Nitrogen Oxyanion-dependent Dissociation of a Two-component Complex That Regulates Bacterial Nitrate Assimilation

Nitrogen is an essential nutrient for growth and is readily available to microbes in many environments in the form of ammonium and nitrate. Both ions are of environmental significance due to sustained use of inorganic fertilizers on agricultural soils. Diverse species of bacteria that have an assimilatory nitrate/nitrite reductase system (NAS) can use nitrate or nitrite as the sole nitrogen source for growth when ammonium is limited. In Paracoccus denitrificans, the pathway-specific two-component regulator for NAS expression is encoded by the nasT and nasS genes. Here, we show that the putative RNA-binding protein NasT is a positive regulator essential for expression of the nas gene cluster (i.e. nasABGHC). By contrast, a nitrogen oxyanion-binding sensor (NasS) is required for nitrate/nitrite-responsive control of nas gene expression. The NasS and NasT proteins co-purify as a stable heterotetrameric regulatory complex, NasS-NasT. This protein-protein interaction is sensitive to nitrate and nitrite, which cause dissociation of the NasS-NasT complex into monomeric NasS and an oligomeric form of NasT. NasT has been shown to bind the leader RNA for nasA. Thus, upon liberation from the complex, the positive regulator NasT is free to up-regulate nas gene expression.     

Cloning ofCandida boidinii DNA fragments promoting autonomous replication of plasmids inSaccharomyces cerevisiae

Fragments of Candida boidinii chromosomal DNA were inserted into the integrative vector YIp-kanr and examined for the presence of sequences promoting autonomous replication of plasmids in Saccharomyces cerevisiae. Restriction maps of two plasmids, designated S6/4 and S6/5, originating from the same S. cerevisiae transformant, were constructed. Southern hybridization data confirmed that the plasmids carry sequences from the C. boidinii chromosome. Both plasmids transform S. cerevisiae strains at 4-5-fold higher frequency than cloning vectors based on the replication origin of the 2 microns plasmid. Mitotic stability of the constructed plasmids is similar to that of the 2 mu-based vector pNF2 in S. cerevisiae.     

Influence of lipid headgroup on the specificity and exchange dynamics in lipid-protein interactions. A spin-label study of myelin proteolipid apoprotein-phospholipid complexes

The pH and salt dependences of the interaction of phosphatidic acid, phosphatidylserine, and stearic acid with myelin proteolipid apoprotein (PLP) in dimyristoylphosphatidylcholine (DMPC) recombinants have been studied by electron spin resonance spectroscopy, using spin-labeled lipids. The two-component spin-label spectra have been analyzed both by spectral subtraction and by simulation using the exchange-coupled Bloch equations to give the fraction of lipids motionally restricted by the protein and the rate of lipid exchange between the fluid and motionally restricted lipid populations. For stearic acid, phosphatidic acid, and phosphatidylserine, the fraction of motionally restricted spin-label increases with increasing pH, with pKa's of 7.7, 7.6, and ca. 9.4, respectively. The corresponding pKa's for the bulk lipid regions of the bilayer are estimated, from changes in the ESR spectra, to be 6.7, 7.4, and 11, respectively. In the dissociated state at pH 9.0, the fraction of motionally restricted component decreases with increasing salt concentration, reaching an approximately constant value at [NaCl] = 0.5-1.0 M for all three negatively charged lipids. The net decreases for stearic acid and phosphatidic acid are considerably smaller (by ca. 30%) than those obtained on protonating the two lipids, whereas for phosphatidylserine the fraction of motionally restricted lipid in high salt is reduced to that corresponding to phosphatidylcholine. For a fixed lipid/protein ratio, the on-rate for exchange at the lipid-protein interface is independent of the degree of selectivity and has a shallow temperature dependence, as expected for a diffusion-controlled process.(ABSTRACT TRUNCATED AT 250 WORDS)