A single transcription factor regulates evolutionarily diverse but functionally linked metabolic pathways in response to nutrient availability

Amy K Schmid¹, David J Reiss¹, Min Pan¹, Tie Koide^1,a & Nitin S Baliga^1,2

^aPresent address: E-mail: Email: tiekoide@gmail.com

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Synopsis

Several lateral gene transfers and homologous gene replacement events are speculated to have an important function in evolution of archaeal metabolic networks (Galperin and Koonin, 1999; Siebers and Schonheit, 2005). If so, then this raises important questions regarding the evolution and the architecture of gene regulatory networks (GRNs) that integrate and coordinate these enzymes in the face of unique environmental challenges. Metabolism of sugars in Halobacterium salinarum NRC-1 represents one such central process in which several enzymes and entire segments seem to have been acquired through lateral gene transfers. Taking a systems approach, we have characterized global regulation of these core processes by a single regulator TrmB. Specifically, we integrated data from classical physiology and genetics experiments with orthogonal sources of genome-wide evidence, including (i) protein–DNA interactions measured globally with ChIP-chip; (ii) transcriptional responses of genetically and environmentally perturbed strains using microarray analysis; (iii) genome-wide distribution of a conserved TF-binding motif signature); (iv) a reconstructed metabolic network (Figure 6).

Figure 6

TrmB is a global bifunctional regulator, which coordinates the expression of evolutionarily diverse metabolic enzyme genes. Pathway diagram represents the reconstructed TrmB-specified metabolic network for H. salinarum (see Supplementary Figure 3 and Supplementary information for reconstruction method). Numbered, colored boxes represent mRNA expression level for each enzyme-encoding gene in the trmB mutant in the absence of glucose. Numbers correspond to gene names listed to the right. Gene names shown in bold black type represent those whose encoded enzymes have been biochemically characterized in archaea (see also Supplementary Table 6 for details on enzyme functions). Gene names in bold red indicate those that are indicated by the literature to be unique to the archaeal domain (Supplementary Table 6). Asterisks next to gene names denote a gene in the second or higher position in an operon directly controlled by TrmB. Red boxes represent those that are induced in the trmB knockout background in the absence of glucose; green, repressed; the intensity of the color corresponds to the extent of change (see legend). Boxes bounded by black lines are direct targets of TrmB (according to ChIP-chip data), and those bounded by blue lines represent targets for which a TrmB-binding motif was identified. Magenta dots indicate central metabolic intermediates. Dotted lines between glycerol and glyceraldehyde-3-phosphate represent putative alternative pathways for entry of glycerol into the trunk portion of glycolysis (Gonzalez et al, 2008). Not all 113 TrmB targets are listed in the figure for the sake of clarity. Explanations for abbreviations of metabolic intermediates are listed in Supplementary Table 6. Purple and orange shaded areas represent examples of pathways with mixed evolutionary ancestry (see Results).

Full figure and legend (1,111K)Figures & Tables index

Deletion of this regulator resulted in severe growth defects in diverse environments including nutrient replete or limitation, metal excess or limitation, and oxidative stress conditions. This generalized growth phenotype was likely because of a reduced NAD⁺/NADH ratio relative to the parent strain and readily complemented by glucose or glycerol. Global transcription analysis of the trmB strain revealed perturbed regulation of 182 genes (16 down- and 166 upregulated) with significant overrepresentation of carbohydrate metabolism genes. Surprisingly, the deletion of TrmB also resulted in defective regulation of amino acid, cofactor, vitamin, and purine biosynthesis genes. These observations were further refined through ChIP-chip experiments, which showed that TrmB was physically associated with the promoters of many of these genes in a glucose- or glycerol-dependent manner (P=5.5 10^-11). We have verified the requirement of a conserved cis-regulatory motif within many of these promoters to be essential for TrmB function. Together these results support the hypothesis that, depending on the carbon source (glucose or glycerol), TrmB acts as both a transcriptional activator and a repressor to directly coordinate enzymes of central metabolism with associated pathways.

Upon integration of the gene regulatory network with a reconstructed metabolic network, we observed several instances of direct TrmB-mediated transcriptional control of metabolic enzymes with the biosynthesis of their cognate cofactors. For example, TrmB directly controls over 10 enzymes that require adenosine phosphates (AXP; Figure 6, e.g. reactions 3, 7, 10, 11, 27, 33, 63) and six genes that encode the biosynthesis of these cofactors (Figure 6, reactions 43, 44, 45, 46, 47, 52). Our data suggest that TrmB represses the semi-phosphorylative Entner–Doudoroff (E–D) glycolytic pathway (Danson et al, 2007; Kanai et al, 2007; van der Oost and Siebers, 2007; Pfeiffer et al, 2008) (e.g. gap, pykA, VNG0442G) (Figure 6), and induces gluconeogenesis (e.g. ppsA; Figure 6). Thus, a deletion in trmB would lead to an inability to generate energy through gluconeogenesis in the absence of glucose. Furthermore, trmB cultures grown in the absence of glucose upregulate enzymes that reduce NAD(P)⁺ to NAD(P)H, potentially forcing the cell toward an oxidized state (e.g. Figure 6, reactions 19, 31). If so, then this would lead to a shortage of reducing equivalents. The hypersensitivity to oxidative stress and reduced NAD⁺/H ratio observed in trmB mutant cells are consistent with this hypothesis. We conclude that TrmB acts to maintain redox and energy balance in response to nutrient availability in H. salinarum.

The TrmB-specified regulatory network coordinates the transcription of enzymes of mixed evolutionary lineage (Figure 6). For example, in the shikimate biosynthesis pathway, only one gene encoding shikimate kinase is of archaeal origin, whereas all other genes are conserved throughout evolution (orange shaded area, Figure 6). Strikingly, all genes of this pathway except shikimate kinase are direct TrmB targets (Figure 6), suggesting that shikimate kinase may have been acquired by homologous gene replacement (Galperin and Koonin, 1999). In this regard, the TrmB regulatory network might be a specific example of an active evolutionary process, because several lateral gene transfer or homologous gene replacement events are thought to have occurred in the evolutionary compilation of metabolic networks (Galperin and Koonin, 1999). In summary, this study provides insight into how the architecture of a large metabolic network and an associated GRN may have co-evolved using components of diverse origins, and how this assembly may be conserved across the archaeal lineage.

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