An in-depth analysis of the intracellular metabolite concentrations, metabolic fluxes, and gene expression (metabolome, fluxome, and transcriptome, respectively) of lysine-producing Corynebacterium glutamicum ATCC 13287 was performed at different stages of batch culture and revealed distinct phases of growth and lysine production. For this purpose, 13 C flux analysis with gas chromatography-mass spectrometry-labeling measurement of free intracellular amino acids, metabolite balancing, and isotopomer modeling were combined with expression profiling via DNA microarrays and with intracellular metabolite quantification. The phase shift from growth to lysine production was accompanied by a decrease in glucose uptake flux, the redirection of flux from the tricarboxylic acid (TCA) cycle towards anaplerotic carboxylation and lysine biosynthesis, transient dynamics of intracellular metabolite pools, such as an increase of lysine up to 40 mM prior to its excretion, and complex changes in the expression of genes for central metabolism. The integrated approach was valuable for the identification of correlations between gene expression and in vivo activity for numerous enzymes. The glucose uptake flux closely corresponded to the expression of glucose phosphotransferase genes. A correlation between flux and expression was also observed for glucose-6-phosphate dehydrogenase, transaldolase, and transketolase and for most TCA cycle genes. In contrast, cytoplasmic malate dehydrogenase expression increased despite a reduction of the TCA cycle flux, probably related to its contribution to NADH regeneration under conditions of reduced growth. Most genes for lysine biosynthesis showed a constant expression level, despite a marked change of the metabolic flux, indicating that they are strongly regulated at the metabolic level. Glyoxylate cycle genes were continuously expressed, but the pathway exhibited in vivo activity only in the later stage. The most pronounced changes in gene expression during cultivation were found for enzymes at entry points into glycolysis, the pentose phosphate pathway, the TCA cycle, and lysine biosynthesis, indicating that these might be of special importance for transcriptional control in C. glutamicum.

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A c~enzyme-F~~,, non-reducing [NiFe] hydrogenase was isolated from Methanococcus voltae. It consists of three subunits. They are the products of the previously identified genes vhuA, vhuG and vhuU. The vhuU gene product is of only 25 amino acids. This novel very small hydrogenase subunit contains selenocysteine within a conserved amino-acid sequence previously shown to be involved in Ni coordination. The subunit is shorter than the predicted primary gene product and is therefore apparently post-translationally processed.[NiFe] hydrogenases are widespread in prokaryotes. At least their functional domains, carrying Fe-S clusters or the nickel-containing reaction center are believed to be descendants of a common prototype since strong similarities can be seen in all known enzymes of this type [l, [4]. One of the enzymes (the fruA,G,B product), which is able to reduce the deazaflavine Fdz0, has previously been purified [5]. It contains selenium. In the gene of the largest subunit (fruA) a single internal TGA codon is found in the region coding for the assumed Ni-binding site. It most likely encodes selenocysteine. This notion is supported by the fact that the TGA codon is followed by a sequence capable of forming a moderately stable secondary structure in the transcript. These two features are known to be prerequisites for the incorporation of selenocysteine into formate dehydrogenase in Escherichia coli [6].A second hydrogenase gene group (vhuG,A, U) has been predicted to encode a selenium containing F420 non-reducing hydrogenase. Again a TGA codon and an adjacent downstream sequence capable of secondary-structure formation are seen in the homologous position to the one of the previously mentioned hydrogenase [4]. The DNA sequence analysis led to the prediction of a very small subunit containing the selenocysteine, which would carry at least part of the Ni-coordination site, whereas the homologous domain would be part of the largest subunit of the putative selenium-free vhc hydrogenase (Fig. 1). Such a small subunit has never been found in other [NiFe] hydrogenases and might be a very useful tool for the study of the primary reaction center of the enzyme.In this study we describe this new [NiFe] hydrogenase from M. voltae which does comprise three subunits. One of them is indeed very small and contains selenocysteine. To our surprise the primary translation product of the small gene appears to be processed. Removal of a predicted C-terminal portion of 44 amino-acid residues leads to the final length of only 25 amino acids and a molecular mass of only 2884 Da if the N-terminal methionine is removed. EXPERIMENTAL PROCEDURES Cell growth and enyzme isolationM. voltae cells were grown to late exponential phase as described previously [7] in a 300-1 fermenter. After harvesting under aerobic conditions they were frozen in liquid nitrogen. All enzyme purification steps were performed under anaerobic conditions. Crude lysate and centrifugation10 g cells were lysed in 30 ml buffer A (50 mM TrisMCl, pH 7.5) containing 10...

The selenium-containing F ,,-non-reducing hydrogenase from Methanococcus voltae was anaerobically purified. The enzyme as isolated showed an EPR spectrum with g,,_ = 2.21, 2.15 and 2.01. Upon illumination this spectrum disappeared and a new signal with the lowest g value at 2.05 arose. EPR studies were carried out either with the enzyme containing natural selenium or enriched in the nuclear isotope "Se. The hype&e splitting caused by "Se in the 'dark' signal is shown to be highly anisotropic. In contrast the splitting is nearly isotropic after illumination. A new model for the nickel site is proposed to explain these observations.

Methanococcus voltae carries genetic information for four [NiFe] hydrogenases. Two of the hydrogenases are predicted to contain selenocysteine on the basis of in-frame TGA codons, while the genes encoding the two other enzymes contain cysteine codons at homologous positions. Their predicted subunit compositions and their electron acceptor specificities are similar to those of the respective selenium-containing enzymes. The selenium-containing hydrogenases have been purified and characterized. Only one of them reduces the deazaflavin F(420). The activity of the F(420)-nonreducing enzyme is exceptionally high. The selenium atom has been shown by EPR spectroscopy to be a ligand to the Ni atom in the primary reaction centers in both enzymes. The spectroscopic analyses also yielded a description of the electronic configuration around the NiFe center at different oxidation states and in the presence of the competitive inhibitor, CO. The genes encoding the selenium-free hydrogenases are expressed only in the absence of selenium. They are linked by an intergenic region in which regulatory cis elements were defined by employing reporter gene constructs and site-directed mutagenesis.