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First Characterization of an Archaeal GTP-Dependent Phosphoenolpyruvate Carboxykinase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1. Wakao Fukuda, 2004.Phosphoenolpyruvate carboxykinase (PCK), which catalyzes the nucleotide-dependent, reversible decarboxylation of oxaloacetate to yield phosphoenolpyruvate and CO2, is one of the important enzymes in the interconversion between C3 and C4 metabolites . This study focused on the first characterization of the enzymatic properties and expression profile of an archaeal PCK from the hyperthermophilic archaeon Thermococcus kodakaraensis (PckTk) . PckTk showed 30 to 35% identities to GTP-dependent PCKs from mammals and bacteria but was located in a branch distinct from that of the classical enzymes in the phylogenetic tree, together with other archaeal homologs from Pyrococcus and Sulfolobus spp . Several catalytically important regions and residues, found in all known PCKs irrespective of their nucleotide specificities, were conserved in PckTk . However, the predicted GTP-binding region was unique compared to those in other GTP-dependent PCKs . The recombinant PckTk actually exhibited GTP-dependent activity and was suggested to possess dual cation-binding sites specific for Mn2+ and Mg2+ . The enzyme preferred phosphoenolpyruvate formation from oxaloacetate, since the Km value for oxaloacetate was much lower than that for phosphoenolpyruvate . The transcription and activity levels in T . kodakaraensis were higher under gluconeogenic conditions than under glycolytic conditions . These results agreed with the role of PckTk in providing phosphoenolpyruvate from oxaloacetate as the first step of gluconeogenesis in this hyperthermophilic archaeon . Additionally, under gluconeogenic conditions, we observed higher expression levels of PckTk on pyruvate than on amino acids, implying that it plays an additional role in the recycling of excess phosphoenolpyruvate produced from pyruvate, replacing the function of the anaplerotic phosphoenolpyruvate carboxylase that is missing from this archaeon . Generation of Enhanced Competitive Root-Tip-Colonizing Pseudomonas Bacteria through Accelerated Evolution. Sandra de Weert, 2004.A recently published procedure to enrich for efficient competitive root tip colonizers (I . Kuiper, G . V . Bloemberg, and B . J . J . Lugtenberg, Mol . Plant-Microbe Interact . 14:1197-1205) after bacterization of seeds was applied to isolate efficient competitive root tip colonizers for both the dicotyledenous plant tomato and the monocotyledenous plant grass from a random Tn5luxAB mutant bank of the good root colonizer Pseudomonas fluorescens WCS365 . Unexpectedly, the best-colonizing mutant, strain PCL1286, showed a strongly enhanced competitive root-tip-colonizing phenotype . Sequence analyses of the Tn5luxAB flanking regions showed that the transposon had inserted in a mutY homolog . This gene is involved in the repair of A · G mismatches caused by spontaneous oxidation of guanine . We hypothesized that, since the mutant is defective in repairing its mismatches, its cells harbor an increased number of mutations and therefore can adapt faster to the environment of the root system . To test this hypothesis, we constructed another mutY mutant and analyzed its competitive root tip colonization behavior prior to and after enrichment . As a control, a nonmutated wild type was subjected to the enrichment procedure . The results of these analyses showed (i) that the enrichment procedure did not alter the colonization ability of the wild type, (ii) that the new mutY mutant was strongly impaired in its colonization ability, but (iii) that after three enrichment cycles it colonized significantly better than its wild type . Therefore it is concluded that both the mutY mutation and the selection procedure are required to obtain an enhanced root-tip-colonizing mutant . Growth Characteristics and Intraspecies Host Specificity of a Large Virus Infecting the Dinoflagellate Heterocapsa circularisquama. Keizo Nagasaki, 2003.The growth characteristics and intraspecies host specificity of Heterocapsa circularisquama virus (HcV), a large icosahedral virus specifically infecting the bivalve-killing dinoflagellate H . circularisquama, were examined . Exponentially growing host cells were more sensitive to HcV than those in the stationary phase, and host cells were more susceptible to HcV infection in the culture when a higher percent of the culture was replaced with fresh medium each day, suggesting an intimate relationship between virus sensitivity and the physiological condition of the host cells . HcV was infective over a wide range of temperatures, 15 to 30°C, and the latent period and burst size were estimated at 40 to 56 h and 1,800 to 2,440 infective particles, respectively . Transmission electron microscopy revealed that capsid formation began within 16 h postinfection, and mature virus particles appeared within 24 h postinfection at 20°C . Compared to Heterosigma akashiwo virus, HcV was more widely infectious to H . circularisquama strains that had been independently isolated in the western part of Japan, and only 5.3% of the host-virus combinations (53 host and 10 viral strains) showed resistance to viral infection . The present results are helpful in understanding the ecology of algal host-virus systems in nature .
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