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The 2HCT and ESS families are two families of secondary transporters. Members of the two families are unrelated in amino acid sequence but share similar hydropathy profiles, which suggest a similar folding of the proteins in membranes. Structural models show two homologous domains containing five transmembrane segments (TMSs) each, with a reentrant or pore loop between the fourth and fifth TMSs in each domain. Here we show that GGXG sequence motifs present in the putative reentrant loops are important for the activity of the transporters. Mutation of the conserved Gly residues to Cys in the motifs of the Na(+)citrate transporter CitS in the 2HCT family and the Na(+)-glutamate transporter GltS in the ESS family resulted in strongly reduced transport activity. Similarly, mutation of the variable residue "X" to Cys in the N-terminal half of GltS essentially inactivated the transporter. The corresponding mutations in the N- and C-terminal halves of CitS reduced transport activity to 60 and 25% of that of the Wild type, respectively. Residual activity of any of the mutants could be further reduced by treatment with the membrane permeable thiol reagent N-ethylmaleimide (NEM). The X to Cys mutation (S405C) in the cytoplasmic loop in the C-terminal half of CitS rendered the protein sensitive to the bulky, membrane impermeable thiol reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AmdiS) added at the periplasmic side of the membrane, providing further evidence that this part of the loop is positioned between the transmembrane segments. The putative reentrant loop in the C-terminal half of the ESS family does not contain the GGXG motif, but a conserved stretch rich in Gly residues. Cysteine-scanning mutagenesis of a stretch of 18 residues in the GltS protein revealed two residues important For function. Mutant N356C was completely inactivated by treatment with NEM, and mutant P351C appeared to be the counterpart of mutant S405C of CitS; the mutant was inactivated by AmdiS added at the periplasmic side of the membrane. The data support, in general, the structural and mechanistic similarity between the ESS and 2HCT transporters and, more particularly, the two-domain structure of the transporters and the presence and functional importance of the reentrant loops present in each domain. It is proposed that the GGXG motifs are at the vertex of the reentrant loops.

Glutathione and gamma-glutamylcysteine were produced in Lactococcus lactis using a controlled expression system and the genes gshA and gshB from Escherichia coli encoding the enzymes gamma-glutamylcysteine synthetase and glutathione synthetase. High levels of gamma-glutamylcysteine were found in strains growing on chemically defined medium and expressing either gshA alone or both gshA and gshB. As anticipated, glutathione was found in a strain expressing gshA and gshB. The level of glutathione production could be increased by addition of the precursor amino acid cysteine to the medium. The addition of cysteine led to an increased activity of glutathione synthetase, which is remarkable because the amino acid is not a substrate of this enzyme. The final intracellular glutathione concentration attained was 358 nmol mg(-1) protein, which is the highest concentration reported for a bacterium, demonstrating the suitability of engineered L. lactis for fine-chemical production and as a model for studies of the impact of glutathione on flavour formation and other properties of food.

Using uranine as a physical tracer, this study assessed the sampling efficiencies of four bioaerosol samplers (Andersen 6-stage impactor, all glass impinger “AGI-30,” OMNI-3000, and Airport MD8 with gelatin filter) for collecting Gram-positive bacteria (Enterococcus faecalis), Gram-negative bacteria (Escherichia coli and Campylobacter jejuni), and bacteria without cell wall (Mycoplasma synoviae) which were aerosolized in a HEPA isolator. In addition, the half-life times of these bacteria in aerosols were estimated. The uranine concentrations collected by the samplers were used for calculating the physical efficiencies, and the bacteria/uranine ratios were used for calculating the biological efficiencies. The results show the Airport MD8 had the highest physical efficiency. Compared with the Airport MD8, the physical efficiencies of the AGI-30 and the OMNI-3000 were 74% and 49%, respectively. A low physical efficiency of the Andersen impactor (18%) was obtained, but it was mainly caused by the incomplete recovery of uranine when handling the air samples, so could not be ascribed to the sampler efficiency. Both the Andersen impactor and the AGI-30 showed high biological efficiencies for all four bacterial species. The biological efficiencies of the OMNI-3000 for C. jejuni (1%) and of the Airport MD8 for E. coli (38%) and C. jejuni (2%) were significantly lower than 100%, indicating that their sampling stresses inactivated the bacterial culturability. The half-life times at 21-23°C temperature and 80-85% relative humidity were 43.3 min for E. faecalis, 26.7 min for M. synoviae, 21.2 min for E. coli, and 4.0 min for C. jejuni in the air.

Amino acid transport in membrane vesicles of Bacillus stearothermophilus was studied. A relatively high concentration of sodium ions is needed for uptake of L-alanine (K(t) = 1.0 mM) and L-leucine (K(t) = 0.4 mM). In contrast, the Na+-H+-L-glutamate transport system has a high affinity for sodium ions (K(t)

Amino acid transport in membrane vesicles of Bacillus stearothermophilus was studied. A relatively high concentration of sodium ions is needed for uptake of L-alanine (K(t) = 1.0 mM) and L-leucine (K(t) = 0.4 mM). In contrast, the Na+-H+-L-glutamate transport system has a high affinity for sodium ions (K(t)