (http://itol.embl.de/index.shtml)
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(Letunic and Bork 2011). Gene essentiality data was extracted from the Database of Essential
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Genes (DEG) database (Luo et al. 2014) (http://www.essentialgene.org/). Structures were
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visualized with the Protein Data Bank (PDB) tools (www.rcsb.org) (Berman et al. 2000). The
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distribution
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(http://bioinformatics.unipr.it/B6db) (Percudani and Peracchi 2009). The id-mapping tools of
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Ecogene
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(http://www.uniprot.org/) (Li et al. 2015) where routinely used..The positions of regulatory sites
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for the PdxR family were extracted from RegPrecise 3.0 (http://regprecise.lbl.gov) (Novichkov et
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al. 2013). The subcellular localization of plant COG0325 proteins was predicted using TargetP
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(Emanuelsson et al. 2007). Prediction of E. coli promoters was done using RegulonDB (Salgado
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et al. 2013).
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Growth conditions and media. Bacteria were grown on Luria Bertani (LB) medium (BD
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Diagnostics Systems) at 37 °C or on M9 minimal medium (Sambrook et al. 1989) unless
of
PLP
enzymes
in
(http://www.ecogene.org/)
E.
coli
(Zhou
was
and
extracted
Rudd
from
2013)
the
B6
and
database
Uniprot
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otherwise stated. Growth media were solidified with agar (15 g/l) (BD Diagnostics Systems) for
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the preparation of plates. Transformation and P1 transductions were performed following
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standard procedures (Moore 2011). The sensitivity to P1 phage of all recipient strains used was
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verified. Anhydrotretracycline (aTet, 50 ng/ml), Ampicillin (Amp, 100 µg/ml), Kanamycin (Kan,
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50 µg/ml), Spectinomycin (Sp, 50 µg/ml) and Chloramphenicol (Cm, 30 µg/ml) were used as
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appropriate. ilvE avtA::KanR (LSP5001) and ilvE::KanR avtA (LSP5001) were grown on
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VBE minimal medium 0.5% glucose (w/v) (Whalen and Berg 1982).
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Strain and plasmid constructions. All strains and plasmids used in this study are listed in Table
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S1 and all oligonucleotides in Table S2. Details of the constructions are described in the
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supplemental methods.
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Effect of PN on yggS. yggS (VDC6594) cells were grown overnight in 5 ml of M9 glucose
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0.2% (w/v), diluted 500-fold in 5 ml of M9 medium and plated on M9 glucose 0.2%; 20 l drops
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of PN at concentrations of 5.9 mM, 590 M, 295 M or 59M were set on the top of the agar
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and plates were incubated overnight at 37 °C. For complementation experiments, the yggS
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strain was transformed freshly for every experiment. The plasmids used are listed in Table S1.
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Cells transformed with pBAD18, pBAD24yggSEc (pBY291.3) were plated on M9 glucose 0.2%
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(w/v) arabinose 0.2% (w/v), ampicillin 100 g/ml. Cells transformed with pBAD18
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LOC100191932 were plate on M9 glucose 0.2% (w/v) in presence or in absence of arabinose
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0.2% (w/v) ampicillin 100 g/ml. Finally, yggS were transformed with pBEY YBL036C
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(pBEY329.12), pBEY279.1 (empty vector) in the presence or absence of aTet 50 ng/ml.
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Bioscreen growth curves. Cells were grown at 37 °C overnight in M9 supplemented with 0.2%
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glucose (w/v) and 1 mM glycine, starting from an optical density (OD) of 0.05 at 600 nm. Cells
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were then diluted 50-fold into sterile 100-well honeycomb plates with cover (Labsystems)
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containing the corresponding medium: M9 glucose 0.2% (w/v) supplemented or not with glycine
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1 mM, LB supplemented or not with methionine (0.13 mM) and 0.16 mM thymidine (dT), M9
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glucose 0.2% (w/v) and glycine 1 mM supplemented or not with 0.4% casamino acids, or with
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0.13 mM methionine and 0.16 mM thymidine. The OD at 600 nm was measured using a
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Labsystems Bioscreen C plate reader. Cells were grown at 37 °C with vigorous shaking. Time
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points were recorded every 30 minutes. For growth in presence of 20 different amino acids, cells
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were grown in M9 glucose 0.2% (w/v) supplemented with 1 mM glycine and 0.2 mg/ml or 0.1
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mg/ml of the corresponding amino acids. All growth experiments were performed in 10
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replicates and standard deviations (SD) were determined
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Extraction and vitamin B6 analysis. B6 species (PLP, PNP, PMP, PL, PN and PM) were
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extracted from wild-type and yggS pellets (1.0 ml culture of OD600=1.0) in 0.9 ml of 5% (w/v)
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metaphosphoric acid and 0.1 ml of internal standard 4-deoxypyridoxine (4-dPN, 73 nmol/ml in
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5% metaphosphoric acid). The suspension was vortexed and sonicated. After centrifugation at
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10,000 g for 15 min, the supernatant was filtered and a 50-µl sample was taken for HPLC
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analysis using fluorescence detection (excitation 328 nm, emission 393 nm). The separation was
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performed on a Microsorb-MV C18 column (150 × 4.6 mm, 5 µm particle size) using a gradient
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program described by Sampson et al with some modifications (Sampson and O'Connor 1989).
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Mobile phase A (0.033 M phosphoric acid and 0.008 M 1-octanesulfonic acid, adjusted to pH 2.2
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with KOH), B (0.033 M phosphoric acid, adjusted to pH 2.2 with KOH) and C (acetonitrile)
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were used and the gradient program was as follows: 98% A and 2% C for 10 minutes; a linear
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gradient to 78% B and 22% C for 8 minutes; a linear gradient to 98% A and 2% C for 2 min;
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column equilibration for 5.0 min with 98% A and 2% C. Total running time was 25 min and the
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flow rate was 1.2 ml/min. A post-column reagent (1.0 mg/ml sodium bisulfite in 1.0 M
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potassium phosphate buffer adjusted to pH 7.5 with KOH) was used to enhance fluorescence of
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PLP. All activities were measured at least three times and standard deviations were determined.
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Chemicals. PNP was provided by the Vanderbilt Institute of Chemical Biology, Chemical
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Synthesis Core, Vanderbilt University, Nashville, TN 37232‐0412.
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Results
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yggS is widely distributed and clusters strongly with genes in diverse metabolic and cellular
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pathways.
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To gain further insight in the role of the COG0325 family, a subsystem was constructed in the
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SEED database to capture all COG0325 members (named YggS_2015_Minimal). The yggS gene
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is widely distributed in Bacteria and Eukaryotes but is quite rare in Archaea where only
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Methanosarcinales and Aciduliprofundum sp. MAR08-339 harbor yggS orthologs. Members of
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this family are present almost universally among Bacteria with >90% of the bacterial species in
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the SEED database containing at least one yggS homolog (see Subsystem and Fig. 2A). A few
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Bacteria harbor yggS paralogs and these are scattered around the phylogenetic tree (Fig. 2A).
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COG0325 genes are present in most (but not all) Eukaryotes including yeast, Caenorhabditis
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elegans, fruit fly, Arabidopsis, Z. mays, zebrafish, chicken, and mammals including humans [see
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group OG5_127174 in the OrthoMCL database].
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Physical clustering was first analyzed using the STRING and SEED databases and is
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summarized in Table 1. The clustering between COG0325 genes and proC observed decades ago
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(De Wergifosse et al. 1994) was confirmed; clustering between yggS and proC was observed in
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~20% of the genomes analyzed (3% in the 1000 genome set) (Table 1). However, our data
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showed that the link between yggS and proline synthesis is not robust (Fig. S1A). First, although
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proABC genes are quite often clustered, such clusters never include yggS and some Bacteria such
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as Helicobacter pylori have proAB homologs but lack yggS homologs (Fig. S1A). Second,
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several bacteria have two proC genes, one that clusters with proAB and another that clusters with
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yggS (Fig. S1B).
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Our analysis uncovered other physical clustering associations. The strongest association
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was with cell division/cell wall related genes (Table 1). The yggS gene is often located near the
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end of the well-known division and cell wall (dcw) cluster (Tamames et al. 2001) and also
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clusters independently with two genes also often located at the end of the dcw cluster yggT
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(YlmG) and sepF (Table 1). Physical clustering associations also linked YggS to PLP salvage
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and to the PLP dependent enzyme GlyA (Table 1 and Fig. S2). Indeed, yggS genes were found
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associated with pdxK, glyA and B6 transporter genes in operons predicted to be under the control
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of various PdxR-type PLP-responsive transcription regulators (Jochmann et al. 2011; Suvurova
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and Rodionov 2015; Tramonti et al. 2015) (Fig. S1B and Fig. S2). Finally, genes encoding
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essential ATP-dependent enzymes (with essential lysines) were found to cluster strongly with
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yggS (Table 1). Examples include the leucyl- and isoleucyl-tRNA synthetase genes that in
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combination clustered with yggS in ~25-30% of the genomes.
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Vitamin B6 homeostasis defects are observed in the yggS strain.
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YggS is a PLP-binding protein (Eswaramoorthy et al. 2003), and our comparative genomic
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analyses linked members of the YggS family to vitamin B6 synthesis (Table 1, Fig. S2). To
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explore a potential role of this protein family in vitamin B6 homeostasis in vivo, we tested
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whether the absence of yggS led to phenotypes in conditions of B6 excess. An E.coli K12 yggS
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derivative VDC6594 was constructed and tested for response to B6 vitamers. Excess PN led to a
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toxicity ring on minimal medium in the yggS but not in the wild-type strain (Fig. 3, Table 2 and
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Table 3). PLP, PM or PL were not toxic in any background (data not shown). This PN toxicity
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ring was very reproducible (Table 2) and was complemented by expressing the E. coli yggS gene
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in trans (Fig. 3C) and suppressed by the presence of PL (Table 2). The presence of ring, and not
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a halo, couldsuggest that suppressors appeared. To discriminate between the two hypotheses, we
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re-isolated yggS cells from the inside or from the outside of the PN toxicity ring and both
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retained the PN sensitivity phenotype (Fig. S3A). We also showed that overexpressing pdxK led
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to PN toxicity in the WT strain (Fig. S3B), the absence of yggS did not seem to increase this
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toxicity (data not shown).
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The universality of the YggS function first reported by Ito et al. (Ito et al. 2013) was
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confirmed here, as the PN toxicity phenotype was complemented by expressing the COG0325
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gene from Z. mays, LOC100191932, in the presence of inducer (Fig. 3D). No complementation
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was observed in absence of inducer (Fig. 3E). Similar results were found with the COG0325
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gene from A. thaliana, At1g11930 (Table 2) but in addition it appeared that overexpression of
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that gene was toxic (data not shown). Finally, it was found that expression of the yeast COG0325
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gene, YBL036C, also complemented the PN toxicity phenotype (Fig. 3F).
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As the growth phenotypes were consistent with a role of YggS in vitamin B6
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homeostasis, we analyzed the B6 pools in the wild-type and yggS strains grown in M9 glucose
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at the end of the exponential phase (Fig. 4A). The mutant accumulated PNP 71.64 ± 6.10
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pmol/mg of proteins and no PNP is detected in the wild-type (Fig. 4B). No difference in the PLP
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levels was observed between the yggS and the wild-type cells. Of note the total pool of PLP is
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measured here not the free pool. Because the free PLP pool is only a small proportion of the total
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pool (di Salvo et al. 2011), changes in the free pool will not be detected with this method, hence
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it still to be determined if the absence of yggS affects the free PLP pool. Methods to measure the
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PLP-ome or the PLP bound to proteins exists but they are all semi-quantitative (Whittaker et al.
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2015). We still decided to compare the bound PLP (PLP to proteins) in the WT and yggS
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strains using an antibody that detects PLP bound to proteins (Whittaker et al. 2015). The soluble
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fractions of both strains were separated by 2D gels (Fig. S4A and B) and PLP-bound proteins
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were detected using the anti-PLP antibody. 17 major protein spots were detected in both strains
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and no major differences were observed (Fig. S4C and S4D). The absence of yggS does not have
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a major impact on the bound PLP pool, but clearly the method is not quantitative enough to
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detect small differences.
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YggS is a monomeric PLP protein that has no direct effect on PdxH enzymatic activity.
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The structure of E. coli YggS shows that PLP is covalently bound to lysine residue 36
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through a Schiff base linkage (PDB: 1W8G). The role of PLP in YggS quaternary structure or
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stability has yet to be determined. Recombinant YggS purified by Ni-affinity as described in the
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supplemental methods and gel filtration chromatography migrated in SDS-PAGE as a single
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band with a molecular mass of ~30 kDa (Fig. S5A). The monomeric state observed in the crystal
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structure was confirmed by gel filtration chromatography, which indicated a native molecular
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mass of 22.6 kDa (data not shown). The presence of PLP in the sample was detected by optical
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spectroscopy (Fig. S5B); 32% of the purified YggS contained bound PLP.
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Two forms of YggS were generated: apo-YggS with no detectable bound PLP, and native
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holo-YggS in which PLP was reincorporated to the purified YggS (82% PLP). The holo- and
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apo-YggS gave a symmetrical peak upon gel filtration that corresponded to the monomeric state
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(data not shown). Thus, PLP had no effect on YggS oligomerization. The ability of apo-YggS to
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bind PLP was confirmed by ITC with a Kd value was 0.37 ±0.49 (SD) M.
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To determine whether YggS can bind PNP, a comparative analysis of thermal stability of
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the purified YggS (with 32% of PLP binding sites occupied by PLP) was carried out by
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thermofluor assay (Ericsson et al. 2006; Lavinder et al. 2009; Phillips and de la Pena 2011) in the
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absence or presence of PNP or PLP (Fig. S5C and S5D). Only PLP was found to stabilize YggS
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as the apparent Tm of the YggS was 86 °C with PLP compared to 78 °C without it. In presence of
316
PNP the apparent Tm of YggS was 78 °C. That PNP did not affect the thermal stability of YggS
317
even at high concentration (data not shown) indicates that YggS does not bind PNP.
318
PdxH (pyridoxine 5'-phosphate oxidase) mediates a key step in the PLP biosynthesis
319
pathway using PNP as substrate (Zhao and Winkler 1995; di Salvo et al. 2011) (Fig. 1). Free PLP
320
inhibits both human and E. coli PdxH and interacts with FMN when it is present (Musayev et al.
321
2003). (Fig. 4) A defect of the PNP oxidase activity could lead to the accumulation of PNP in
322
the yggS strain (Fig.4). This hypothesis was tested by measuring PNP oxidase specific activity
323
in the soluble fraction of the wild-type and the yggS strains as described in the supplemental
324
methods. No difference in PdxH specific activity was observed. The specific activities of
325
theyggS and wild-type were 2.9 ±0.02 (SD) and 2.11 ± 0.12 (SD) nmol h-1 mg-1.
326
327
328
329
Pleotropic effects of yggS depletion.
Previous studies have linked the absence of yggS to amino acid pool imbalances,
particularly the secretion of valine in E.coli MG1655 (Ito et al. 2013).
330
A full analysis of the intracellular and extracellular metabolite pools extracted from the
331
wild-type and yggS strains grown in minimal medium was therefore performed and the
332
metabolomes of the two strain compared by gas chromatography time-of-flight mass
333
spectrometry as described in the supplemental methods (Table S3 and Table S4). Among the
334
most striking differences observed were increases in putrescine, N-acetylputrescine, and
335
putrescine-related metabolites such as γ-aminobutyric acid (and its cyclized form γ-
336
butyrolactam) and 5’-deoxy-5’-thiomethyladenosine in the yggS strain (Table S3). Putrescine is
337
the major polyamine in E. coli (Cohen 1998) and the regulation of its catabolism is very
338
complex, being induced by different stresses including nitrogen limitation (Schneider et al.
339
2013); several enzymes in these pathways are PLP-dependent (Samsonova et al. 2003; Kurihara
340
et al. 2005; Samsonova et al. 2005). In the conditions used in our study, we did not observe
341
significant accumulation of valine in the yggS strain or valine release to the medium (Table S4,
342
and Table 3). Nor could valine secretion by the yggS mutant be detected by feeding the valine
343
requirements of the valine auxotrophs LSP5001 or 5002 (data not shown).
344
Another link between YggS, PLP, and amino acid metabolism was observed with the
345
synthetic lethality phenotype of the E. coli ΔyggS::CmR and ΔglyA::KanR alleles on the rich
346
medium LB (Nichols et al. 2011). GlyA is a PLP-dependent enzyme involved in glycine and
347
serine metabolism (Fig. 1). As this phenotype had only been observed in a high throughput
348
experiment, a yggSglyA derivative was constructed in a BW25113 background. The double
349
mutants did not grow on LB (Fig. S6 and Table 3) but grew on M9 glucose 0.2% (w/v)
350
supplemented with 1 mM glycine (Fig. 5A). The ΔglyA strain also showed a growth phenotype
351
in LB (Fig. S6) as previously reported (Nichols et al. 2011) but it was not as severe as the double
352
mutant. It was then shown that the LB sensitivity phenotype is caused by the presence of amino
353
acids in the medium, as addition of casamino acids 0.4% (w/v) (Fig. 5B and Table 3). Casamino
354
acids also affected growth of the glyA strain (VDC6664) but not of the yggS (VDC6594) or
355
wild-type (WT) (BW25113) strains (Fig. 5B). Growth was then followed with all 20 protein L-
356
amino acids (added individually in presence of 1 mM glycine). Out of these 20, threonine (0.84
357
mM) and alanine (2.24 mM) delayed growth of the double mutant compared to the glyA, yggS
358
and wild-type strains (Fig. S7A and S7B) even if the final growth rates were similar (~0.04
359
OD600nm.h-1). The growth defects with single amino acids were not as pronounced as with LB or
360
casamino acids, suggesting additive or synergistic effects. The addition of dT and methionine
361
also partially suppressed the phenotype (Fig. 5C).
362
Finally, because of these links with amino acid metabolism, we tested whether amino
363
acids affected the PN toxicity phenotype of the yggS. We found that the PN toxicity ring
364
observed in the yggS strain (Fig. 5D) was suppressed by the presence of casamino acids (Fig.
365
S8B and Table 3) and that out of the 20 amino acids tested individually, only leucine (Fig. 5D),
366
isoleucine (Fig. 5E) or threonine (Fig. 5F) suppressed the ring of toxicity (Table 3, Fig. S8).
367
Valine could not be tested because it is toxic. This suppression was not due to an increase growth
368
rate as adding L-leucine, L-isoleucine or L-threonine did not affect the growth rate of the yggS
369
mutant (data not shown). Thr is a precursor or Ile and the last step in the synthesis of Ile and Leu
370
is catalyzed by the PLP-dependent transaminase B or IlvE (Fig. 1). The levels of IlvE were
371
analyzed by Western blot and a slight but reproducible increase in levels of IlvE protein in the
372
yggS strain was observed (Fig. S9).
373
The strongest physical clustering observed in our comparative genomic analysis was
374
between yggS genes and cell division and murein synthesis genes (Table 1). Scanning electron
375
microscopy supported this functional link, indeed yggS glyA cells grown in minimal medium
376
were found to be 23% longer than wild-type cells and a few filaments were observed (Fig. S10
377
and Table 3). The morphology and length of yggS or glyA cells were similar to those of wild-type
378
cells and the addition of D-alanine (1 mM), PL (1 mM), or the combination of methionine (0.13
379
mM) and dT (0.16 mM) did not suppress the observed phenotype in the conditions tested (data
380
not shown).
381
Discussion
382
This study clearly connects YggS, the E. coli member of the COG0325 family, with B6
383
metabolism. First, comparative genomic associations firmly linked the COG0325 family to PLP
384
salvage (Fig. S2). Second, we confirmed that YggS binds PLP as previously reported (Ito et al.
385
2013), and determined the Kd value. We also showed that PLP binding does not affect the
386
quaternary structure of the protein. Finally, we show that the E. coli yggS strain accumulates
387
PNP and that high levels of exogenous PN (but not PL, PM or PLP) are toxic to the mutant
388
strain. In the toxicity tests, the formation of a toxicity ring was very reproducible, however the
389
transformedyggS cells could not be stored for more than a week at 4°C without losing the
390
phenotype. Moreover, bacterial growth was observed in the center of the ring of toxicity. We
391
showed that these were not suppressors but further work is needed to understand concentration
392
dependence of the PN sensitivity phenotype.
393
YggS is widely distributed among bacteria, fungi, and eukaryotes and the universality of
394
YggS function is strengthened by the fact that expressing orthologs from yeast and plants
395
complement the PN toxicity phenotype of the E. coli yggS mutant. However, both the cause of
396
PN toxicity and the exact role of YggS in vitamin B6 homeostasis remain to be elucidated. PN
397
neurotoxicity has been previously reported in mammals (rats, dogs and humans) (Schaeppi and
398
Krinke 1982; Albin et al. 1987; Perry et al. 2004) and we show also here that PN is toxic when
399
PdxK is overproduced in E.coli also suggestive of PNP toxicity (Fig. S3B). However, the
400
mechanism of this toxicity is unknown although it was suggested that PNP competitively inhibits
401
PLP-dependent enzymes in the brain (Albin et al. 1987). That PN (or PNP) could inhibit PLP
402
enzymes in E. coli in the absence of YggS fits with the suppression of the PN toxicity phenotype
403
by PL (if this allows to rebalance the PNP/PLP pools) or casamino acids (if this bypasses the
404
need for specific PLP enzymes). One candidate PLP enzyme is IlvE as adding Ile, Val or Thr
405
suppresses the phenotype and as -ketoglutarate a product of IlvE activity was found to be
406
reduced in the metabolomics analysis (Table S3). How YggS would protect from this PN toxicity
407
is not clear. Indeed, YggS does not protect by sequestering PNP as we showed that it does not
408
bind PNP. We found that PdxH specific activity was not affected by the absence of YggS so
409
other type of regulations must occur and further studies are required to elucidate the causes of
410
PNP increase.
411
As summarized Table 3, YggS depletion has a pleotropic effect on metabolism and
412
diverse phenotypes were observed. We propose that most of the phenotypes observed in E. coli
413
in the absence of yggS are caused by lower activities of PLP-dependent enzymes (Fig. 1).
414
Because PLP participates in nearly sixty reactions in E. coli (http://bioinformatics.unipr.it/B6db),
415
physiological studies in strains with defects in PLP metabolism are difficult to interpret, mainly
416
because: 1) several independent phenotypes are observed; 2) these phenotypes can be due to
417
primary and secondary effects; 3) causes cannot be distinguished from effects. Such difficulties
418
are why it took decades to decipher the molecular basis of phenotypes caused by the absence of
419
ridA that, possibly like yggS, perturbs whole cellular networks by inhibiting PLP enzymes
420
(Downs and Ernst 2015). In addition, even if the core function of YggS is conserved, as the
421
cross-kingdom complementations observed here and in previous studies suggest, the network
422
perturbations caused by its absence may vary from one organism to another. For example, YggS
423
is dispensable in most organisms studied so far but is essential in Pseudomonas aeruginosa
424
(Rusmini et al. 2014) and possibly in the pathogenic bacteria Haemophilus influenzae,
425
Helicobacter pylori, Streptococcus pneumoniae and Staphylococcus aureus (data extracted from
426
the DEG database (Luo et al. 2014)). These combinations of issues make the reproducibility and
427
interpretation of physiological and metabolomic data in mutants affected in PLP metabolism
428
very difficult, as discussed below.
429
According to Ito et al., the absence of yggS leads to a 10% decrease of CoA that triggers
430
accumulation of -ketobutyrate and an increase in the L-valine pool (Ito et al. 2013). These
431
results were not reproduced in our study as no significant increase in L-valine was observed and
432
the -ketobutyrate pool was found to decrease and not increase in most conditions. The
433
differences between the two studies could be due to the different genetic backgrounds
434
(BW25133 or MG1655), the different growth temperatures (30 °C or 37 °C) or to the growth
435
phase at which the analyses were made. Indeed, metabolite pools varied greatly with the stage of
436
growth (Table S3). Further analytical studies are required to reconcile these discrepancies.
437
The growth defect of the yggS glyA strain in the presence of exogenous amino acids is a
438
strong phenotype, but remains unexplained. The partial suppression of this phenotype by dT and
439
methionine suggests a link to THF metabolism that fits with the previously reported sensitivity to
440
sulfonamides of a yggS mutant (Nichols et al. 2011). Our data suggested a strong link of YggS to
441
amino acid metabolism, and indeed metabolomics analysis does reveal changes in levels between
442
wild type and yggS mutant for six amino acids. Finally, our metabolomics data suggested that
443
several PLP-dependent enzymes involved in the putrescine metabolism could have been affected
444
in the yggS strain.
445
Another phenotype observed was the cell division defect of the glyA yggS strain. This
446
could be due to lower levels of D-alanine if the PLP-dependent alanine racemase activity is
447
lowered in this background as shown in numerous studies on B6 limitation (Grogan 1988; Kim et
448
al. 2010). As the addition of D-alanine did not suppress the phenotype it could also have more
449
complex causes. Indeed, it was previously reported that cells containing a pdxH mutation placed
450
under PL limitation were elongated and contained nucleoids that could not segregate and this
451
phenotype was not suppressed by PN or D-alanine, suggesting that it was caused by an early cell
452
division defect (Lam and Winkler 1992). The strong clustering of yggS with genes involved in
453
cell division combined with the observed phenotypes point to a regulatory role in this process
454
that will require further investigation.
455
Finally, our comparative genomics data suggest that the strong linkage of yggS with proC
456
is almost certainly due to a function of proC not directly related to proline synthesis. We propose
457
a role of ProC in PLP metabolism, as in humans as the ProC deficiency depletes the PLP pool
458
through the formation of PLP-P5C adducts (Farrant et al. 2001)(Fig. 1). In Bacteria, ProC could
459
be a mechanism to detoxify excess P5C and we are currently exploring this hypothesis.
460
This work has laid the foundation to elucidate the molecular function of the COG0325
461
family. Possibilities include a carrier function to deliver PLP to the target enzymes or a
462
protective function so that PLP does not inactivate essential lysines in proteins. More generally,
463
like the predatory genetic studies of the rid family (Downs and Ernst 2015), our work
464
underscores the power of genetics to uncover, and to start unraveling, complex biochemical
465
processes.
466
467
Acknowledgments
468
We thank S. Shanker and the staff at UF ICBR for DNA sequencing and the ICBR
469
members Cecilia Silva-Sanchez and Sixue Chen for help with 2D-gel analyses. We gratefully
470
acknowledge the staff at UF ICBR for microscopy. We thank the Cambillau laboratory for the E.
471
coli yggS expression plasmid and Robert McKenna, Antonette Bennett, and the University Of
472
Florida Center Of Structural Biology for help with data acquisition and interpretation of the
473
thermal denaturation and titration calorimetry studies. This work was funded by US National
474
Science Foundation (NSF) grants MCB-1153413 and IOS-1025398. MB was a recipient of a
475
postdoctoral fellowship from the Human Frontier Scientific Program.
476
477
478
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694
695
696
697
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698
Table 1. Summary of physical clustering analysis. Set 1: 1536 representative genome set. Set
699
2: 1000 representative genome set. Total: all 11411 organisms in PubSeed at the time of the
700
analysis. Genes encoding for: * PLP enzymes; proteins with
701
bind GTP or ATP. #no current name for this gene.
&
Essential lysines; proteins that
$
702
Gene
%Set1 %Set2 %Total
Function
Pyrroline-5-carboxylate reductase (EC
1.5.1.2)
SepF, FtsZ-interacting protein related
to cell division
Cell division initiation protein
DivIVA
COG0762, cell division protein
YlmG/Ycf19 (putative), YggT family
Septum formation protein Maf
Cell division protein FtsZ (EC
3.4.24.-)
SepF, FtsZ-interacting protein related
to cell division
UDP-N-acetylglucosamine--Nacetylmuramyl-(pentapeptide)
pyrophosphoryl-undecaprenol Nacetylglucosamine transferase (EC
2.4.1.227)
proC
21.7
3.0
18.4
sepF
22.8
0.8
24.3
divIVA
20.1
1.8
26.1
yggT
19.2
0.5
36.3
maf
2.0
1.5
0.9
ftsZ$
17.3
0.1
29.0
sepF
22.8
0.8
24.3
murG
11.6
0.1
18.0
pilT$
19.2
0.8
30.9
lsp
14.8
1.4
13.5
pdxR
1.2
0.4
0.8
pdxK$
0.9
PLP
0.7
transporter#
0.3
0.5
Lipoprotein signal peptidase (EC
3.4.23.36)
Predicted transcriptional regulator of
pyridoxine metabolism
Pyridoxal kinase (EC 2.7.1.35)
0.4
0.5
B6 transport protein
yqgF
12.0
0.2
23.5
ileS&$
16.7
0.3
25.6
leuS&$
7.6
6.4
5.5
rluD
7.3
0.1
8.7
Twitching motility protein PilT
YqgF, involved in pre-16S RNA
processing
Isoleucyl-tRNA synthetase (EC
6.1.1.5)
Leucyl-tRNA synthetase (EC 6.1.1.4)
Ribosomal large subunit
pseudouridine synthase D (EC
4.2.1.70)
Process
Proline
synthesis
Cell division
Cell division
Cell division
Cell division
Cell division
Cell division
Cell wall
synthesis
Surface
motility
Secretion
PLP salvage
PLP salvage
PLP salvage
Translation
Translation
Translation
Translation
703
704
rdgB
12.0
ND
23.1
yggW
12.4
0.6
23.3
yqgE
11.1
0.3
22.8
topoI
1.3
0.9
0.4
glyA*
0.8
0.1
0.5
aOT3*
0.3
0.5
0.3
Nucleoside 5-triphosphatase RdgB
(dHAPTP, dITP, XTP-specific) (EC
3.6.1.15)
Radical SAM family enzyme, similar
to coproporphyrinogen III oxidase,
oxygen-independent, clustered with
nucleoside-triphosphatase RdgB
UPF0301 protein YqgE
DNA topoisomerase I (EC 5.99.1.2)
Serine hydroxymethyltransferase (EC
2.1.2.1)
Acetylornithine aminotransferase (EC
2.6.1.11)
Nucleotide
metabolism
?
?
Replication
Amino acid
metabolism
Amino acid
metabolism
705
Table 2. Pyridoxine toxicity phenotypes on M9 glucose 0.2% (w/v). % Antibiotics Cm or Amp
706
were added when required; the three plasmids were used as controls and gave similar results.
707
*Arabinose 0.2 % was added. & plate was supplemented with aTet 50 ng/ml.
Strain
Wild-type BW25113
glyA (VDC6664)
yggS pBAD18/pBAD24/pBAD33%*
yggS pBAD24 yggSEC*
yggS pBAD18: LOC100191932*
yggS pBAD18: At1g11930
yggS pBEY YBL036C &
yggS pBAD18 + PL 50 M*
708
709
710
Diameter of the zone of inhibition
(cm)
0.1 mg/ml
1 mg/ml PN
PN
None
None
None
None
4.15 ±0.05
5.25±0.1
None
None
None
None
None
None
None
None
None
None
711
712
Table 3. Phenotypes discussed in this study. M9 Casa is M9 glucose 0.2% (w/v) supplemented
713
with 0.4% (w/v) casamino acids. M9 is M9 glucose 0.2% (w/v) supplemented with isoleucine 50
714
g/ml, leucine 30g/ml , or threonine 30g/ml. $metabolomics data presented in Table 1 and
715
Table S3. *in the conditions used in this study, no significant accumulation of valine was
716
observed in the yggS strain. nd is non determined; - : no phenotype observed; + : phenotype
717
observed. Leu: L-leucine; Ile: L-isoleucine; Thr: L-threonine.
Phenotype
PN sensitivity
PN sensitivity on M9 Casa, M9 Leu, Ile or
Thr
Growth on LB
Growth on M9 Casa
Valine secretion
Longer cells
718
719
720
glyA
-
yggS
+
yggS glyA
+
nd
-
nd
+
+
nd
-
+
+
-$*
-
nd
+
721
Figures legends
722
Figure 1. PLP synthesis and a subset of pathways influenced by PLP levels in E.coli. In E.
723
coli PLP can be synthesized via the de novo PdxFBAJ dependent pathway or salvaged using
724
PdxK, PdxY and PdxH (see review in (Fitzpatrick et al. 2007)). PLP is a cofactor for enzymes
725
involved in cell wall synthesis, amino acids, and one-carbon metabolism. P5C can sequester free
726
PLP.
727
Tetrahydrofolate; CH2-THF: 5,10-methylene-tetrahydrofolate; Gcv: Glycine-cleavage complex;
728
TA: L-threonine aldolase. P5C is formed spontaneously and reversibly from GSA. Dashed
729
arrows indicate that several enzymes are required for a conversion. Proteins in bold with an
730
asterisk are enzymes that require PLP. This figure is based on E. coli literature (see introduction)
731
except for formation of the P5C-PLP adduct that has been reported in human but not E. coli to
732
date Abbreviations for enzymes names are given in the text.
733
Figure 1. PLP synthesis and a subset of pathways influenced by PLP levels in E.coli. In E.
734
coli PLP can be synthesized via the de novo PdxFBAJ dependent pathway or salvaged using
735
PdxK, and PdxH (see review in (Fitzpatrick et al. 2007)). PLP is a cofactor for enzymes involved
736
in cell wall synthesis, amino acids, and one-carbon metabolism. P5C can sequester free PLP.
737
GSA: Glutamic 5-semialdehyde; P5C: Δ1-pyrroline-5 carboxylic acid; THF: Tetrahydrofolate;
738
CH2-THF: 5,10-methylene-tetrahydrofolate; Gcv: Glycine-cleavage complex; TA: L-threonine
739
aldolase; TyrB: aromatic amino acid transferase. P5C is formed spontaneously and reversibly
740
from GSA. Dashed arrows indicate that several enzymes are required for a conversion. Proteins
741
in bold with an asterisk are enzymes that require PLP. This figure is based on E. coli literature
742
(see introduction) except for formation of the P5C-PLP adduct that has been reported in human
743
but not E. coli to date Abbreviations for enzymes names are given in the text.
GSA:
Glutamic
5-semialdehyde;
P5C:
Δ1-pyrroline-5
carboxylic
acid;
THF:
744
Figure 2. Distribution of yggS genes among bacteria. (A) The tree was constructed in iTol
745
(itol.embl.de/). Tree branches are colored by phylum. The presence of yggS homologs are
746
displayed in rings around the tree. The presence of at least one yggS homolog in the specific
747
organism in the tree is shown by the innermost (blue) ring. The presence of two or three yggS
748
paralogs in one genome are shown by the middle (green) ring and the outer (orange) ring
749
respectively. (B) Physical clustering of yggS genes in Pseudomonas sp. GM79 and
750
Rhodopseudomonas palustris TIE-1 with PLP synthesis, salvage genes and PLP dependent
751
enzymes. The red arrows indicate predicted PdxR binding sites.
752
753
Figure 3. Toxic effect of PN on the E. coli yggS strain and the universality of YggS
754
function. The cells were plated as described in the material and methods section. (A) yggS
755
(VDC6594) lawn and drops of PN at concentrations of 5.9 mM (a), 590 M (b), 295 M (c) or
756
59M (d); PN at the concentrations (a) or (b) were used in experiments (B-F). (B) yggS
757
pBAD18 lawn; in presence of arabinose 0.2% (C) yggSin pBAD24yggSEc (pBY291.3) lawn in
758
presence of arabinose 0.2%; (D)yggS transformed with pBAD18 LOC100191932 (pBY298.7)
759
lawn in presence of arabinose 0.2%; (E) yggS transformed pBAD18 LOC100191932 in absence
760
of arabinose 0.2%; (F) yggS transformed with pBEY YBL036C (pBEY329.12) in presence of
761
aTet 50 ng/ml. The arrows indicate the presence of the ring of toxicity.
762
763
Figure 4. Analysis of B6 derivatives in wild-type and yggS E. coli. The B6 vitamers were
764
analyzed by HPLC with fluorometric detection. (A) B6 vitamer profile; 4-dPN is the internal
765
standard 4-deoxypyridoxine. (B) Determination of PNP, PMP, PLP, concentrations (pmol/mg
766
protein) in wild-type (dark gray) and yggS(light gray) strains.
767
768
Figure 5. Effects of amino acids in a yggS background. Wild-type (yellow), yggS
769
(VDC6594) (blue), glyA (orange), and yggS glyA (grey) strains were grown in the Bioscreen
770
C at 37 °C in different media. (M9 glucose 0.2% (w/v) supplemented with 1 mM glycine;
771
(M9 glucose 0.2% (w/v) supplemented with 1 mM glycine and 0.4% casamino acids; (C) LB
772
supplemented with methionine (0.13 mM) and 0.16 mM dT. The average of 10 independent
773
growth curves was plotted, and errors bars represent standard deviations. PN at the
774
concentrations 5.9 mM (a), 590 M (b) were used in experiments (D-F). (D) yggSin pBAD33
775
lawn on M9 supplemented with L-leucine 30 g/mL. (E) yggSin pBAD33 lawn on M9
776
supplemented with L-isoleucine 50 g/mL; (F) (E) yggSin pBAD33 lawn on M9 supplemented
777
with L-threonine 30 g/mL. Rings of toxicity were observed for yggSin pBAD33 lawn on M9
778
(Fig. S7A) for PN at concentrations of 5.9 mM (a), 590 M (b). M9 was supplemented with each
779
20 amino acids individually. Any other amino acid than L-leucine, L-isoleucine and L-threonine
780
gave the same phenotype as Fig. S7C. dT: thymidine; Met: methionine.
781
.
782
783
784