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Products encoded in the trans-acting factor (TAF) region are necessary for the biosynthesis of anguibactin and for maximal expression of iron transport and biosynthesis genes in the plasmid-encoded iron-scavenging system of Vibrio anguillarum. Here we identify angB, a locus located in the TAF region, which encodes products essential for anguibactin biosynthesis. We demonstrate that a 287-amino-acid polypeptide, encoded by angB and designated AngB, has an isochorismate lyase activity necessary for the synthesis of 2,3-dihydroxybenzoic acid, an anguibactin biosynthesis intermediate. Complementation of various angB mutations provided evidence that an additional, overlapping gene exists at this locus. This second gene, designated angG, also has an essential biosynthetic function. The angG gene directs the expression of three polypeptides when overexpressed in Escherichia coli, all of which are translated in the same frame as AngB. The results of site-directed mutagenesis and in vivo phosphorylation experiments suggest that the carboxy-terminal end of AngB and the AngG polypeptide(s) function as aryl carrier proteins involved in the assembly of the anguibactin molecule. Our results also show that the regulatory functions of the TAF are encoded in a region, TAFr, which is distinct from and independent of the angB and angG genes.

The low-copy-number and broad-host-range pSM19035-derived plasmid pBT233 is stably inherited in Bacillus subtilis cells. Two distinct regions, segA and segB, enhance the segregational stability of the plasmid. Both regions function in a replicon-independent manner. The maximization of random plasmid segregation is accomplished by the recombination proficiency of the host or the presence of the pBT233 segA region. The segA region contains two open reading frames (orf) [alpha and beta]. Inactivation or deletion of orf beta results in SegA- plasmids. Better than random segregation requires an active segB region. The segB region contains two orfs (orf epsilon and orf zeta). Inactivation of either of the orfs does not lead to an increase in cell death, but orf zeta- plasmids are randomly segregated. These results suggest that pBT233 stabilization relies on a complex system involving resolution of plasmid oligomers (segA) and on the function(s) encoded by the segB region.

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Acid-sensing ion channels (ASICs) are voltage-independent Na ؉ channels activated by extracellular protons. ASIC1a is expressed in neurons in mammalian brain and is implicated in long term potentiation of synaptic transmission that contributes to learning and memory. In ischemic brain injury, however, activation of this Ca 2؉ -permeable channel plays a critical role in acidosis-mediated, glutamate-independent, Ca 2؉ toxicity. We report here the identification of insulin as a regulator of ASIC1a surface expression. In modeled ischemia using Chinese hamster ovary cells, serum depletion caused a significant increase in ASIC1a surface expression that resulted in the potentiation of ASIC1a activity. Among the components of serum, insulin was identified as the key factor that maintains a low level of ASIC1a on the plasma membrane. Neurons subjected to insulin depletion increased surface expression of ASIC1a with resultant potentiation of ASIC1a currents. Intracellularly, ASIC1a is predominantly localized to the endoplasmic reticulum in Chinese hamster ovary cells, and this intracellular localization is also observed in neurons. Under conditions of serum or insulin depletion, the intracellular ASIC1a is translocated to the cell surface, increasing the surface expression level. These results reveal an important trafficking mechanism of ASIC1a that is relevant to both the normal physiology and the pathological activity of this channel.Acid-sensing ion channels belong to the epithelial sodium channel and degenerin family of ion channels and primarily transport Na ϩ into cells. ASICs 2 are activated by the presence of extracellular protons, which serve as ligands for these channels. So far six isoforms of ASICs (ASIC1a, 1b, 2a, 2b, 3, and 4) have been found in the mammalian central and peripheral nervous system. ASIC1a is expressed in various regions of brain including hippocampus, cerebral cortex, cerebellum, and amygdala (1-3). The role of ASIC1a in brain function is well characterized, in particular by electrophysiological and behavioral studies of ASIC1a knock-out (ASIC1aH ϩ -evoked currents are involved in synaptic transmission that contributes to important normal brain functions such as learning and memory in hippocampus and fear-related behaviors in the amygdala (3-5). Like other ASIC isoforms, the amino acid sequence of ASIC1a reveals a structure highly conserved among the epithelial sodium channel family (6). The crystal structure of a truncated chicken ASIC1a channel determined that this two-transmembrane protein is assembled as a trimer (7). Cerebral neurons express native ASIC1a as an assembly of homomultimers as well as heteromultimers in association with ASIC2a (8). Although ASIC1a and ASIC2a share high homology in their amino acid sequences, these proton-activated channels exhibit distinct sensitivity to extracellular pH. ASIC1a is more sensitive to changes in extracellular proton levels than ASIC2a and thus activates at a higher pH (pH of halfmaximal channel activation pH 0.5 ϭ 6.2), whereas ASIC2a activate...