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PubMed1992

Enzymatic synthesis and isolation of thymidine diphosphate-6-deoxy-D-xylo-4-hexulose and thymidine diphosphate-L-rhamnose. Production using cloned gene products and separation by HPLC.

A two-step enzymatic synthesis of dTDP-L-rhamnose is developed using enzymes from sonicated extracts of cultures of Escherichia coli K12 strains harboring plasmids containing different parts of the rfb gene cluster of Salmonella enterica LT2. The intermediate dTDP-6-deoxy-D-xylo-4-hexulose was isolated after a 1-h reaction, using only dTDP-D-glucose and dTDP-D-glucose 4,6-dehydratase, followed by protein precipitation and desalting by gel chromatography (yield 89%). In a two-step reaction using dTDP-D-glucose and dTDP-D-glucose 4,6-dehydratase in the first step, and with NADPH, dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase and NADPH:dTDP-6-deoxy-L-lyxo-4-hexulose-4-reductase in the second hour of incubation, the dTDP-D-glucose was fully converted to dTDP-L-rhamnose. The hexoses of both products were identified by mass spectroscopy. The molar yield of dTDP-L-rhamnose, after protein precipitation, anion-exchange chromatography and desalting by gel chromatography, was 62%, corresponding to more than 150 mg, starting from 250 mg of dTDP-D-glucose. When stored lyophilysed under nitrogen, these products were found to be stable for several months. Both dTDP-6-deoxy-D-xylo-4-hexulose and dTDP-L-rhamnose have light absorption maxima at 267 nm, with molar absorption coefficients close to that of dTMP. However, the absorption coefficient of dTDP-6-deoxy-D-xylo-4-hexulose at the absorption maximum of 320 nm (specific for sugars containing keto groups) was found to be approximately 20% higher than values presented earlier. Furthermore, an HPLC technique is presented for determining the net activity of dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase and NADPH:dTDP-6-deoxy-L-lyxo-4-hexulose-4-reductase, based on separation of dTDP-6-deoxy-D-xylo-4-hexulose and dTDP-L-rhamnose. The HPLC technique is also suitable for determination of all the nucleotide components involved in the synthesis.

Bacterial Proteins↗
PubMed1999

Regulation of bacteriophage lambda development by guanosine 5'-diphosphate-3'-diphosphate.

On infection of its host, Escherichia coli, bacteriophage lambda can follow one of two alternative developmental pathways: lytic or lysogenic. Here we demonstrate that the "lysis-versus-lysogenization" decision is influenced by guanosine tetraphosphate (ppGpp), a nucleotide that is synthesized in E. coli cells in response to amino acid or carbon source starvation. We found that the efficiency of lysogenization is the highest at ppGpp concentrations somewhat higher than the basal level; too low and too high levels of ppGpp result in less efficient lysogenization. Maintenance of the already integrated lambda prophage and phage lytic development were not significantly influenced in the host lacking ppGpp. We found that the level of HflB/FtsH protease, responsible for degradation of the CII protein, an activator of "lysogenic" promoters, depends on ppGpp concentration. The highest levels of HflB/FtsH was found in bacteria lacking ppGpp and in cells bearing increased concentrations of this nucleotide. Using lacZ fusions, we investigated the influence of ppGpp on activities of lambda promoters important at the stage of the lysis-versus-lysogenization decision. We found that each promoter is regulated differentially in response to the abundance of ppGpp. Moreover, our results suggest that the cAMP level may influence ppGpp concentration in cells. The mechanism of the ppGpp-mediated control of lambda development at the stage of the lysis-versus-lysogenization decision may be explained on the basis of differential influence of guanosine tetraphosphate on activities of p(L), p(R), p(E), p(I), and p(aQ) promoters and by dependence of HflB/FtsH protease level on ppGpp concentration.

ATP-Dependent Proteases↗
PubMed1981

Transcription of the E. coli tufB gene: cotranscription with four tRNA genes and inhibition by guanosine-5'-diphosphate-3'-diphosphate.

The transcription of the tufB gene by purified RNA polymerase holoenzyme was studied using the transducing phage lambda rifd 18 DNA and the hybrid plasmid pTUB1 DNA (Miyajima et al. 1979) as templates. The size of tufB mRNA synthesized in this system was about 1,700 nucleotides, and the same strand as for rrnB was transcribed. By electron microscopic examination of the R-loop formed between lambda fus3 DNA and tufB mRNA synthesized under the direction of pTUB1 DNA, it was found that the untranslated sequence of about 500 nucleotides is at the 5' end of tufB mRNA. The sequencing of the 5' region of tufB mRNA synthesized on the truncated template has revealed that the tufB gene is cotranscribed with its upstream genes for four tRNAs (thrU, tyrU, glyT, and thrT). The synthesis of this mRNA molecule is completely abolished by low concentrations of ppGpp. Neither pppGpp, ppGp, nor pGpp was effective as inhibitor in this cell-free system.

Depression, Chemical↗