Indolepyruvate ferredoxin oxidoreductase from Pyrococcus sp. KOD1 possesses a mosaic structure showing features of various oxidoreductases

Indolepyruvate ferredoxin oxidoreductase (IOR) catalyzes the oxidative decarboxylation of arylpyruvates. Gene cloning and sequencing analysis of the IOR gene from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was performed. Two genes, iorA and iorB, encoding α and β subunits of IOR were found to be tandemly arranged, which suggests that gene expression is translationaly coupled. Sequence analysis showed the C-terminal region of the α subunit to have a typical ferredoxin-type [4Fe-4S] cluster motif (CXXCXXCXXCXXXCP), which is similar to that present in the δ subunits of other oxidoreductases such as pyruvate ferredoxin oxidoreductase (POR) and 2-ketoisovalerate ferredoxin oxidoreductase (VOR). We suggest that the α subunit of KOD1-IOR has a mosaic structure composed of features characteristic of the α, β and δ subunits from POR and VOR. KOD1-IOR was overproduced in anaerobically incubated Escherichia coli cells and the crude enzyme was extracted under anaerobic conditions. The optimal temperature for activity of recombinant IOR was 70°?C and the half-life of this enzyme in the presence of air was 15 min at 25°?C.     

Gene cloning, sequencing and enzymatic properties of glutamate synthase from the hyperthermophilic archaeon Pyrococcus sp. KOD1

The gltA gene encoding a glutamate synthase (GOGAT) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned as a 6.6 kb HindIII-BamHI fragment. Sequence analysis indicates that gltA encodes a 481- amino acid protein (53 269 Da). The deduced amino acid sequence of KOD1-GltA includes conserved regions that are found in the small subunits of bacterial GOGAT: two cysteine clusters, an adenylate-binding consensus sequence and an FAD-binding consensus sequence. However, no sequences homologous to the large subunit of bacterial GOGAT were found in the upstream or downstream regions. In order to examine whether GltA alone can act as a functional GOGAT, GltA was overexpressed in Escherichia coli BL21 (DE3) cells using an expression plasmid. GltA was purified to homogeneity and shown to be functional as a homotetramer of approximately 205 kDa, which is equivalent to the molecular weight of the native GOGAT from KOD1, thus indicating that KOD1-GOGAT is the smallest known active GOGAT. GltA is capable of both glutamine-dependent and ammonia-dependent synthesis of glutamate. Synthesis of glutamate by KOD1-GltA required NADPH, indicating that this enzyme is an NADPH-GOGAT (EC 1.4.1.13). The optimum pH for both activities was 6.5. However, GltA exhibited different optimum temperatures for activity depending on the reaction assayed (glutamine-dependent reaction, 80° C; ammonia-dependent reaction, 90° C). Received: 30 October 1996 / Accepted: 13 January 1997     

Between-tree variations in leaf δ13C of Quercus pubescens and Quercus ilex among Mediterranean habitats with different water availability

In this study, sun leaf carbon isotope composition (δ¹³C) of two co-occurring woody Mediterranean species (Quercus pubescens Willd., a deciduous oak, and Q. ilex L., an evergreen one) was investigated on four sites with different water availability. The total range of δ¹³C values was 4.4 and 3.1‰ for Q. pubescens and Q. ilex respectively. The intra-site variability was about 3‰. Total mean per species was equal. There were significant differences among sites, but at each site means of δ¹³C were not significantly different between species. A simple physiological model predicts no difference in intrinsic water-use efficiency (WUE_i) between evergreen and deciduous oaks. The relationship between site means of δ¹³C and water parameters suggests that there is a leaf functional adjustment with respect to available water resource. No correlation was found between δ¹³C and the contents of any mass-based biochemical constituent. Nevertheless there was a significant correlation between δ¹³C and leaf mass per area of Q. ilex. For both species, there is also a positive correlation between leaf δ¹³C and individual crown area, i.e. a structural characteristic at tree level. Causal relations between δ¹³C and plant-environment interactions are discussed. Received: 25 October 1996 / Accepted: 19 January 1997     

Magnesium chelatase: association with ribosomes and mutant complementation studies identify barley subunit Xantha-G as a functional counterpart of Rhodobacter subunit BchD

Magnesium chelatase catalyses the insertion of Mg²⁺ into protoporphyrin and is found exclusively in organisms which synthesise chlorophyll or bacteriochlorophyll. Soluble protein preparations containing >10 mg protein/ml, obtained by gentle lysis of barley plastids and Rhodobacter sphaeroplasts, inserted Mg²⁺ into deuteroporphyrin IX in the presence of ATP at rates of 40 and 8 pmoles/mg protein per min, respectively. With barley extracts optimal activity was observed with 40 mM Mg²⁺. The activity was inhibited by micromolar concentrations of chloramphenicol. Mutations in each of three genetic loci, Xantha-f, -g and -h, in barley destroyed the activity. However, Mg-chelatase activity was reconstituted in vitro by combining pairwise the plastid stroma protein preparations from non-leaky xantha-f, -g and -h mutants. This establishes that, as in Rhodobacter, three proteins are required for the insertion of magnesium into protoporphyrin IX in barley. These three proteins, Xantha-F, -G and -H, are referred to as Mg-chelatase subunits and they appear to exist separate from each other in vivo. Active preparations from barley and Rhodobacter yielded pellet and supernatant fractions upon centrifugation for 90 min at 272 000 × g. The pellet and the supernatant were inactive when assayed separately, but when they were combined activity was restored. Differential distribution of the Mg-chelatase subunits in the fractions was established by in vitro complementation assays using stroma protein from the xantha-f, -g, and -h mutants. Xantha-G protein was confined to the pellet fraction, while Xantha-H was confined to the supernatant. Reconstitution assays using purified recombinant BchH, BchI and partially purified BchD revealed that the pellet fraction from Rhodobacter contained the BchD subunit. The pellet fractions from both barley and Rhodobacter contained ribosomes and had an A₂₆₀:A₂₈₀ ratio of 1.8. On sucrose density gradients both Xantha-G and BchD subunits migrated with the plastid and bacterial ribosomal RNA, respectively. Received: 9 September 1996 / Accepted: 22 October 1996     

Subunit Regulation of the Human Brain α1E Calcium Channel

The α₁ subunit coding for the human brain type E calcium channel (Schneider et al., 1994) was expressed in Xenopus oocytes in the absence, and in combination with auxiliary α₂δ and β subunits. α_1E channels directed with the expression of Ba²⁺ whole-cell currents that completely inactivated after a 2-sec membrane pulse. Coexpression of α_1E with α_2bδ shifted the peak current by +10 mV but had no significant effect on whole-cell current inactivation. Coexpression of α_1E with β_2a shifted the peak current relationship by −10 mV, and strongly reduced Ba²⁺ current inactivation. This slower rate of inactivation explains that a sizable fraction (40 ± 10%, n= 8) of the Ba²⁺ current failed to inactivate completely after a 5-sec prepulse. Coinjection with both the cardiac/brain β_2a and the neuronal α_2bδ subunits increased by ≈10-fold whole-cell Ba²⁺ currents although coinjection with either β_2a or α_2bδ alone failed to significantly increase α_1E peak currents. Coexpression with β_2a and α_2bδ yielded Ba²⁺ currents with inactivation kinetics similar to the β_2a induced currents, indicating that the neuronal α_2bδ subunit has little effect on α_1E inactivation kinetics. The subunit specificity of the changes in current properties were analyzed for all four β subunit genes. The slower inactivation was unique to α_1E/β_2a currents. Coexpression with β_1a, β_1b, β₃, and β₄, yielded faster-inactivating Ba²⁺ currents than currents recorded from the α_1E subunit alone. Furthermore, α_1E/α_2bδ/β_1a; α_1E/α_2bδ/β_1b; α_1E/α_2bδ/β₃; α_1E/α_2bδ/β₄ channels elicited whole-cell currents with steady-state inactivation curves shifted in the hyperpolarized direction. The β subunit-induced changes in the properties of α_1E channel were comparable to modulation effects reported for α_1C and α_1A channels with β₃≈β_1b > β_1a≈β₄≫β_2a inducing fastest to slowest rate of whole-cell inactivation. Received: 27 March 1997/Revised: 10 July 1997     

Evidence for NO-dependent vasodilation in the trout (Oncorhynchus mykiss ) coronary system

The effects of l-arginine, and its analogues N ^ω-nitro-l-arginine methyl ester and N ^ω-nitro-l-arginine on vascular resistance were investigated in the intact coronary system of an isolated non-working trout heart preparation. l-Arginine, at 10^–8 mol · l^–1induced a slight vasodilatory effect (max 10%). N ^ω-nitro-l-arginine methyl ester and N ^ω-Nitro-l-arginine in the range 10^–8–10^–4 mol · l^–1 caused dose-dependent increases in coronary resistance. The vasodilatory action of l-arginine was abolished when the preparation was pretreated with 10^–4 mol · l^–1 N ^ω-nitro-l-arginine or N ^ω-nitro-l-arginine methyl ester. Nitroprusside alone at 1 mmol · l^–1 induced a maximum vasodilation (30%) of the coronary system. Methylene blue a known inhibitor of guanylate cyclase, induced a strong vasoconstriction (already significant at 10^–5 mol · l^–1) and was able to overcome the vasodilative effect of nitroprusside. The endothelial nitric oxide agonists acetylcholine and serotonin, established in mammalian vessels, also mediate vasodilation in trout coronary system. In 50% of preparations, acetylcholine induced a biphasic response with vasodilation at low concentration (max 15% at 10^–8 mol · l^–1). Serotonin displayed a dose-response vasodilation in the range 10^–8–10^–4 mol · l^–1 (max 20%). These vasodilative effects were reduced or abolished by 10^–4 mol · l^–1 l-NA. These data support the existence of NO-mediated vasodilation mechanisms in the trout coronary system. Accepted: 1 July 1996     

A novel yeast gene, RHK1 , is involved in the synthesis of the cell wall receptor for the HM-1 killer toxin that inhibits β-1,3-glucan synthesis

The HM-1 killer toxin from Hansenula mrakii is known to inhibit cell wall β-1,3-glucan synthase of Saccharomyces cerevisiae and other sensitive strains of yeast. A number of mutants of Saccharomyces cerevisiae that show resistance to this toxin were isolated in order to clarify the killing mechanism of the toxin. These mutants, designated rhk (resistant to Hansenula killer), were classified into three complementation groups. A novel gene RHK1, which complements the killer-resistant phenotype of the largest complementation group rhk1, was isolated. DNA sequence analysis revealed an open reading frame that encodes a hydrophobic protein composed of 458 amino acids. Gene disruption followed by tetrad analysis showed that RHK1 is not essential and loss of RHK1 function endowed S. cerevisiae cells with complete killer resistance. A biochemical analysis suggested that RHK1 does not participate directly in the synthesis of β-1,3-glucan but is involved in the synthesis of the receptor for the HM-1 killer toxin. Received: 27 June 1996 / Accepted: 14 October 1996     

In vitro studies on calcium absorption from the gastrointestinal tract in␣small ruminants

Unidirectional flux rates of Ca²⁺ across gastrointestinal tissues from sheep and goats were measured in vitro by applying the Ussing-chamber technique. Except for the sheep duodenum, mucosal to serosal Ca²⁺ flux rates (J _ms) exceeded respective flux rates in the opposite direction (J _sm) in both species and in all segments of the intestinal tract. This resulted in net Ca²⁺ flux rates␣(J _net = J _ms − J _sm) ranging between −2 and 9 nmol · cm⁻² · h⁻¹ in sheep and between 10 and 15 nmol cm⁻² · h⁻¹ in goats. In sheep, only J _net in jejunum, and in goats, J _netin duodenum and jejunum were significantly different from zero. Using sheep rumen wall epithelia, significant J _net of Ca²⁺ of around 5 nmol · cm⁻² · h⁻¹ could be detected. Since the experiments were carried out in the absence of an electrochemical gradient, significant net Ca²⁺ absorption clearly indicates the presence of active mechanisms for Ca²⁺ transport. Dietary Ca depletion caused increased calcitriol plasma concentrations and induced significant stimulations of net Ca²⁺ absorption in goat rumen. J _net of Ca²⁺ across goat rumen epithelia was significantly reduced by 1 mmol · l ⁻¹ verapamil in the mucosal buffer solution. In conclusion, there is clear evidence for the rumen as a main site for active Ca²⁺ absorption in small ruminants. Stimulation of active Ca²⁺ absorption by increased plasma calcitriol levels and inhibition by mucosal verapamil suggest mechanistic and regulatory similarities to active Ca²⁺ transport as described for the upper small intestines of monogastric species. Accepted: 31 July 1996     

Light response characteristics of a morphologically diverse group of southern hemisphere conifers as measured by chlorophyll fluorescence

Unlike northern hemisphere conifer families, the southern family, Podocarpaceae, produces a great variety of foliage forms ranging from functionally broad-, to needle-leaved. The production of broad photosynthetic surfaces in podocarps has been linked qualitatively to low-light-environments, and we undertook to assess the validity of this assumption by measuring the light response of a morphologically diverse group of podocarps. The light response, as apparent photochemical electron transport rate (ETR), was measured by modulated fluorescence in ten species of this family and six associated species (including five Cupressaceae and one functionally needle-leaved angiosperm) all grown under identical glasshouse conditions. In all species, ETR was found to increase as light intensity increased, reaching a peak value (ETR_max) at saturating quantum flux (PPFD_sat), and decreasing thereafter. ETR_max ranged from 217 μmol electrons · m⁻² · s⁻¹ at a PPFD_sat of 1725 μmol photons · m⁻² · s⁻¹ in Actinostrobus acuminatus to an ETR of 60 μmol electrons · m⁻² · s⁻¹ at a PPFD_sat of 745 μmol electrons · m⁻² · s⁻¹ in Podocarpus dispermis. Good correlations were observed between ETR_max and both PPFD_sat and maximum assimilation rate measured by gas-exchange analysis. The effective quantum yield at light saturation remained constant in all species with an average value of 0.278 ± 0.0035 determined for all 16 species. Differences in the shapes of light response curves were related to differences in the response of non-photochemical quenching (q _n), with q _n saturating faster in species with low PPFD_sat. Amongst the species of Podocarpaceae, the log of average shoot width was well correlated with PPFD_sat, wider leaves saturating at lower light intensities. This suggests that broadly flattened shoots in the Podocarpaceae are an adaptation to low light intensity. Received: 15 April 1996 / Accepted: 30 September 1996     

Influence of stand structure on carbon-13 of vegetation, soils, and canopy air within deciduous and evergreen forests in Utah, United States

Carbon isotope ratios (δ¹³C) were studied in evergreen and deciduous forest ecosystems in semi-arid Utah (Pinus contorta, Populus tremuloides, Acer negundo and Acer grandidentatum). Measurements were taken in four to five stands of each forest ecosystem differing in overstory leaf area index (LAI) during two consecutive growing seasons. The δ¹³C_leaf (and carbon isotope discrimination) of understory vegetation in the evergreen stands (LAI 1.5–2.2) did not differ among canopies with increasing LAI, whereas understory in the deciduous stands (LAI 1.5–4.5) exhibited strongly decreasing δ¹³C_leaf values (increasing carbon isotope discrimination) with increasing LAI. The δ¹³C values of needles and leaves at the top of the canopy were relatively constant over the entire LAI range, indicating no change in intrinsic water-use efficiency with overstory LAI. In all canopies, δ¹³C_leaf decreased with decreasing height above the forest floor, primarily due to physiological changes affecting c _i/c _a (> 60%) and to a minor extent due to δ¹³C of canopy air (< 40%). This intra-canopy depletion of δ¹³C_leaf was lowest in the open stand (1‰) and greatest in the denser stands (4.5‰). Although overstory δ¹³C_leaf did not change with canopy LAI, δ¹³C of soil organic carbon increased with increasing LAI in Pinus contorta and Populus tremuloides ecosystems. In addition, δ¹³C of decomposing organic carbon became increasingly enriched over time (by 1.7–2.9‰) for all deciduous and evergreen dry temperate forests. The δ¹³C_canopy of CO₂ in canopy air varied temporally and spatially in all forest stands. Vertical canopy gradients of δ¹³C_canopy, and [CO₂]_canopy were larger in the deciduous Populus tremuloides than in the evergreen Pinu contorta stands of similar LAI. In a very wet and cool year, ecosystem discrimination (Δ_e) was similar for both deciduous Populus tremulodies (18.0 ± 0.7‰) and evergreen Pinus contorta (18.3 ± 0.9‰) stands. Gradients of δ¹³C_canopy and [CO₂]_canopy were larger in denser Acer spp. stands than those in the open stand. However, ¹³C enrichment above and photosynthetic draw-down of [CO₂]_canopy below tropospheric baseline values were larger in the open than in the dense stands, due to the presence of a vigorous understory vegetation. Seasonal patterns of the relationship δ¹³C_canopy versus 1/[CO₂]_canopy were strongly influenced by precipitation and air temperature during the growing season. Estimates of Δ_e for Acer spp. did not show a significant effect of stand structure, and averaged 16.8 ± 0.5‰ in 1933 and 17.4 ± 0.7‰ in 1994. However, Δ_e varied seasonally with small fluctuations for the open stand (2‰), but more pronounced changes for the dense stand (5‰). Received: 15 April 1996 / Accepted: 19 October 1996     

Identification and genetic characterization of the Pseudomonas aeruginosaleuB gene encoding 3-isopropylmalate dehydrogenase

The Pseudomonas aeruginosa leuB gene, encoding 3-isopropylmalate dehydrogenase, was identified upstream of asd, encoding aspartate-β-semialdehyde dehydrogenase. Genetic analysis indicated that leuB is identical to the previously mapped gene defined by the leu-10 allele. The chromosomal leuB locus was inactivated by gene replacement. The insertions had no adverse effect on expression of the downstream asd gene but resulted in leucine auxotrophy. The leuB gene encodes a protein containing 360 amino acids (with a molecular weight of 39153), which was expressed in Escherichia coli as a M, 42000 protein. The results suggested that, in contrast to the situation in other bacteria (E. coli, Salmonella typhimurium and Bacillus subtilis) the P. aeruginosa leuB gene is physically separated from the genes encoding the other enzymes of the isopropylmalate pathway. Received: 15 August 1996 / Accepted: 23 October 1996     

Wound-inducible and organ-specific expression of ORF13 from Agrobacterium rhizogenes 8196 T-DNA in transgenic tobacco plants

Tissue-specific expression of the ORF13 promoter from Agrobacterium rhizogenes 8196 was assessed throughout the development of transgenic tobacco plants using a GUS reporter gene. ORF13 exhibited high activity in roots but with different patterns of expression. The activity of the ORF13 promoter in vascular tissues increased from the base to the tip of the stem. The ORF13 promoter is wound inducible in a limited area adjacent to the wound site. The time course of wound induction of ORF13 in transgenic tobacco containing an ORF13 promoter-GUS translational fusion was similar to that previously described for genes involved in plant defense responses. A series of 5′ deletions of the ORF13 promoter fused to the β-glucuronidase gene was examined for expression in roots and leaves of transgenic plants. Cis-acting elements that modulate quantitative expression of the transgene after wounding were detected. Received: 11 July 1996 / Accepted: 19 November 1996     

Crosstalk between cAMP and pheromone signalling pathways in Ustilago maydis

In the phytopathogenic basidiomycete Ustilago maydis mating and dikaryon formation are controlled by a pheromone/receptor system and the multiallelic b locus. Recently, a gene encoding a G protein α subunit, gpa3, was isolated and has subsequently been implicated in pheromone signal transduction. Mutants deleted for gpa3 are sterile and nonpathogenic, and exhibit a morphology that is similar to that of mutants with defects in the adenylate cyclase gene uac1. We have found that the sterility and mutant morphology of gpa3 deletion strains can be rescued by exogenous cAMP. In these mutants and in the corresponding wild-type strains, exogenous cAMP stimulates pheromone gene expression to a level comparable to that seen in the pheromone-stimulated state. In addition, we demonstrate that uac1 is epistatic to gpa3. We conclude that Gpa3 controls the cAMP signalling pathway in U.maydis and discuss how this pathway feeds into the pheromone response. Received: 4 May 1998 / Accepted: 24 July 1998     

An Aspergillus nidulans nuclear protein, AnCP, involved in enhancement of Taka-amylase A gene expression, binds to the CCAAT-containing taaG2 , amdS , and gatA promoters

Using AnCP (Aspergillus nidulans CCAAT-binding protein) as a CCAAT-specific binding factor model, the possibility that one factor is able to recognize CCAAT sequences in several different genes in A.␣nidulans was examined. DNase I protection analysis showed that AnCP specifically bound to CCAAT sequence-containing regions comprising 21 to 36 bp of the taa, amdS and gatA genes. Furthermore, replacement of the CCAAT sequence with CGTAA was found to abolish the binding of AnCP and to have an inhibitory effect on taa promoter activity. This clearly demonstrates a positive function of the CCAAT element. However, amylase was induced by starch and repressed by glucose in a CCAAT-box disruptant, as in wild-type cells. Received: 28 June 1996 / Accepted: 7 October 1996     

Rhythmic components of photoperiodic time measurement in the pitcher-plant mosquito, Wyeomyia smithii

Photoperiodic time measurement regulating larval diapause in the pitcher-plant mosquito, Wyeomyia smithii, varies in a close relationship with latitude. The critical photoperiod mediating the maintenance and termination of diapause is positively correlated with latitude (r ² = 0.977) among six populations from southern (30–31° N), intermediate (40° N), and northern (46–49° N) latitudes in North America. The developmental response to unnaturally short and to unnaturally long photoperiods declines with increasing latitude, so that longer critical photoperiods are associated with a downward rather than a lateral shift in the photoperiodic response curve. Exotic light and dark cycles of varying period (T) with a short (10 h) photophase and a scotophase ranging from 14 (T = 24) to 62 (T = 72) h, reveal two geographic patterns: a decline in perturbability of the photoperiodic clock with increasing latitude, and no change with latitude in the 21-h period of rising and falling development with increasing T. These results show (1) that there is a rhythmic component to photoperiodic time measurement in W. smithii, (2) that the period of this rhythm is about 21 h in all populations, and (3) that more northern populations show decreasing responsiveness to photoperiod and increasing stability against perturbation by exotic period lengths (T > 24). Previous studies on W.␣smithii indicate that this single temperate species of a tropical and subtropical genus has evolved from south to north. We therefore conclude that the evolution of increasing critical photoperiod in W. smithii during its adaptive radiation into North America has more likely involved the amplitude and not the period of the underlying circadian pacemaker. Received: 22 July 1996 / Accepted: 30 September 1996