No abstract

L-Lysine catabolism in Pseudomonas putida KT2440 was generally thought to occur via the aminovalerate pathway. In this study we demonstrate the operation of the alternative aminoadipate pathway with the intermediates D-lysine, L-pipecolate, and aminoadipate. The simultaneous operation of both pathways for the use of L-lysine as the sole carbon and nitrogen source was confirmed genetically. Mutants with mutations in either pathway failed to use L-lysine as the sole carbon and nitrogen source, although they still used L-lysine as the nitrogen source, albeit at reduced growth rates. New genes were identified in both pathways, including the davB and davA genes that encode the enzymes involved in the oxidation of L-lysine to ␦-aminovaleramide and the hydrolysis of the latter to ␦-aminovalerate, respectively. The amaA, dkpA, and amaB genes, in contrast, encode proteins involved in the transformation of Pseudomonas putida KT2440, a derivative of P. putida mt-2 cured of the TOL plasmid, can grown on proline, lysine, glutamate, and other amino acids as the sole carbon and nitrogen source. The ability to assimilate these compounds confers on the strain a selective advantage to grow in the rhizosphere of a number of plants where these amino acids are part of the exudates (2,11,28,29).The catabolism of L-lysine by P. putida mainly involves the following steps: L-lysine 3 ␦-aminovaleramide 3 ␦-aminovalerate (AMV) 3 glutarate semialdehyde 3 glutarate, which is then channeled to the Krebs cycle. This pathway is known as the AMV pathway ( Fig. 1) and was well characterized at the biochemical level in the late 1970s (6,7,13). In this route, the first step involves the oxidative decarboxylation of the amino acid to yield ␦-aminovaleramide, which is hydrolyzed to produce ammonium and ␦-aminovalerate. Thereafter, ␦-aminovalerate is converted into glutarate via glutarate semialdehyde (6, 7) in reactions catalyzed by the products of the davD and the davT genes, the only genes of the pathway identified so far (11, 38). The davD gene forms an operon with davT, the gene order being davDT (38). The rei-2 mutant is a KT2440 derivative that is unable to use L-lysine as a carbon source and which was isolated after mutagenesis of the wild-type strain with mini-Tn5-Јlux. Mini-Tn5-Јlux was inserted within the davT gene, giving rise to a davDT:Јlux transcriptional fusion. The relevance of this pathway during the colonization of the root system of corn by P. putida is evidenced by the fact that rei-2 cells emitted light in response to root exudates. In agreement with the pathway described above, both davD and davT mutants were unable to use L-lysine or ␦-aminovalerate as a carbon source. The davD promoter was expressed at a certain level in the absence of L-lysine, but its expression increased about fourfold in response to the addition of exogenous Llysine to the culture medium. However, the real inducer of this operon seems to be AMV because in a mutant unable to metabolize L-lysine to ␦-aminovalerate, this compound, and not L-lysine, acted as ...

The Bacillus subtilis ferric uptake regulator (Fur) protein is the major sensor of cellular iron status. When iron is limiting for growth, derepression of the Fur regulon increases the cellular capacity for iron uptake and mobilizes an iron-sparing response mediated in large part by a small noncoding RNA named FsrA. FsrA functions, in collaboration with three small basic proteins (FbpABC), to repress many "low-priority" iron-containing enzymes. We have used transcriptome analyses to gain insights into the scope of the iron-sparing response and to define subsets of genes dependent for their repression on FsrA, FbpAB, and/or FbpC. Enzymes of the tricarboxylic acid (TCA) cycle, including aconitase and succinate dehydrogenase (SDH), are major targets of FsrA-mediated repression, and as a consequence, flux through this pathway is significantly decreased in a fur mutant. FsrA also represses the DctP dicarboxylate permease and the iron-sulfur-containing enzyme glutamate synthase (GltAB), which serves as a central link between carbon and nitrogen metabolism. Allele-specific suppression analysis was used to document a direct RNA-RNA interaction between the FsrA small RNA (sRNA) and the gltAB leader region. We further demonstrated that distinct regions of FsrA are required for the translational repression of the GltAB and SDH enzyme complexes.T he tricarboxylic acid (TCA) cycle is a central pathway of Bacillus subtilis metabolism. The flux of carbon through the TCA cycle generates ATP through substrate-level phosphorylation and by the generation of reducing equivalents that feed the electron transport chain. TCA cycle intermediates also serve as biosynthetic precursors for numerous amino acids, heme, and other key metabolites (4,9,18,21,43). Of particular note, ␣-ketoglutarate is transaminated by glutamate synthase using glutamine as a donor to generate two glutamate molecules. Thus, glutamate synthase serves as a direct link between central carbon metabolism and nitrogen metabolism. Citric acid, another key intermediate in the TCA cycle, can also play a role in metal ion homeostasis by facilitating the uptake of cations, including Fe(III), Mg(II), and Mn(II) (25,30).Since the TCA cycle is central to many biosynthetic and metabolic processes, several regulators exact tight control over the expression of TCA cycle enzymes, including both global (CcpA, CodY, and TnrA) and pathway-specific (CcpC, RocR, and GltC) regulators (39). To this list, we can now add the ferric uptake regulator (Fur). Fur has dual roles in iron homeostasis. Under iron-limiting growth conditions, derepression of numerous Furregulated operons (2) allows expression of siderophore biosynthesis and uptake pathways (30). Many other genes are downregulated in the fur mutant (2), including several with roles in central metabolism. Many of the genes downregulated in the fur mutant may be the targets of a Fur-regulated sRNA A (FsrA) and three small, Fur-regulated basic proteins (FbpABC) that can act as coregulators with FsrA. These effectors repress the transla...

Although a whole arsenal of mechanisms are potentially involved in metabolic regulation, it is largely uncertain when, under which conditions, and to which extent a particular mechanism actually controls network fluxes and thus cellular physiology. Based on 13 C flux analysis of Escherichia coli mutants, we elucidated the relevance of global transcriptional regulation by ArcA, ArcB, Cra, CreB, CreC, Crp, Cya, Fnr, Hns, Mlc, OmpR, and UspA on aerobic glucose catabolism in glucose-limited chemostat cultures at a growth rate of 0.1 h ؊1 . The by far most relevant control mechanism was cyclic AMP (cAMP)-dependent catabolite repression as the inducer of the phosphoenolpyruvate (PEP)-glyoxylate cycle and thus low tricarboxylic acid cycle fluxes. While all other mutants and the reference E. coli strain exhibited high glyoxylate shunt and PEP carboxykinase fluxes, and thus high PEP-glyoxylate cycle flux, this cycle was essentially abolished in both the Crp and Cya mutants, which lack the cAMP-cAMP receptor protein complex. Most other mutations were phenotypically silent, and only the Cra and Hns mutants exhibited slightly altered flux distributions through PEP carboxykinase and the tricarboxylic acid cycle, respectively. The Cra effect on PEP carboxykinase was probably the consequence of a specific control mechanism, while the Hns effect appears to be unspecific. For central metabolism, the available data thus suggest that a single transcriptional regulation process exerts the dominant control under a given condition and this control is highly specific for a single pathway or cycle within the network.Some parts of metabolic networks are organism specific, but the core network is highly conserved. Almost all aerobic bacteria have a similar set of about 100 enzymes that catalyze the formation of biosynthetic building blocks, energy, and cofactors. This core network is ubiquitous because all specialized catabolic pathways finally merge into one or more of the common intermediates. Obviously, not all core reactions are simultaneously active and the evolved regulatory structure of an organism ensures appropriate and flexible activity of the various enzymes under the conditions normally encountered. One key regulatory task is to direct carbon fluxes such that all of the necessary biomass components are synthesized at the appropriate stoichiometry and rate from a wide range of substrates.Transcriptional regulation is generally considered the main microbial control mechanism, and a complicated network of global and specific transcription factors could potentially manage this distribution of fluxes (31). Such transcriptional control of metabolic activity is firmly established for the degradation and biosynthesis branches of the network. A typical example is aromatic amino acid biosynthesis with fine tuning of flux into the various branches by allosteric feedback inhibition of key enzymes but transcriptional regulation as the general control mechanism for absence or presence of the pathway (41). The situation is much less clear, ...