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Identification of the LIV-I/LS System as the Third Phenylalanine Transporter in Escherichia coli K-12. Takashi Koyanagi, 2004.In Escherichia coli, the active transport of phenylalanine is considered to be performed by two different systems, AroP and PheP . However, a low level of accumulation of phenylalanine was observed in an aromatic amino acid transporter-deficient E . coli strain (  aroP
pheP
mtr
tna
tyrP) .
The uptake of phenylalanine by this strain was significantly
inhibited in the presence of branched-chain amino acids . Genetic
analysis and transport studies revealed that the LIV-I/LS system,
which is a branched-chain amino acid transporter consisting of two
periplasmic binding proteins, the LIV-binding protein (LIV-I system)
and LS-binding protein (LS system), and membrane components, LivHMGF,
is involved in phenylalanine accumulation in E . coli cells .
The Km values for phenylalanine in the LIV-I and LS
systems were determined to be 19 and 30 µM, respectively . Competitive
inhibition of phenylalanine uptake by isoleucine, leucine, and valine
was observed for the LIV-I system and, surprisingly, also for the
LS system, which has been assumed to be leucine specific on the
basis of the results of binding studies with the purified LS-binding
protein . We found that the LS system is capable of transporting
isoleucine and valine with affinity comparable to that for leucine
and that the LIV-I system is able to transport tyrosine with affinity
lower than that seen with other substrates . The physiological
importance of the LIV-I/LS system for phenylalanine accumulation was
revealed in the growth of phenylalanine-auxotrophic E . coli
strains under various conditions .
A Monocarboxylate Permease of Rhizobium leguminosarum Is the First Member of a New Subfamily of Transporters. A. H. F. Hosie, 2002.Amino acid transport by Rhizobium leguminosarum is dominated by two ABC transporters, the general amino acid permease (Aap) and the branched-chain amino acid permease (Bra) . However, mutation of these transporters does not prevent this organism from utilizing alanine for growth . An R . leguminosarum permease (MctP) has been identified which is required for optimal growth on alanine as a sole carbon and nitrogen source . Characterization of MctP confirmed that it transports alanine (Km = 0.56 mM) and other monocarboxylates such as lactate and pyruvate (Km = 4.4 and 3.8 µM, respectively) . Uptake inhibition studies indicate that propionate, butyrate,  -hydroxybutyrate, and acetate are also transported by MctP, with the apparent affinity for solutes demonstrating a preference for C3-monocarboxylates . MctP has significant sequence similarity to members of the sodium/solute symporter family . However, sequence comparisons suggest that it is the first characterized permease of a new subfamily of transporters . While transport via MctP was inhibited by CCCP, it was not apparently affected by the concentration of sodium . In contrast, glutamate uptake in R . leguminosarum by the Escherichia coli GltS system did require sodium, which suggests that MctP may be proton coupled . Uncharacterized members of this new subfamily have been identified in a broad taxonomic range of species, including proteobacteria of the ß-subdivision, gram-positive bacteria, and archaea . A two-component sensor-regulator (MctSR), encoded by genes adjacent to mctP, is required for activation of mctP expression .
Identification of XcpZ Domains Required for Assembly of the Secreton of Pseudomonas aeruginosa. Viviane Robert, 2002.Most of the exoproteins secreted by Pseudomonas aeruginosa are transported via the type II secretion system . This machinery, which is widely conserved in gram-negative bacteria, consists of 12 Xcp proteins organized as a multiprotein complex, also called the secreton . We previously reported that the mutual stabilization of XcpZ and XcpY plays an important role in the assembly of the secreton . In this study, we engineered variant XcpZ proteins by using linker insertion mutagenesis . We identified three distinct regions of XcpZ required for both the stabilization of XcpY and the functionality of the secreton . Interestingly, we also demonstrated that another component of the machinery, XcpP, can modulate the stabilizing activity of XcpZ on XcpY . Tri1 Encodes the Cytochrome P450 Monooxygenase for C-8 Hydroxylation during Trichothecene Biosynthesis in Fusarium sporotrichioides and Resides Upstream of Another New Tri Gene. Isaac B. Meek, 2003.Many Fusarium species produce one or more agriculturally important trichothecene mycotoxins, and the relative level of toxicity of these compounds is determined by the pattern of oxygenations and acetylations or esterifications on the core trichothecene structure . Previous studies with UV-induced Fusarium sporotrichioides NRRL 3299 trichothecene mutants defined the Tri1 gene and demonstrated that it was required for addition of the oxygen at the C-8 position during trichothecene biosynthesis . We have cloned and characterized the Tri1 gene from NRRL 3299 and found that it encodes a cytochrome P450 monooxygenase . The disruption of Tri1 blocks production of C-8-oxygenated trichothecenes and leads to the accumulation of 4,15-diacetoxyscirpenol, the same phenotype observed in the tri1 UV-induced mutants MB1716 and MB1370 . The Tri1 disruptants and the tri1 UV-induced mutants do not complement one another when coinoculated, and the Tri1 gene sequence restores T-2 toxin production in both MB1716 and MB1370 . The DNA sequence flanking Tri1 contains another new Tri gene . Thus, Tri1 encodes a C-8 hydroxylase and is located either in a new distal portion of the trichothecene gene cluster or in a second separate trichothecene gene cluster .
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