The 3-Hydroxy-2-Butanone Pathway Is Required for Pectobacterium carotovorum Pathogenesis

Pectobacterium species are necrotrophic bacterial pathogens that cause soft rot diseases in potatoes and several other crops worldwide. Gene expression data identified Pectobacterium carotovorum subsp. carotovorum budB, which encodes the α-acetolactate synthase enzyme in the 2,3-butanediol pathway, as more highly expressed in potato tubers than potato stems. This pathway is of interest because volatiles produced by the 2,3-butanediol pathway have been shown to act as plant growth promoting molecules, insect attractants, and, in other bacterial species, affect virulence and fitness. Disruption of the 2,3-butanediol pathway reduced virulence of P. c. subsp. carotovorum WPP14 on potato tubers and impaired alkalinization of growth medium and potato tubers under anaerobic conditions. Alkalinization of the milieu via this pathway may aid in plant cell maceration since Pectobacterium pectate lyases are most active at alkaline pH

Functional and structural determinants of reverse operation in the pH-dependent oligopeptide transporter PepT1

The functional and structural basis of reverse operation of PepT1 has been studied in Xenopus oocytes expressing the wild-type and mutated forms of this protein. Using brief pulses from a negative holding potential, wild-type and Arg282 mutants exhibit outward currents in the presence of Gly-Gln. The reversal potential of these currents is affected by both pH and substrate concentration, confirming coupled transport in the wild type and in the mutants as well. Long-lasting voltage and current-clamp experiments show that the outward currents are only temporary, and reflect accumulation and/or depletion effects near the membrane. The ability to operate in reverse mode was confirmed in all isoforms by intracellular injection of substrate. The role of Arg282 and Asp341 in the reverse transport was also investigated using charged substrates. Positive Lys-Gly (but not Gly-Lys) showed enhanced transport currents in the Arg282 mutants. In contrast, negative Gly-Asp and Asp-Gly elicited modest currents in all isoforms

Unified modeling of the mammalian and fish proton-dependent oligopeptide transporter PepT1.

Electrophysiological and biophysical analyses were used to compare the partial and complete transport cycles of the intestinal oligopeptide transporter PepT1 among three species (seabass, zebrafish and rabbit). On the whole, the presteady-state currents of the fish transporters were similar to each other. Rabbit PepT1 differed from the fish transporters by having slower-decaying currents, and the charge vs. potential (Q/V) and time constant vs. potential (\u3a4/V) curves shifted to more positive potentials. All of the isoforms were similarly affected by external pH, showing acidity-induced slowing of the transients and positive shifts in the Q/V and \u3a4/V curves. Analysis of the pH-dependence of the unidirectional rates of the intramembrane charge movement suggested that external protonation of the protein limits the speed of this process in both directions. The complete cycle of the transporter was studied using the neutral dipeptide Gly-Gln. Michaelis-Menten analysis confirmed that, in all species, acidity significantly increases the apparent affinity for the substrate but does not strongly impact maximal transport current. Simulations using a kinetic model incorporating the new findings showed good agreement with experimental data for all three species, both with respect to the presteady-state and the transport currents

Residues R282 and D341 act as electrostatic gates in the proton-dependent oligopeptide transporter PepT1.

The oligopeptide transporter PepT1 is a protein found in the membrane of the cells of the intestinal walls, and represents the main route through which proteic nutrients are absorbed by the organism. Along the polypeptidic chain of this protein, two oppositely charged amino acids, an arginine in position 282 and an aspartate in position 341 of the sequence, have been hypothesised to form a barrier in the absorption pathway. In this paper we show that appropriate mutations of these amino acids change the properties of PepT1 in a way that confirms that these parts of the protein indeed act as an electrostatic gate in the transport process. The identification of the structural basis of the functional mechanism of this transporter is important because, in addition to its role in nutrient uptake, PepT1 represents a major pathway for the absorption of several therapeutic drug

U–Pb isotopic ages and Hf isotope composition of zircons in Variscan gabbros from central Spain: evidence of variable crustal contamination

Ion microprobe U–Pb analyses of zircons from three gabbroic intrusions from the Spanish Central System (SCS) (Talavera, La Solanilla and Navahermosa) yield Variscan ages (300 to 305 Ma) in agreement with recent studies. Only two zircon crystals from La Solanilla massif gave slightly discordant Paleoproterozoic ages (1,848 and 2,010 Ma). Hf isotope data show a relatively large variation with the juvenile end-members showing ɛHfi values as high as +3.6 to +6.9 and +1.5 to +2.9 in the Navahermosa and Talavera gabbros, respectively. These positive ɛHfi values up to +6.9 might represent the composition of the subcontinental mantle which generates these SCS gabbros. This ɛHfi range is clearly below depleted mantle values suggesting the involvement of enriched mantle components on the origin of these Variscan gabbros, and is consistent with previous whole-rock studies. The presence of zircons with negative ɛHfi values suggest variable, but significant, crustal contamination of the gabbros, mainly by mixing with coeval granite magmas. Inherited Paleoproterozoic zircons of La Solanilla gabbros have similar trace element composition (e.g. Th/U ratios), but more evolved Hf-isotope signatures than associated Variscan zircons. Similar inherited ages have been recorded in zircons from coeval Variscan granitoids from the Central Iberian Zone. Granitic rocks have Nd model ages (TDM) predominantly in the range of 1.4 to 1.6 Ga, suggesting a juvenile addition during the Proterozoic. However, Hf crustal model ages of xenocrystic Proterozoic zircons in La Solanilla gabbro indicate the presence of reworked Archean protoliths (TDM2 model ages of 3.0 to 3.2 Ga) incorporated into the hybridized mafic magma