Search

In our endeavor to improve the nitrogen fixation efficiency of a soil diazotroph that would be unaffected by synthetic nitrogenous fertilizers, we have deleted a part of the negative regulatory gene nifL and constitutively expressed the positive regulatory gene nifA in the chromosome of Azotobacter chroococcum CBD15, a strain isolated from the local field soil. No antibiotic resistance gene or other foreign gene was present in the chromosome of the engineered strain. Wheat seeds inoculated with this engineered strain, which we have named Azotobacter chroococcum HKD15, were tested for 3 years in pots and 1 year in the field. The yield of wheat was enhanced by ~60% due to inoculation of seeds by A. chroococcum HKD15 in the absence of any urea application. Ammonium only marginally affected acetylene reduction by the engineered Azotobacter strain. When urea was also applied, the same wheat yield could be sustained by using seeds inoculated with A. chroococcum HKD15 and using ~85 kg less urea (~40 kg less nitrogen) than the usual ~257 kg urea (~120 kg nitrogen) per hectare. Wheat plants arising from the seeds inoculated with the engineered Azotobacter strain exhibited far superior overall performance, had much higher dry weight and nitrogen content, and assimilated molecular 15N much better. A nitrogen balance experiment also revealed much higher total nitrogen content. Indole-3-acetic acid (IAA) production by the wild type and that by the engineered strain were about the same. Inoculation of the wheat seeds with A. chroococcum HKD15 did not adversely affect the microbial population in the field rhizosphere soil. [ABSTRACT FROM AUTHOR]

The bacterial Sec protein translocation system catalyzes the transport of unfolded precursor proteins across the cytoplasmic membrane. Using a recently developed real time fluorescence-based transport assay, the effects of the number and distribution of positive charges on the transport time and transport efficiency of proOmpA were examined. As expected, an increase in the number of lysine residues generally increased transport time and decreased transport efficiency. However, the observed effects were highly dependent on the polylysine position in the mature domain. In addition, a string of consecutive positive charges generally had a more significant effect on transport time and efficiency than separating the charges into two or more charged segments. Thirty positive charges distributed throughout the mature domain resulted in effects similar to 10 consecutive charges near the N terminus of the mature domain. These data support a model in which the local effects of positive charge on the translocation kinetics dominate over total thermodynamic constraints. The rapid translocation kinetics of some highly charged proOmpA mutants suggest that the charge is partially shielded from the electric field gradient during transport, possibly by the co-migration of counter ions. The transport times of precursors with multiple positively charged sequences, or "pause sites," were fairly well predicted by a local effect model. However, the kinetic profile predicted by this local effect model was not observed. Instead, the transport kinetics observed for precursors with multiple polylysine segments support a model in which translocation through the SecYEG pore is not the rate-limiting step of transport. [ABSTRACT FROM AUTHOR]

The plant vacuolar H+-translocating ATPase (V-ATPase, EC 3.6.1.34) generates a H+ electro-chemical gradient across the tonoplast membrane. We isolated two full-length cDNA clones (VHA-A1 and VHA-A2) from tomato (Lycopersicon esculentumMill. cv. Large Cherry Red) coding for two isoforms of the V-ATPase catalytic subunit (V-ATPases A1 and A2). The cDNA clones encoding the two isoforms share 90% identity at the nucleotide level and 96% identity at the amino acid level. The 5'- and 3'-untranslated regions, however, are highly diverse. Both V-ATPase A1 and A2 isoforms encode polypeptides of 623 amino acids, with calculated molecular masses of 68,570 and 68,715, respectively. The expression of VHA-A1 and accumulation of V-ATPase A1 polypeptide were ubiquitous in all tissues examined. In response to salinity, the abundances of both transcript (VHA-A1) and protein (V-ATPase A1) of the A1 isoform in leaves were nearly doubled. In contrast to the A1 isoform, VHA-A2 transcript and V-ATPase A2 polypeptide were only detected in abundance in roots, and in minor quantities in mature fruit. In roots, accumulation of transcripts and polypeptides did not change in response to salinity for either isoform. Subcellular localization indicated that the highest levels of both V-ATPase A1 and A2 isoforms were in the tonoplast. However, significant quantities of both isoforms were detected in membranes associated with endoplasmic reticulum and/or Golgi. Immunoprecipitation of dissociated V1 domains using isoform-specific antibodies showed that V1 domains consist of either V-ATPase A1 or A2 catalytic subunit isoforms. [ABSTRACT FROM AUTHOR]

An extracellular α-type carbonic anhydrase (dCAII) from the salt-tolerant alga Dunaliella salina differs from its mesophilic counterparts in remaining active from zero to multimolar salt concentrations. To gain insight into the outstanding salt tolerance of dCAII, the enzyme was functionally overexpressed in Escherichia coli, purified by affinity chromatography and crystallized by the hanging-drop method. The crystals belonged to space group P2[SUB1], with unit-cell parameters a = 47.0, b = 119.9, c = 58.5 Å β = 94.2°. Data from a single crystal were collected to 2.4 Å resolution under cryogenic conditions (120 K) using an R-AXIS IV[SUP++] detector mounted on a Rigaku RU-H3R rotating-anode generator. The asymmetric unit contains two molecules of the protein, which corresponds to V[SUBM] = 2.65 Å[SUP3] Da[SUP-1] and a solvent content of 52.7%. [ABSTRACT FROM AUTHOR]

A 60-kDa, salt-inducible, internally duplicated alpha-type carbonic anhydrase (Dca) is associated with the plasma membrane of the extremely salt-tolerant, unicellular, green alga Dunaliella salina. Unlike other carbonic anhydrases, Dca remains active over a very broad range of salinities (0-4M NaCl), thus representing a novel type of extremely halotolerant enzyme. To elucidate the structural principles of halotolerance, structure-function investigations of Dca have been initiated. Such studies require considerable amounts of the enzyme, and hence, large-scale algal cultivation. Furthermore, the purified enzyme is often contaminated with other, co-purifying algal carbonic anhydrases. Expression in heterologous systems offers a means to produce, and subsequently purify, sufficiently large amounts of Dca required for activity and structural studies. Attempts to over-express Dca in the Escherichia coli BL21(DE3)pLysS strain, after optimizing various expression parameters, produced soluble, but weakly active protein, composed of fully reduced and variably -S-S- cross-linked chains (each of the Dca repeats contains a pair of cysteine residues, presumably forming a disulfide bond). However, when the E. coli Origami B(DE3)pLysS strain was used as a host, a functionally active enzyme with proper disulfide bonds was formed in good yield. Affinity-purified recombinant Dca resembled the native enzyme from D. salina in activity and salt tolerance. Hence, this expression system offers a means of pursuing detailed studies of this extraordinary protein using biochemical, biophysical, and crystallographic approaches.