Cloning of the ribokinase gene of Staphylococcus hyicus subsp. hyicus in Staphylococcus carnosus

Summary A gene library with DNA of Staphylococcus hyicus subsp. hyicus was established in S. carnosus by using the plasmid vector pCT20. Two clones of S. carnosus were isolated which were able to ferment d-ribose. The two hybrid plasmids (pRib 1) and (pRib 2) were isolated and characterized. They contained inserted DNA fragments of S. hyicus subsp. hyicus with sizes of 10.2 and 8.2 kb, respectively. d-Ribose uptake and enzyme activities were studied. All strains tested [S. hyicus subsp. hyicus, S. carnosus (wild type) and the two S. carnosus clones] possessed an inducible uptake system for d-ribose. S. hyicus subsp. hyicus possessed in addition enzyme activities of d-ribokinase and d-ribose-5-P isomerase. None of these enzyme activities could be detected in S. carnosus (wildtype). Only in the S. carnosus clones containing (pRib 1) or (pRib 2) could a d-ribokinase activity be demonstrated, indicating that the gene for d-ribokinase of S. hyicus subsp. hyicus was cloned in S. carnosus.Abbreviations bp base pairs - C-TLC cellulose-thin layer chromoatography - kb kilo base pairs - pR_ib 1 and 2 ribokinase activity conferring hybridplasmids - MBq megabequerel - wt wild type     

Cloning and characterization of a gene encoding trehalose phosphorylase (TP) from Pleurotus sajor-caju

Complementary DNA for a gene encoding trehalose phosphorylase (TP) that reversibly catalyzes trehalose synthesis and degradation from alpha-glucose-1-phosphate (alpha-Glc-1-P) and glucose was cloned from Pleurotus sajor-caju. The cDNA of P. sajor-caju TP (designated PsTP, GenBank Accession No. AF149777) encodes a polypeptide of 751 amino acids with a deduced molecular mass of 83.7 kDa. The PsTP gene is expressed in mycelia, pilei, and stipes of fruiting bodies. Trehalose phosphorylase PsTP was purified from PsTP-transformed Escherichia coli. The enzyme catalyzes both the phosphorolysis of trehalose to produce alpha-Glc-1-P and glucose, and the synthesis of trehalose. The apparent K(m) values for trehalose and Pi in phosphorolytic reaction at pH 7.0 were 74.8 and 5.4 mM, respectively. The PsTP gene complemented Saccharomyces cerevisiae Deltatps1, Deltatps2 double-mutant cells, allowing their growth on glucose medium. Furthermore, yeast transformed with PsTP produced 2-2.5-fold more trehalose than non-transformants or cells transformed with empty vector only.     

Studies on lipase directed export of Escherichia coli beta-lactamase in Staphylococcus carnosus

Summary The lipase (lip) gene of Staphylococcus hyicus was used to study the expression of the Escherichia coli -lactamase (bla) gene in S. carnosus. The bla gene, devoid of its promotor and most of the signal sequence, was fused to the lip structural gene at various positions. A set of 11 secretion vectors (pLL1 to pLL11) was isolated and analysed. All secretion vectors caused -lactamase production and activity in S. carnosus. However, the amount of hybrid proteins secreted was influenced by the length of the NH₂-terminal lipase portion. An increased concentration, comparable to that of the native lipase, of secreted lipase/-lactamase hybrid proteins was only found when the lipase portion of the construct comprised more than 101 amino acids of the NH₂-terminal region of the lipase preprotein; the proposed lipase signal peptide is 36 amino acids long. If the hybrid proteins constructed contained 101 or less amino acids of the NH₂-terminal lipase preprotein, only low amounts of secreted hybrid proteins were detectable and a significant portion of the hybrid proteins and -lactamase activity was found in the cellular fraction. The results indicate that the lipase possesses adjacent to the signal peptide a peptide domain that is essential for the secretion of the lipase/-lactamase hybrid proteins.Abbreviations Cm chloramphenicol - bla gene beta lactamase coding gene of Escherichia coli - lip gene lipase-coding gene of Staphylococcus hyicus - PA polyacrylamide - PAGE PA gel electrophoresis - SDS sodium dodecyl sulphate - [] indicates plasmid-carrier state     

Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana

Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrite uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.Abbreviations EDTA ethylenediaminetetraacetic acid - FAD flavine-adenine dinucleotide - IgG immunoglobulin G - NR nitrate reductase - PM plasma membrane - TX-100 Triton X-100     

Diverse effects of formate on the dissimilatory metabolism of nitrate in Pseudomonas denitrificans ATCC 13867: Growth,nitrite accumulation in culture,cellular activities of nitrate and nitrite reductases

The growth of Pseudomonas denitrificans ATCC 13867 under denitrifying conditions was significantly stimulated by adding an appropriate amount of formate (2.5 mM or above) to the growth medium. The accumulation of nitrite in the culture was markedly depressed so long as formate remained in the culture above a certain level. Cellular activities of enzymes participating in denitrification also changed. The cells grown in the presence of formate exhibited a lower nitrate reductase activity and, in contrast, a higher nitrite reductase activity than the cells grown without added formate.     

Isolation,sequencing and mutational analysis of a gene cluster involved in nitrite reduction inParacoccus denitrificans

By using the gene encoding the C-terminal part of thecd ₁-type nitrite reductase ofPseudomonas stutzeri JM300 as a heterologous probe, the corresponding gene fromParacoccus denitrificans was isolated. This gene,nirS, codes for a mature protein of 63144 Da having high homology withcd ₁-type nitrite reductases from other bacteria. Directly downstream fromnirS, three othernir genes were found in the ordernirECF. The organization of thenir gene cluster inPa. denitrificans is different from the organization ofnir clusters in some Pseudomonads.nirE has high homology with a S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (uro'gen III methylase). This methylase is most likely involved in the hemed ₁ biosynthesis inPa. denitrificans. The third gene,nirC, codes for a small cytochromec of 9.3 kDa having high homology with cytochromec _55X ofPs. stutzeri ZoBell. The 4th gene,nirF, has no homology with other genes in the sequence databases and has no relevant motifs. Inactivation of either of these 4 genes resulted in the loss of nitrite and nitric oxide reductase activities but not of nitrous oxide reductase activity.nirS mutants lack thecd ₁-type nitrite reductase whilenirE, nirC andnirF mutants produce a small amount ofcd ₁-type nitrite reductase, inactive due to the absence of hemed ₁. Upstream from thenirS gene the start of a gene was identified which has limited homology withnosR, a putative regulatory gene involved in nitrous oxide reduction. A potential FNR box was identified between this gene andnirS.Abbreviations SDS sodium dodecyl sulfate - NBT nitroblue tetrazolium - PAGE polyacrylamide gel electrophoresis     

Regulation of assimilatory nitrate reduction at the level of nitrite in Chlorella fusca

Batch cultures of Chlorella fusca excreted nitrite into the medium if gassed with air (0.03% CO₂), but they did not if supplied with air containing 5% CO₂. After a change from high to low CO₂ concentration in the gas stream, nitrite excretion started immediately. After an increase in CO₂ concentration to 5%, nitrite uptake started within only 30 min. Changes of in-vitro activities of nitrate reductase, nitrite reductase and glutamine synthetase did not correspond to changes of nitrite concentration in the medium and therefore could not explain these observations. A nitrite-binding site, whose activity corresponded with both nitrite excretion and uptake, was detected at the chloroplast envelope. From these data an additional regulatory step in the assimilatory nitrate-reduction sequence is suggested. This includes an envelopeprotein fraction probably regulating the availability of nitrite within the chloroplast.Abbreviations FMN riboflavin 5-phosphate - GS glutamine synthetase - NIR nitrite reductase - NR nitrate reductase     

Interference by S-nitroso compounds with the measurement of nitrate in methods requiring reduction of nitrate to nitrite by cadmium

Various methods suited for the measurement of nitrate require its reduction to nitrite by cadmium under acidic or alkaline conditions. N^G-Nitroarginine analogs have been shown to interfere with the measurement of nitrate by such assays. In the present work we show by gas chromatography−mass spectrometry that under alkaline reduction conditions the S-nitroso compounds S-nitrosoglutathione and S-nitrosohomocysteine but not S-nitroso-N-acetylcysteine and S-nitroso-N-acetylpenicillamine can considerably contribute to nitrate and thus interfere with its measurement. Our results suggest that S-nitroso compounds may interfere with the measurement of nitrate in methods requiring cadmium-catalyzed reduction of nitrate to nitrite.     

Cloning and characterization of a tomato GTPase-like gene related to yeast and Arabidopsis genes involved in vesicular transport

The reduced translation product of a tomato cDNA derived from a gene expressed in a number of tomato tissues of different developmental stages contained sequence motifs characteristic of the GTPase superfamily of proteins. The sequence was closely related to the Sar1 protein of Saccharomyces cerevisiae, a protein essential for the formation of protein transport vesicles at the endoplasmic reticulum (ER) (A. Nakano and M. Muramatsu, Cell Biol 109 (1989): 2677–2691). From analysis of the GTPase superfamily gene sequences, including the tomato SAR-like gene, it is proposed that the SAR genes comprise a distinct GTPase subfamily, presumably with a common, essential function in vesicular transport.     

Ammonium and nitrate uptake in gap,generalist and understory species of the genus Piper

Summary We studied root net uptake of ammonium (NH ₄ ⁺ ) and nitrate (NO ₃ ^– ) in species of the genus Piper (Piperaceae) under high, intermediate and low photosynthetically active photon flux densities (PFD). Plants were grown hydroponically, and then transferred to temperature controlled (25° C) root cuvettes for nutrient uptake determinations. Uptake solutions provided NH ₄ ⁺ and NO ₃ ^– simultaneously (both) or separately (single). In the first experiment, seven species of Piper, from a broad range of rainforest light habitats ranging from gap to understory, were screened for mineral nitrogen preference (100 M NH ₄ ⁺ and/or 100 M NO ₃ ^– ) at intermediate PFD (100 mol m^–2 s^–1). Preference for NH ₄ ⁺ relative to NO ₃ ^– , defined as the ratio of NH ₄ ⁺ (both):NO ₃ ^– (both) net uptake, was higher in understory species than in gap species. Ammonium repression of NO ₃ ^– uptake, defined as the ratio of NO ₃ ^– (single): NO ₃ ^– (both) net uptake, was also higher in understory species as compared to gap species. In a second set of experiments, we examined the effect of nitrogen concentration (equimolar, 10 to 1000 M) on NH ₄ ⁺ preference and NH ₄ ⁺ repression of NO ₃ ^– net uptake at high (500 mol m^–2 s^–1) and low (50 mol m^–2 s^–1) PFD in a gap (P. auritum), generalist (P. hispidum) and understory species (P. aequale). All species exhibited negligible NH ₄ ⁺ repression of NO ₃ ^– net uptake at high PFD. At low PFD, NH ₄ ⁺ preference and repression of NO ₃ ^– net uptake occurred in all species (understory > generalist > gap), but only at intermediate nitrogen concentrations, i.e. between 10 and 200 M. Ammonium repression of net NO ₃ ^– uptake decreased or increased rapidly (in < 48 h) after transitions from low to high or from high to low PFD respectively. No significant diurnal patterns in NO ₃ ^– or NH ₄ ⁺ net uptake were observed.CIWDPB publication # 1130     

The roles of nitrate and ammonium in the regulation of the development of nitrate reductase in Chlamydomonas reinhardii

The regulation of the development of nitrate reductase (NR) activity in Chlamydomonas reinhardii has been compared in a wild-type strain and in a mutant (nit-A) which possesses a modified nitrate reductase enzyme that is non-functional in vivo. The modified enzyme cannot use NAD(P)H as an electron donor for nitrate reduction and it differs from wild-type enzyme in that NR activity is not inactivated in vitro by incubation with NAD(P)H and small quantities of cyanide; it is inactivated when reduced benzyl viologen or flavin mononucleotide is present. After short periods of nitrogen starvation mutant organisms contain much higher levels of terminal-NR activity than do similarly treated wild-type ones. Despite the inability of the mutant to utilize nitrate, no nitrate or nitrite was found in nitrogen-starved cultures; it is therefore concluded that the appearance of NR activity is not a consequence of nitrification. After prolonged nitrogen starvation (22 h) the NR level in the mutant is low. It increases rapidly if nitrate is then added and this increase in activity does not occur in the presence of ammonium, tungstate or cycloheximide. Disappearance of preformed NR activity is stimulated by addition of tungstate and even more by addition of ammonium. The results are interpreted as evidence for a continuous turnover of NR in cells of the mutant with ammonium both stimulating NR breakdown and stopping NR synthesis. Nitrate protects the enzyme from breakdown. Reversible inactivation of NR activity is thought to play an insignificant rôle in the mutant.Abbreviations NR nitrate reductase - BV benzyl viologen     

Effect of nitrate pulses on the nitrate-uptake rate,synthesis of mRNA coding for nitrate reductase,and nitrate-reductase activity in the roots of barley seedlings

Using pulses of nitrate, instead of the permanent presence of external nitrate, to induce the nitrate-assimilating system in Hordeum vulgare L., we demonstrated that nitrate can be considered as a trigger or signal for the induction of nitrate uptake, the appearance of nitratereductase activity and the synthesis of mRNA coding for nitrate reductase. Nitrate pulses stimulated the initial rate of nitrate uptake, even after subsequent cultivation in N-free medium, and resulted in a higher acceleration of the uptake rate in the presence of nitrate than in its absence.Abbreviations NR nitrate reductase     

Cloning, sequencing and expression of a dextransucrase gene (dexYG) from Leuconostoc mesenteroides

The gene dexYG encoding the dextransucrase from an industrial strain of Leuconostoc mesenteroides 0326 was isolated by PCR. The nucleotide sequence of the dexYG gene consists of an open reading frame (ORF) of 4,584 bp, coding for a 1,527 aa protein with a Mr of 170 kDa. The results were analysed by a BLAST similarity search of the GenBank database, which revealed the amino acid sequence was similiar to dsrD derived from L. mesenteroides Lcc4. The dexYG gene was subcloned into the plasmid pET28a(+) and was expressed in E. coli BL21 (DE3) by IPTG induction. The pH value was one of the main reasons which caused the degradation of enzyme activity in the later stage of induction. The highest activity was reached 36 U/ml after 5 h induction in medium at pH 6.0. Biotransformation yield of the enzyme reached 65% and the molecular weight of transformed dextran was more than 68 kDa in 2 h.     

Cloning, sequencing and expression of a recA gene from Amycolatopsis mediterranei

The 3.5 kb nucleotide fragment, including the recA gene and its downstream recX-like gene, has been isolated from a genomic library by dot-blot hybridization with the Mycobacterium smegmatis recA gene. The recA gene, consisting of 1047 base pairs (bp), encodes a polypeptide of 348 amino acids while the recX-like gene, consisting of 450 bp, encodes a shorter polypeptide of 149 amino acids. Both the deduced amino acid sequences of recA and recX resemble those of the recA and recX genes from other bacteria. The cloned Amycolatopsis mediterranei U32 recA gene conferred partial resistance to ethyl methane sulfonate when expressed in E. coli with the lacZ promoter.     

The effect of endogenous and externally supplied nitrate on nitrate uptake and reduction in sugarbeet seedlings

The pericarp of the dormant sugarbeet fruit acts as a storage reservoir for nitrate, ammonium and -amino-N. These N-reserves enable an autonomous development of the seedling for 8–10 d after imbibition. The nitrate content of the seed (1% of the whole fruit) probably induces nitrate-reductase activity in the embryo enclosed in the pericarp. Nitrate that leaks out of the pericarp is reabsorbed by the emerging radicle. Seedlings germinated from seeds (pericarp was removed) without external N-supply are able to take up nitrate immediately upon exposure via a low-capacity uptake system (v_max = 0.8 mol NO ₃ ^- ·(g root FW)^–1·h^–1; K_s = 0.12 mM). We assume that this uptake system is induced by the seed nitrate (10 nmol/seed) during germination. Induction of a high-capacity nitrate-uptake system (v_max = 3.4 mol NO ₃ ^- ·(g root FW)^–1·h^–1; K_s = 0.08 mM) by externally supplied nitrate occurs after a 20-min lag and requires protein synthesis. Seedlings germinated from whole fruits absorb nitrate via a highcapacity uptake mechanism induced by the pericarp nitrate (748 nmol/pericarp) during germination. The uptake rates of the high-capacity system depend only on the actual nitrate concentration of the uptake medium and not on prior nitrate pretreatments. Nitrate deprivation results in a decline of the nitrate-uptake capacity (t_1/2 of v_max = 5 d) probably caused by the decay of carrier molecules. Small differences in K_s but significant differences in v_max indicate that the low- and high-capacity nitrate-uptake systems differ only in the number of identical carrier molecules.Abbreviations NR nitrate reductase - pFPA para-fluorophenylalanine This work was supported by a grant from Bundesministerium für Forschung und Technologie and by Kleinwanzlebener Saatzucht AG, Einbeck.