Response of extreme precipitation to uniform surface warming in quasi-global aquaplanet simulations at high resolution.

Projections of precipitation extremes in simulations with global climate models are very uncertain in the tropics, in part because of the use of parameterizations of deep convection and model deficiencies in simulating convective organization. Here, we analyse precipitation extremes in high-resolution simulations that are run without a convective parameterization on a quasi-global aquaplanet. The frequency distributions of precipitation rates and precipitation cluster sizes in the tropics of a control simulation are similar to the observed distributions. In response to climate warming, 3 h precipitation extremes increase at rates of up to [Formula: see text] in the tropics because of a combination of positive thermodynamic and dynamic contributions. The dynamic contribution at different latitudes is connected to the vertical structure of warming using a moist static stability. When the precipitation rates are first averaged to a daily timescale and coarse-grained to a typical global climate-model resolution prior to calculating the precipitation extremes, the response of the precipitation extremes to warming becomes more similar to what was found previously in coarse-resolution aquaplanet studies. However, the simulations studied here do not exhibit the high rates of increase of tropical precipitation extremes found in projections with some global climate models. This article is part of a discussion meeting issue 'Intensification of short-duration rainfall extremes and implications for flash flood risks'.

Maltose/maltodextrin system of Escherichia coli: transport, metabolism, and regulation.

The maltose system of Escherichia coli offers an unusually rich set of enzymes, transporters, and regulators as objects of study. This system is responsible for the uptake and metabolism of glucose polymers (maltodextrins), which must be a preferred class of nutrients for E. coli in both mammalian hosts and in the environment. Because the metabolism of glucose polymers must be coordinated with both the anabolic and catabolic uses of glucose and glycogen, an intricate set of regulatory mechanisms controls the expression of mal genes, the activity of the maltose transporter, and the activities of the maltose/maltodextrin catabolic enzymes. The ease of isolating many of the mal gene products has contributed greatly to the understanding of the structures and functions of several classes of proteins. Not only was the outer membrane maltoporin, LamB, or the phage lambda receptor, the first virus receptor to be isolated, but also its three-dimensional structure, together with extensive knowledge of functional sites for ligand binding as well as for phage lambda binding, has led to a relatively complete description of this sugar-specific aqueous channel. The periplasmic maltose binding protein (MBP) has been studied with respect to its role in both maltose transport and maltose taxis. Again, the combination of structural and functional information has led to a significant understanding of how this soluble receptor participates in signaling the presence of sugar to the chemosensory apparatus as well as how it participates in sugar transport. The maltose transporter belongs to the ATP binding cassette family, and although its structure is not yet known at atomic resolution, there is some insight into the structures of several functional sites, including those that are involved in interactions with MBP and recognition of substrates and ATP. A particularly astonishing discovery is the direct participation of the transporter in transcriptional control of the mal regulon. The MalT protein activates transcription at all mal promoters. A subset also requires the cyclic AMP receptor protein for transcription. The MalT protein requires maltotriose and ATP as ligands for binding to a dodecanucleotide MalT box that appears in multiple copies upstream of all mal promoters. Recent data indicate that the ATP binding cassette transporter subunit MalK can directly inhibit MalT when the transporter is inactive due to the absence of substrate. Despite this wealth of knowledge, there are still basic issues that require clarification concerning the mechanism of MalT-mediated activation, repression by the transporter, biosynthesis and assembly of the outer membrane and inner membrane transporter proteins, and interrelationships between the mal enzymes and those of glucose and glycogen metabolism.

Learning new tricks from an old dog: MalT of the Escherichia coli maltose system is part of a complex regulatory network.

The regulation of the maltose system in Escherichia coli has traditionally been viewed as a simple positive feedback loop. Today, we know that there are cross connections to several, seemingly unrelated, metabolic pathways. MalT, the central activator of the mal genes, is the key element in this complex regulatory network and integrates the different signals to give an appropriate transcriptional response.

Formation of large, ion-permeable membrane channels by the matrix protein (porin) of Escherichia coli.

One of the major proteins of the outer membrane of Escherichia coli, the matrix protein (porin), has been isolated by detergent solubilisation. When the protein is added in concentrations of the order 10 ng/cm3 to the outer phases of a planar lipid bilayer membrane, the membrane conductance increases by many orders of magnitude. At lower protein concentrations the conductance increases in a stepwise fashion, the single conductance increment being about 2 nS (1 nS = 10(-9) siemens = 10(-9) omega -1) in 1 MKCl. The conductance pathway has an ohmic current vs. voltage character and a poor selectivity for chloride and the alkali ions. These findings are consistent with the assumption that the protein forms large aqueous channels in the membrane. From the average value of the single-channel conductance a channel diameter of about 0.9 nm is estimated. This channel size is consistent with the sugar permeability which has been reported for lipid vesicles reconstituted in the presence of the protein.

The crystal structure of a liganded trehalose/maltose-binding protein from the hyperthermophilic Archaeon Thermococcus litoralis at 1.85 A.

We report the crystallization and structure determination at 1.85 A of the extracellular, membrane-anchored trehalose/maltose-binding protein (TMBP) in complex with its substrate trehalose. TMBP is the substrate recognition site of the high-affinity trehalose/maltose ABC transporter of the hyperthermophilic Archaeon Thermococcus litoralis. In vivo, this protein is anchored to the membrane, presumably via an N-terminal cysteine lipid modification. The crystallized protein was N-terminally truncated, resulting in a soluble protein exhibiting the same binding characteristics as the wild-type protein. The protein shows the characteristic features of a transport-related, substrate-binding protein and is structurally related to the maltose-binding protein (MBP) of Escherichia coli. It consists of two similar lobes, each formed by a parallel beta-sheet flanked by alpha-helices on both sides. Both are connected by a hinge region consisting of two antiparallel beta-strands and an alpha-helix. As in MBP, the substrate is bound in the cleft between the lobes by hydrogen bonds and hydrophobic interactions. However, compared to maltose binding in MBP, direct hydrogen bonding between the substrate and the protein prevails while apolar contacts are reduced. To elucidate factors contributing to thermostability, we compared TMBP with its mesophilic counterpart MBP and found differences known from similar investigations. Specifically, we find helices that are longer than their structurally equivalent counterparts, and fewer internal cavities.

Crystal structure of the effector-binding domain of the trehalose-repressor of Escherichia coli, a member of the LacI family, in its complexes with inducer trehalose-6-phosphate and noninducer trehalose.

The crystal structure of the Escherichia coli trehalose repressor (TreR) in a complex with its inducer trehalose-6-phosphate was determined by the method of multiple isomorphous replacement (MIR) at 2.5 A resolution, followed by the structure determination of TreR in a complex with its noninducer trehalose at 3.1 A resolution. The model consists of residues 61 to 315 comprising the effector binding domain, which forms a dimer as in other members of the LacI family. This domain is composed of two similar subdomains each consisting of a central beta-sheet sandwiched between alpha-helices. The effector binding pocket is at the interface of these subdomains. In spite of different physiological functions, the crystal structures of the two complexes of TreR turned out to be virtually identical to each other with the conformation being similar to those of the effector binding domains of the LacI and PurR in complex with their effector molecules. According to the crystal structure, the noninducer trehalose binds to a similar site as the trehalose portion of trehalose-6-phosphate. The binding affinity for the former is lower than for the latter. The noninducer trehalose thus binds competitively to the repressor. Unlike the phosphorylated inducer molecule, it is incapable of blocking the binding of the repressor headpiece to its operator DNA. The ratio of the concentrations of trehalose-6-phosphate and trehalose thus is used to switch between the two alternative metabolic uses of trehalose as an osmoprotectant and as a carbon source.

Convenient transfer of lacZ-gene fusions to phage M13 by in vivo recombination and their use for nucleotide sequencing.

We describe a method that facilitates the sequencing of lacZ fusion joints based on in vivo subcloning onto phage M13. The method is useful for lacZ fusions that are isolated with the transposable lambda placMu phage into plasmids carrying the pBR322 bla gene. In vivo cloning of lacZ fusions is accomplished by recombination with two M13 phages carrying 5' or 3' segments of the bla gene, adjacent but differing in orientation to lacZ'. The presented method allows rapid sequencing of many fusion joints without subcloning in vitro.

Glycerol uptake in Escherichia coli is sensitive to membrane lipid composition.

In Escherichia coli, a functional GlpF protein is necessary for efficient uptake of glycerol at low concentrations. Here we show that GlpF-mediated glycerol uptake was sensitive to a variety of lipid alterations. Overproduction or mutation of the genes coding for enzymes involved in lipid biosynthesis resulted in changed membrane composition and fluidity. The strains with altered lipid composition had a substrate affinity for glycerol (Km) similar to that of wild-type cells, but the Vmax for glycerol uptake was affected. Experiments with glpF::lacZ and glpK::lacZ protein fusions showed that the expression of these two genes was not changed under these conditions. In addition, we observed that mutations in glpF were accompanied by reduced membrane permeability for compounds unrelated to glycerol. Passive diffusion across the membranes of glpF mutants for o-nitrophenyl galactoside was 5-fold slower than in glpF+ cells. The mutants were more resistant to the hydrophobic antibiotic tetracycline, as well as to the membrane perturbants ethanol and dimethylsulphoxide and to the stress of low-osmolarity medium.

Mutations in phoB, the positive gene activator of the pho regulon in Escherichia coli, affect the carbohydrate phenotype on MacConkey indicator plates.

Mutants defective in phoB, the positive gene activator of the Escherichia coli pho regulon, exhibit aberrant behaviour on MacConkey indicator plates. They appear pale in the presence of a fermentable carbon source such as trehalose, maltose or glucose. The addition of at least 5 mM phosphate corrects this defect. Colonies of phoB+ strains turn red on MacConkey indicator plates and derepress the pho regulon when the cells are able to ferment the carbon source. In contrast, the inability to ferment the carbon source maintains the pho regulon in the repressed state.

The repression of trehalose transport and metabolism in Escherichia coli by high osmolarity is mediated by trehalose-6-phosphate phosphatase.

Trehalose transport and metabolism in Escherichia coli are induced by trehalose in the growth medium but only at low osmolarity. In contrast, synthesis of internal trehalose as an osmoprotectant occurs only at high osmolarity, independent of the carbon source. The synthesis of internal trehalose proceeds via the UDP-glucose-mediated transfer of glucose to glucose-6-phosphate, forming trehalose-6-phosphate, which is then hydrolysed to trehalose. We demonstrate that the inducer for the synthesis of the trehalose transport system as well as of amylotrehalase, the key enzyme in trehalose metabolism at low osmolarity, is trehalose-6-phosphate. We found that the inability to induce these proteins at high osmolarity is primarily due to activity of trehalose-6-phosphate phosphatase, the enzyme responsible for the final step in the synthesis of internal trehalose under these conditions. A gene, otsP, necessary for the synthesis of the biosynthetic trehalose-6-phosphate phosphatase, is located at min 42 closely linked to otsA/B the structural genes for the trehalose-6-phosphate synthase. There is another gene locus near 84 min on the chromosome, that we termed otsR, which is involved in the regulation of otsA/B and possibly otsP. The nature of this regulatory gene is unclear at present.

The OmpC protein of Yersinia enterocolitica: purification and properties.

OmpC, one of the major outer membrane proteins of Yersinia enterocolitica, was isolated and purified to homogeneity. When solubilized at room temperature, this protein appeared on SDS polyacrylamide gel electrophoresis as an oligomer. After heating to the temperature of boiling water, the apparent molecular weight of the monomer was 36,000. The incorporation of purified OmpC into black lipid membranes resulted in an increase in membrane conductance demonstrating pore-forming activity. The reconstituted pores exhibited the characteristics of general diffusion pores. They showed cation selectivity and had a single channel conductance of 1.3 nS in 1.0 M KCl. Assuming a constant diameter of the pore, a length of 6 nm (the width of the outer membrane) and the same ion conductivity inside and outside the pore, the diameter of the pore protein was estimated as 1.0 nm. Polyclonal antibodies were raised against the native, pore-forming protein preparation. These antibodies did not recognize the denatured form of the protein, but cross-reacted with native OmpC and OmpF of Escherichia coli. The regulation of OmpC expression in Y. enterocolitica was dependent on the osmolarity of the medium in the same way as in E. coli.

Enzymatic preparation of radiolabeled linear maltodextrins and cyclodextrins of high specific activity from [14C] maltose using amylomaltase, cyclodextrin glucosyltransferase and cyclodextrinase.

Radiolabeled linear and cyclic maltodextrins of high specific radioactivity were prepared using enzymes involved in maltodextrin metabolism. 14C-Labeled maltose was the starting material yielding products of identical specific radioactivity with respect to glucosyl residues. The enzymatic steps involved: i) Formation of linear 14C-labeled maltodexrins (< maltooctaose) using amylomaltase from Escherischia coli; ii) Cyclisation to alpha-cyclodextrin using cyclodextrin-glucosyltransferase of Kiebsiella oxytoca M5a1; iii) Removal of the remaining linear dextrins by amyloglucosidase. The products were purified by paper chromatography, or maltohexaose was specifically obtained from purified alpha-cyclodextrin by the action of cyclodextrinase of K. oxytoca M5a1.

Malto-oligosaccharide homologues of 3,7-anhydro-2-azi-1,2-dideoxy-D-glycero-D-gulo-octitol+ ++: improved photoaffinity reagents for labelling the malto-oligosaccharide-binding protein of Escherichia coli.

3,7-Anhydro-2-azi-1,2-dideoxy-D-glycero-D-gulo-octitol (2) was synthesized as a beta-D-glucopyranosyl analogue, which could be converted into a series of malto-oligosaccharide derivatives (3-7) by cyclodextrinase-catalyzed glucosyl transfer from alpha-cyclodextrin (cyclomaltohexaose). The pure analogues 3-7 containing 1-5 (1----4)-linked alpha-D-glucose residues inhibited the uptake of maltose via the maltose-binding protein-dependent transport system in Escherichia coli. The concentration of half-maximal inhibition of maltose transport at 60nM decreases with increasing chain-length of the analogue, reaching a minimum at 0.02 mM for 6 (4 glucose residues). 3H-Labelled alpha-cyclodextrin was prepared by partial oxidation and reduction of the aldehyde groups with NaB3H4. Radiolabelled 5a was used to photolabel the binding site of the maltose-binding protein.

sn-Glycerol-3-phosphate transport in Escherichia coli and Salmonella typhimurium.

The gene necessary for the synthesis of the active transport system of sn-glycerol-3-phosphate (G3P) is located at 48 min on the Escherichia coli linkage map. Complementation analysis revealed that there is only one gene necessary for G3P transport. The gene was cloned into the multicopy plasmid pBR322. Strains harboring the hybrid plasmid synthesized large amounts of a protein of 33,000 molecular weight that was found in the cytoplasmic membrane. This protein was identified as the G3P permease. In addition, the periplasm of the hybrid plasmid carrying strain contained large amounts of a soluble protein, identical with the previously recognized GLPT-protein of 40,000 molecular weight. The analysis of amber mutants isolated on the hybrid plasmid showed that the gene for the G3P permease is the first gene in an operon that codes for two genes; the distal gene being the structural gene for the periplasmic GLPT-protein. The corresponding gene region from Salmonella typhimurium has been cloned from an EcoRI libary in lambda gt7. The EcoRI fragment containing the gene necessary for G3P transport was subcloned into the multicopy plasmid pACYC184. The hybrid plasmid directed the synthesis of the G3P permease that behaved identically to the protein from Escherichia coli. However, the gene for the GLPT-protein was not intact but truncated by the EcoRI restriction site. The synthesis of the remaining polypeptide of 30,000 prevented the proper assembly of other transport related binding proteins such as the ribose- and galactose-binding protein.

Cirrus cloud seeding: a climate engineering mechanism with reduced side effects?

Climate engineering, the intentional alteration of Earth's climate, is a multifaceted and controversial topic. Numerous climate engineering mechanisms (CEMs) have been proposed, and the efficacies and potential undesired consequences of some of them have been studied in the safe environments of numerical models. Here, we present a global modelling study of a so far understudied CEM, namely the seeding of cirrus clouds to reduce their lifetimes in the upper troposphere, and hence their greenhouse effect. Different from most CEMs, the intention of cirrus seeding is not to reduce the amount of solar radiation reaching Earth's surface. This particular CEM rather targets the greenhouse effect, by reducing the trapping of infrared radiation by high clouds. This avoids some of the caveats that have been identified for solar radiation management, for example, the delayed recovery of stratospheric ozone or drastic changes to Earth's hydrological cycle. We find that seeding of mid- and high-latitude cirrus clouds has the potential to cool the planet by about 1.4 K, and that this cooling is accompanied by only a modest reduction in rainfall. Intriguingly, seeding of the 15% of the globe with the highest solar noon zenith angles at any given time yields the same global mean cooling as a seeding strategy that involves 45% of the globe. In either case, the cooling is strongest at high latitudes, and could therefore serve to prevent Arctic sea ice loss. With the caveat that there are still significant uncertainties associated with ice nucleation in cirrus clouds and its representation in climate models, cirrus seeding appears to represent a powerful CEM with reduced side effects.