Multiple stress signal integration in the regulation of the complex sigma S-dependent csiD-ygaF-gabDTP operon in Escherichia coli

The csiD-ygaF-gabDTP region in the Escherichia coli genome represents a cluster of sigma S-controlled genes. Here, we investigated promoter structures, sigma factor dependencies, potential co-regulation and environmental regulatory patterns for all of these genes. We find that this region constitutes a complex operon with expression being controlled by three differentially regulated promoters: (i) csiDp, which affects the expression of all five genes, is cAMP-CRP/sigma S-dependent and activated exclusively upon carbon starvation and stationary phase; (ii) gabDp1, which is sigma S-dependent and exhibits multiple stress induction like sigma S itself; and (iii) gabDp2[previously suggested by Schneider, B.L., Ruback, S., Kiupakis, A.K., Kasbarian, H., Pybus, C., and Reitzer, L. (2002) J. Bacteriol. 184: 6976-6986], which appears to be Nac/sigma 70-controlled and to respond to poor nitrogen sources. In addition, we identify a novel repressor, CsiR, which modulates csiDp activity in a temporal manner during early stationary phase. Finally, we propose a physiological role for sigma S-controlled GabT/D-mediated gamma-aminobutyrate (GABA) catabolism and glutamate accumulation in general stress adaptation. This physiological role is reflected by the activation of the operon-internal gabDp1 promoter under the different conditions that also induce sigma S, which include shifts to acidic pH or high osmolarity as well as starvation or stationary phase.     

NMR spectroscopic and theoretical study of the complexation of the inhibitor allosamidin in the binding pocket of the plant chitinase hevamine

Based on NMR spectroscopic information about the allosamidin-hevamine complex, ab initio MO calculations of the ring current effect of the aromatic moieties of Trp255, Tyr183 and Tyr6 of hevamine were carried out to investigate the role of these amino acid residues in binding interactions with allosamidin in solution. In addition, the intermolecular steric compression effect on the 13C chemical shifts of the allosamizoline carbon atoms and the hydrogen bonding to Glu127 was identified. It can be inferred that the binding forces are strongest in the allosamizoline moiety of allosamidin.     

Trehalose Is Not Relevant for In Vivo Activity of ςS-Containing RNA Polymerase in Escherichia coli

The ς^S- and ς⁷⁰-associated forms of RNA polymerase core enzyme (E) of Escherichia coli have very similar promoter recognition specificities in vitro. Nevertheless, the in vivo expression of many stress response genes is strongly dependent on ς^S. Based on in vitro assays, it has recently been proposed that the disaccharide trehalose specifically stimulates the formation and activity of Eς^S and thereby contributes to promoter selectivity (S. Kusano and A. Ishihama, J. Bacteriol. 179:3649–3654, 1997). However, we demonstrate here that a trehalose-free otsA mutant exhibits growth phase-related and osmotic induction of various ς^S-dependent genes which is indistinguishable from that of an otherwise isogenic wild-type strain and that stationary-phase cells do not accumulate trehalose (even though the trehalose-synthesizing enzymes are induced). We conclude that in vivo trehalose does not play a role in the expression of ς^S-dependent genes and therefore also not in sigma factor selectivity at the promoters of these genes.     

Distribution of mitochondria within Muller cells – II. Post-natal development of the rabbit retinal periphery in vivo and in vitro: dependence on oxygen supply

The occurrence and localization of mitochondria within glial (Muller) cells and neurons of the peripheral (avascular) rabbit retina was studied electron microscopically and by immunocytochemical demonstration of the mitochondrial enzyme GABA transaminase (GABA-T). Post-natal development in vivo was compared with development of organ cultures from neonatal rabbit retinae, grown over 2 weeks in vitro. The adult pattern of mitochondrial localization (restriction to the sclerad end of the cells) was observed from the beginning of enzyme expression at early post-natal stages. However, when neonatal retinal pieces were grown in vitro with their vitread surface exposed to the air, their Muller cells contained mitochondria along most of their length. When functionally developed retinae from postnatal day 14 were explanted in vitro, they retained their sclerad mitochondrial distribution for almost 24 h but thereafter the inner portions of their cytoplasm became occupied by mitochondria within a few hours. This was achieved mainly by mitochondrial migration rather than by formation of new mitochondria because it was not prevented by cycloheximide-induced inhibition of protein synthesis. These data support the following hypotheses: (1) the mitochondrial distribution in Muller cells is determined by the local cytoplasmic O2 pressure (pO2), (2) existing mitochondria move towards cytoplasmic regions of sufficient pO2 by rather rapid migration and (3) the start of this migration is delayed by almost 24 h due to the action of as yet unknown control mechanisms. In contrast, the mitochondrial content of retinal ganglion and amacrine cells in the vitread retinal layers was virtually independent of the source and level of oxygen supply.     

Geraniol and geranial dehydrogenases induced in anaerobic monoterpene degradation by Castellaniella defragrans

Castellaniella defragrans is a Betaproteobacterium capable of coupling the oxidation of monoterpenes with denitrification. Geraniol dehydrogenase (GeDH) activity was induced during growth with limonene in comparison to growth with acetate. The N-terminal sequence of the purified enzyme directed the cloning of the corresponding open reading frame (ORF), the first bacterial gene for a GeDH (geoA, for geraniol oxidation pathway). The C. defragrans geraniol dehydrogenase is a homodimeric enzyme that affiliates with the zinc-containing benzyl alcohol dehydrogenases in the superfamily of medium-chain-length dehydrogenases/reductases (MDR). The purified enzyme most efficiently catalyzes the oxidation of perillyl alcohol (k(cat)/K(m) = 2.02 × 10(6) M(-1) s(-1)), followed by geraniol (k(cat)/K(m) = 1.57 × 10(6) M(-1) s(-1)). Apparent K(m) values of <10 μM are consistent with an in vivo toxicity of geraniol above 5 μM. In the genetic vicinity of geoA is a putative aldehyde dehydrogenase that was named geoB and identified as a highly abundant protein during growth with phellandrene. Extracts of Escherichia coli expressing geoB demonstrated in vitro a geranial dehydrogenase (GaDH) activity. GaDH activity was independent of coenzyme A. The irreversible formation of geranic acid allows for a metabolic flux from β-myrcene via linalool, geraniol, and geranial to geranic acid.     

Environmental Effects over the First 2½ Rotation Periods of a Fertilised Poplar Short Rotation Coppice

A short rotation coppice (SRC) with poplar was established in a randomised fertilisation experiment on sandy loam soil in Potsdam (Northeast Germany). The main objective of this study was to assess if negative environmental effects as nitrogen leaching and greenhouse gas emissions are enhanced by mineral nitrogen (N) fertiliser applied to poplar at rates of 0, 50 and 75 kg N ha^?1 year^?1 and how these effects are influenced by tree age with increasing number of rotation periods and cycles of organic matter decomposition and tree growth after each harvesting event. Between 2008 and 2012, the leaching of nitrate (NO₃ ^?) was monitored with self-integrating accumulators over 6-month periods and the emissions of the greenhouse gases (GHG) nitrous oxide (N₂O) and carbon dioxide (CO₂) were determined in closed gas chambers. During the first 4 years of the poplar SRC, most nitrogen was lost through NO₃ ^? leaching from the main root zone; however, there was no significant relationship to the rate of N fertilisation. On average, 5.8 kg N ha^?1 year^?1 (13.0 kg CO_2equ) was leached from the root zone. Nitrogen leaching rates decreased in the course of the 4-year study parallel to an increase of the fine root biomass and the degree of mycorrhization. In contrast to N leaching, the loss of nitrogen by N₂O emissions from the soil was very low with an average of 0.61 kg N ha^?1 year^?1 (182 kg CO_2equ) and were also not affected by N fertilisation over the whole study period. Real CO₂ emissions from the poplar soil were two orders of magnitude higher ranging between 15,122 and 19,091 kg CO₂ ha^?1 year^?1 and followed the rotation period with enhanced emission rates in the years of harvest. As key-factors for NO₃ ^? leaching and N₂O emissions, the time after planting and after harvest and the rotation period have been identified by a mixed effects model.     

Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor.

APO-1 (Fas/CD95), a member of the tumor necrosis factor receptor superfamily, induces apoptosis upon receptor oligomerization. In a search to identify intracellular signaling molecules coupling to oligomerized APO-1, several cytotoxicity-dependent APO-1-associated proteins (CAP) were immunoprecipitated from the apoptosis-sensitive human leukemic T cell line HUT78 and the lymphoblastoid B cell line SKW6.4. CAP1-3 (27-29 kDa) and CAP4 (55 kDa), instantly detectable after the crosslinking of APO-1, were associated only with aggregated (the signaling form of APO-1) and not with monomeric APO-1. CAP1 and CAP2 were identified as serine phosphorylated MORT1/FADD. The association of CAP1-4 with APO-1 was not observed with C-terminally truncated non-signaling APO-1. In addition, CAP1 and CAP2 did not associate with an APO-1 cytoplasmic tail carrying the lprcg amino acid replacement. Moreover, no APO-1-CAP association was found in the APO-1+, anti-APO-1-resistant pre-B cell line Boe. Our data suggest that in vivo CAP1-4 are the APO-1 apoptosis-transducing molecules.     

Comparison of protein active site structures for functional annotation of proteins and drug design

Rapid and accurate functional assignment of novel proteins is increasing in importance, given the completion of numerous genome sequencing projects and the vastly expanding list of unannotated proteins. Traditionally, global primary-sequence and structure comparisons have been used to determine putative function. These approaches, however, do not emphasize similarities in active site configurations that are fundamental to a protein's activity and highly conserved relative to the global and more variable structural features. The Comparison of Protein Active Site Structures (CPASS) database and software enable the comparison of experimentally identified ligand-binding sites to infer biological function and aid in drug discovery. The CPASS database comprises the ligand-defined active sites identified in the protein data bank, where the CPASS program compares these ligand-defined active sites to determine sequence and structural similarity without maintaining sequence connectivity. CPASS will compare any set of ligand-defined protein active sites, irrespective of the identity of the bound ligand.     

Assessing the environmental profile of orange production in Brazil

Background, aim, and scope

Brazil is the world’s largest orange producing country, with a total planted area of more than 820,000 hectares. The bulk of the oranges produced in Brazil (70%) is processed into frozen concentrated orange juice (FCOJ) by large processing companies. Exports represent around 97% of the total FCOJ produced, making Brazil the largest world producer and exporter of FCOJ. The Brazilian citrus sector accounts for half of the world’s supply of orange juice and for 80% of the juice traded on the international market. The goal of this study is to characterize the Brazilian orange producers in terms of farm size, cultivated varieties, watering system and tillage practices. In addition, the study presents some aspects of the LCI of oranges grown specifically for the production of FCOJ in the two major orange-growing areas in Brazil (the Northern and Southern regions of the State of São Paulo) during reference crop year 2002/03 in order to generate detailed production inventory data and identify the potential environmental impacts of tillage, both of which are key to enable the formulation of sustainability parameters for the production of FCOJ in Brazil.

Methods

This study was performed in compliance with the guidelines and requirements of the ISO 14040 standard series. All information and data considered and evaluated in this study (the use of water, energy, fertilizers, pesticides and soil correctors) were gathered through in-depth questionnaires either filled in directly by the farm manager or completed by the farm manager and sent in by mail. The data cover a total of 367,200 metric tons of oranges produced by 4 million plants in commercial production from a total evaluated area that accounts for 19.5% of the overall production of oranges in the State of São Paulo. The two major orange-producing areas in Brazil located respectively in the Northern and Southern regions of the State of São Paulo were evaluated. Only the inputs and outputs associated with tillage practices and technology were considered in this paper. The environmental aspects of the production of fertilizers and pesticides were not included within the boundaries. The functional unit selected for this study was 1,000 kg of oranges for FCOJ. Farm-specific data have been combined with agricultural production data to construct an orange cultivation model.

Results

The orange varieties for the production of FCOJ evaluated in this study were Pêra, Valência and Natal. The farms investigated had a cultivated area varying from 22 to 7,000 ha, with a plant density ranging from 170 to 500 plants/ha and an average yield of 30,500 kg/ha for mature trees. Depending on the region, the production of 1,000 kg of oranges requires approx. 120 to 4,400 MJ of energy; 0.3 to 36 kg of diesel; 1,100 to 54,500 kg of water for irrigation; 0.3 to 65 kg of fertilizers (NPK); 0.1 to 13.5 kg of pesticides; 8 to 650 kg of soil correctors. The distribution of some inputs across the farms located in the two main Brazilian orange-producing regions is presented, in addition to the total weighted average.

Discussion

In addition to the variations among the regions evaluated, it was generally observed that there were very considerable differences in order of magnitude between the data collected from farms located in the same region. Although the inputs are directly related to the specific characteristics of each farm and the climatic and topographic conditions of its location, this study has identified some farms that are in a good position to reduce the amounts of some inputs and enhance their environmental and economical performance.

Conclusions

Taking into account the aspects evaluated in this study, only 21% of the orange growers showed good performance, i.e., consumption of pesticides, fertilizers and soil correction compounds equal to or lower than the weighted averages, except for “land use”, which was above the average for the majority of these farms. This study provided important results that allow for a better understanding of the agricultural practices involved and the potential environmental impacts of this product.

Recommendation and perspective

The authors would like to suggest that the five best-performing farms average values be set as the maximum allowable level and point of departure for the discussion on the good environmental performance of Brazilian FCOJ. Besides the total inputs, the role of good agricultural practices to achieve good yields and, at the same time, improve the sustainability of orange growing should also be taken into consideration. Future updates of this study will show the evolution of natural resource management as a result of improved land use, new agricultural practices, reduced use of fertilizers and agrochemicals.