SummaryA system where archaeal gene expression could be controlled by simple manipulation of growth conditions would enable the construction of conditional lethal mutants in essential genes, and permit the controlled overproduction of proteins in their native host. As tools for the genetic manipulation of Haloferax volcanii are well developed, we set out to identify promoters with a wide dynamic range of expression in this organism. Tryptophan is the most costly amino acid for the cell to make, so we reasoned that tryptophan-regulated promoters might be good candidates. Microarray analysis of H. volcanii gene expression in the presence and absence of tryptophan identified a tryptophanase gene (tna) that showed strong induction in the presence of tryptophan. qRT-PCR revealed a very fast response and an up to 100-fold induction after tryptophan addition. This result has been confirmed using three independent reporter genes (cct1, pyrE2 and bgaH). Vectors containing this promoter will be very useful for investigating gene function in H. volcanii and potentially in other halophilic archaea. To demonstrate this, we used the promoter to follow the consequences of depletion of the essential chaperonin protein CCT1, and to determine the ability of heterologous CCT proteins to function in H. volcanii.
We showed recently that antisense plants with decreased activity of the plastidic ATP/ADP-transporter protein exhibit drastically reduced levels of starch and a decreased amylose/amylopectin ratio, whereas sense plants with increased activity of the transporter possessed more starch than wild-type plants and an increased amylose/amylopectin ratio. In this paper we investigate the effect of altered plastidic ATP/ADP-transporter protein expression on primary metabolism and granule morphology in more detail. Tuber tissues from antisense and sense plants exhibited substantially increased respiratory activity compared with the wild type. Tubers from antisense plants contained markedly increased levels of free sugars, UDP-Glc, and hexose phosphates, whereas phosphoenolpyruvate, isocitrate, ATP, ADP, AMP, UTP, UDP, and inorganic pyrophosphate levels were slightly decreased. In contrast, tubers from sense plants revealed a slight increase in adenine and uridine nucleotides and in the levels of inorganic pyrophosphate, whereas no significant changes in the levels of soluble sugars and metabolites were observed. Antisense tubers contained 50% reduced levels of ADP-Glc, whereas sense tubers contained up to 2-fold increased levels of this sole precursor for starch biosynthesis. Microscopic examination of starch grain morphology revealed that the size of starch grains from antisense tubers was substantially smaller (50%) compared with the wild type. The large starch grains from sense tubers appeared of a more angular morphology, which differed to the more ellipsoid shape of wild type grains. The results suggest a close interaction between plastidial adenylate transport and starch biosynthesis, indicating that ADP-Glc pyrophosphorylase is ATP-limited in vivo and that changes in ADP-Glc concentration determine starch yield, as well as granule morphology. Possible factors linking starch synthesis and respiration are discussed.
In order to unravel the role of regulation on transcript level in central carbohydrate metabolism (CCM) of Thermoproteus tenax, a focused DNA microarray was constructed by using 85 open reading frames involved in CCM. A transcriptional analysis comparing heterotrophic growth on glucose versus autotrophic growth on CO 2 -H 2 was performed.The anaerobic, facultative heterotrophic crenarchaeum Thermoproteus tenax shows a sulfur-dependent energy metabolism and grows optimally at 86°C and pH 5.6. In addition to autotrophic growth of this organism on carbon dioxide and hydrogen (CO 2 -H 2 ), heterotrophic growth on various carbohydrates, such as starch, glycogen, and glucose, was reported (39). A combination of genomics-based and biochemical approaches revealed that T. tenax is the only archaeum currently known that uses two different pathways for carbohydrate catabolism in parallel, a reversible Embden-Meyerhof-Parnas (EMP) pathway and the catabolic, so-called "branched" Entner-Doudoroff (ED) pathway, both of which represent modified versions of the known classical bacterial and eukaryotic pathways (1,26, 28,30). In T. tenax, the operation of a reversible citric acid cycle (CAC), which is involved in the oxidation of pyruvate to CO 2 under heterotrophic growth conditions (24) and in CO 2 fixation under autotrophic growth conditions (30), has been suggested. Compared to the available knowledge about the complexity of archaeal central carbohydrate metabolism (CCM) and its various modifications, information on the regulation of the CCM is rather scarce. As shown for the EMP pathway, the allosteric regulation at the protein level, which plays an important role in the classical pathway of bacteria and eucarya, seems to be of minor relevance in archaea. The classical control points at the beginning (hexokinase/glucose-6-phosphatase and ATP-phosphofructokinase/fructose-1,6-bisphosphatase) and at the end (pyruvate kinase [PK]/phosphoenolpyruvate [PEP] synthetase [PEPS]) of the pathway are absent in T. tenax and all archaea studied so far (31). The key reactions are catalyzed by nonallosteric enzymes in archaea. Due to the ability of T. tenax to grow under autotrophic as well as heterotrophic growth conditions, the organism represents an ideal object for the study of the regulation of the glycolytic/gluconeogenic switch of carbon flux.
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