How Do Haloarchaea Synthesize Aromatic Amino Acids? Miriam Kolog Gulko1¤*, Mike Dyall-Smith2, Orland Gonzalez3, Dieter Oesterhelt1 1 Department of Membrane Biochemistry, Max-Planck-Institute of Biochemistry, Martinsried, Germany, 2 School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, Australia, 3 Teaching and Research Unit Bioinformatics, Institut of Informatik Ludwig-Maximilians-University, Munich, Germany Abstract Genomic analysis of H. salinarum indicated that the de novo pathway for aromatic amino acid (AroAA) biosynthesis does not follow the classical pathway but begins from non-classical precursors, as is the case for M. jannaschii. The first two steps in the pathway were predicted to be carried out by genes OE1472F and OE1475F, while the 3rd step follows the canonical pathway involving gene OE1477R. The functions of these genes and their products were tested by biochemical and genetic methods. In this study, we provide evidence that supports the role of proteins OE1472F and OE1475F catalyzing consecutive enzymatic reactions leading to the production of 3-dehydroquinate (DHQ), after which AroAA production proceeds via the canonical pathway starting with the formation of DHS (dehydroshikimate), catalyzed by the product of ORF OE1477R. Nutritional requirements and AroAA uptake studies of the mutants gave results that were consistent with the proposed roles of these ORFs in AroAA biosynthesis. DNA microarray data indicated that the 13 genes of the canonical pathway appear to be utilised for AroAA biosynthesis in H. salinarum, as they are differentially expressed when cells are grown in medium lacking AroAA. Citation: Gulko MK, Dyall-Smith M, Gonzalez O, Oesterhelt D (2014) How Do Haloarchaea Synthesize Aromatic Amino Acids? PLoS ONE 9(9): e107475. doi:10. 1371/journal.pone.0107475 Editor: Nediljko Budisa, Berlin Institute of Technology, Germany Received April 19, 2013; Accepted August 18, 2014; Published September 12, 2014 Copyright: ß 2014 Kolog Gulko et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The Max-Planck Institute of Biochemistry funded the fellowship of Miriam Kolog Gulko as well as all experiments. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected] ¤ Current address: Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, Georg-August-University, Go¨ttingen, Germany Introduction in all available haloarchaeal genomes (40–47% aa similarity; 16 genomes according to NCBI, October 2012). In H. salinarum, Eukaryotes and bacteria synthesize aromatic amino acids these are ORFs OE1472F and OE1475F, which we propose as (AroAA) via the shikimate pathway, which in the well-studied candidates for the first two steps in the biosynthesis of AroAA E.coli system comprises 17 different enzymes [1,2]. In haloarch- (Fig. 1A). aea, 13 recognizable homologs of the shikimate pathway enzymes Thirdly, homologues of all 13 genes of the classical pathway that can be identified, and these cover all of the later steps necessary to are needed to convert DHQ to AroAA are present in all convert 3-dehydroquinate (DHQ) to AroAA. However, homo- sequenced haloarchaeal genomes (Fig. 1B, www.halolex.mpg.de). logues for the initial reactions responsible for the biosynthesis of This supports the idea that downstream of DHQ the synthesis of DHQ are not present, suggesting that a non-canonical pathway AroAA follows the canonical pathway, as is also the case with M. for AroAA biosynthesis is used in haloarchaea. This is supported jannaschii. by other lines of circumstantial evidence. In the alternative pathway suggested by White for M. jannaschii Firstly, the canonical precursor for AroAA biosynthesis is [6], the first step is a transaldolase (TA) reaction between 6-deoxy- erythrose-4-phosphate (E-4-P), which is a product of the pentose 5-ketofructose 1-phosphate (DKFP) and aspartate semialdehyde phosphate pathway (PPP). However, this pathway does not appear to be present in the archaeal domain, and only some orthologs are (ASA). In H. salinarum, this step is proposed to be carried out by present in varying degrees [3]. In halophilic archaea, only two the enzyme specified by ORF OE1472F (homologue of MJ0400). enzymes related to the pentose phosphate pathway have been Hydroxpyruvaldehyde phosphate (HPAP) would be released and proposed [3–5], and unless there is an alternative pathway for the compound I formed. In the second stage, DHQ is proposed to be production of E-4-P, this compound is not available for the formed by oxidative deamination and cyclization catalyzed by the conventional AroAA pathway in haloarchaea. enzyme specified by ORF OE1475F (homologue of MJ1249). Secondly, the methanogenic archaeon M. jannaschii has been From this stage onwards the canonical pathway can be used for shown to use an alternative pathway of DHQ biosynthesis [6], the biosynthesis of AroAA (Fig. 1B). carried out by enzymes that are unrelated to those of the classical In this study, we tested the hypothesis that haloarchaea use the pathway. Methanogens and haloarchaea are both members of the same reaction pathway for AroAA synthesis as shown experimen- phylum Euryarchaeota, and phylogenetic reconstructions fre- tally for M. jannaschii by White [6]. For this, the model quently show haloarchaea originating from within methanogen haloarchaeon H. salinarum was analysed using both in vivo and clades [7,8]. Consistent with this phylogenetic relationship, close in vitro strategies, including targeted mutations of the proposed homologues of M. jannaschii genes MJ0400 and MJ1249, which first two genes, nutrient requirements, phenotypes, AroAA uptake specify enzymes at he beginning of AroAA pathway, are encoded assays, enzyme activities of the purified gene products and global PLOS ONE | www.plosone.org 1 September 2014 | Volume 9 | Issue 9 | e107475 Aromatic Amino Acid Synthesis in Haloarchaea Figure 1. Proposed pathway for the biosynthesis of AroAA in H. salinarum, based on the pathway described for M. jannaschii. A, The initial steps in the de novo pathway were proposed according to White [6]. Note that no transaldolase reaction with ASA+DKFP was detected in this study, whereas the detected aldolase activity of OE1472F suggests that in H. salinarum the precursor might be F-1,6-P rather than DKFP. For details see Fig 10 and discussion. B, Downstream to DHQ, the canonical pathway is followed. Protein homologs found in H. salinarum are indicated above (or to the right of) the arrows, and the genes names are indicated below (or to the left). ASA-L-aspartate semialdehyde, DKFP-6-deoxy-5-ketofructose 1-phosphate, DHQ-dehydroquinate, DHS- dehydroshikimate. doi:10.1371/journal.pone.0107475.g001 examination of AroAA-related genes using a genome-wide DNA An in-frame deletion strain of aroD (OE1477R) was readily microarray. We provide evidence that the proteins OE1472F and obtained, but similar attempts to delete ORFs OE1472F and OE1475F do specify enzymes that provide DHQ to feed synthesis OE1475F were repeatedly unsuccessful. In the latter cases, of AroAA in haloarchaea. For brevity, we will refer to the hypothesized AroAA pathway in H.salinarum as the proposed pathway (i.e. a non-canonical pathway like that of M. jannaschii). Results Gene selection and construction of knock- out mutants Based on available data, and the experimentally validated pathway of AroAA biosynthesis in M. jannaschii [6], the first three steps would be catalyzed by ORFs OE1472F, OE1475F and Figure 2. Schematic representation of ORFs OE1472F, OE1475F OE1477R (aroD), respectively (Fig 1). A diagram of these genes and OE1477R in the H. salinarum R1 genome (accession number AM774415.1), and their relation to surrounding genes. CHY- and their relation to surrounding genes is shown in figure 2. In- conserved hypothetical protein. Arrows show the relative positions and frame deletions of OE1472F, OE1475F and OE1477R should orientations of ORFs (but are not drawn to scale). Coordinates 230688– convert H. salinarum to aromatic amino acid auxotrophy, 237094 bp. confirming the role of these ORFs in the pathway. doi:10.1371/journal.pone.0107475.g002 PLOS ONE | www.plosone.org 2 September 2014 | Volume 9 | Issue 9 | e107475 Aromatic Amino Acid Synthesis in Haloarchaea transformants invariably reverted to the wild type (WT) rather As expected, the deletion mutant DOE1477R was auxotrophic than to a deletion genotype (data not shown). Instead of complete for AroAA, with exogenous supplementation restoring about half deletions of OE1472F and OE1475F, attempts were then made to of the WT growth rate. Surprisingly, further supplementation with reduce their expression by insertional mutagenesis. As shown in DHQ allowed the cells to reach WT levels (Fig. 6K), suggesting Fig. 3A–B, a plasmid-borne terminator sequence was inserted just that the gene product of OE1477R acts on a substrate upstream to upstream of ORF OE1472F and immediately downstream of the DHQ, which would be inconsistent with the proposed pathway adjacent trpA, creating a stable mutant OE1471F::pMG501 (Fig. 1B). However, LC-MS analyses (table S1 in file S1) showed (StopOE1472F). This construct was confirmed by both Southern that 3% dehydroshikimate (DHS) forms spontaneously during blot (Fig. S1A in File S1) and PCR (Fig. 3C). Although pMG501 incubation of DHQ at 37uC in a salt solution (without cells). did not contain an origin of replication for haloarchaea, an Consequently, cells incubated with only DHQ also received at additional PCR was performed to exclude the possibility that least 0.033 mM DHS, explaining the growth promoting effect of pMG501 could survive in R1 without integration into the DHQ addition. These results are consistent with the assignment of chromosome. As expected, WT or plasmid DNA did not produce ORF OE1477R as gene aroD (Fig. 1). Below it is shown that its an amplification product (Fig.