Storage deterioration of maize having pre-harvest infection with Aspergillus flavus

Deteriorative changes during storage of two different lots of field-contaminated (by Aspergillus flavus ), equally matured and freshly harvested (from two different fields) maize grains of the same cultivar (Ganga) and genotype, and stored under the same natural atmospheric conditions prevailing in the store-house, were studied and compared. It was found that the rate and extent or grain deterioration were much less in lot-2 than in lot-1 which had relatively high pre-harvest internal fungal ( A. flavus ) load. However, the deteriorative changes of both the lots could be reduced when the initial A. flavus infection was completely eliminated. Furthermore, of these already contaminated lots, the kernels selected as free of such infection at harvest also suffered less deterioration during storage.     

Lipid profiles of Aspergillus niger and its unsaturated fatty acid auxotroph, UFA2

A comparative study of the mycelial lipid composition of a wild strain (V35) and one unsaturated fatty acid auxotroph (UFA2) of Aspergillus niger has been performed. The lipid composition of both strains are qualitatively the same but quantitatively different. All the strains contain the following phospholipids: cardiolipin, phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylcholine, and phosphatidylserine; and triglycerides, diglycerides, monoglycerides, ergosterol, and sterol esters as the neutral lipids; mono- and di-galactosyl diglyceride as the major glycolipids along with small amounts of the corresponding mannose analogs. Phosphatidylethanolamine and phosphatidylcholine constitute the bulk of the phospholipids. The mutant (UFA2) contains a higher level of glycerides and lower levels of sterol (both free and esterified form), phospholipids, and glycolipids than the wild type. Aspergillus niger contains C16 to C18 saturated and unsaturated fatty acids. Small amounts of long-chain (C20 to C24) and short-chain (C10 to C14) saturated and unsaturated acids are also present. Linoleic, oleic, and palmitic are the major acids, stearic and linolenic acids being minor ones. UFA2 grows only in the presence of unsaturated fatty acid (C16 or C18) and accumulates a higher concentration of supplemented acid which influences its fatty acid profile.     

Structure and function of the repressor of bacteriophage lambda

Summary By mutagenizing a cIts (cI857) lysogen, a mutant has been isolated with a wild-type phenotype. This mutant phage lysogenizes with low efficiency and produces a low burst. Though the initial rates of repressor synthesis in Escherichia coli after infection with wild-type and mutant are the same, the maximum level of repressor that is synthesized in the latter case is only about 30% of that synthesized in the former. Virulent plates on the lysogen of mutant with slightly less efficiency producing very tiny plaques. Operator-binding studies made in vitro with purified mutant and wild-type repressors show that the binding curve of the former repressor is a rectangular hyperbola while that of the latter is sigmoid. The half-lives of the complexes of mutant and wild-type repressors with right operator are 133 and 27 min, respectively. All these results suggest that the mutant repressor possibly has a higher affinity for the operators. This mutant has been named cIha (ha=high affinity).     

Depth profiling in membranes by fluorescence quenching

Membrane penetration depth is an important parameter in relation to membrane structure and organization. A methodology has been developed to analyze the membrane penetration depths of fluorescent molecules or groups utilizing differential fluorescence quenching caused by membrane embedded spin-label probes located at different depths. The method involves determination of the parallax in the apparent location of fluorophores, detected when quenching by phospholipids spin-labelled at two different depths is compared. By use of relatively simple algebraic expressions, the method allows calculation of depth in å. This method has been used to determine the location of fluorophores in NBD-labelled lipids and anthroyloxy-labelled fatty acids in model membranes and of the membrane embedded tryptophan residues in the reconstituted nicotinic acetylcholine receptor.     

Molecular cloning and sequencing of an operon, carRS of Azospirillum brasilense, that codes for a novel two-component regulatory system: demonstration of a positive regulatory role of carR for global control of carbohydrate catabolism.

A pleiotropic carbohydrate mutant, CR17, of Azospirillum brasilense RG (wild type) that assimilates C4 dicarboxylates (succinate and malate) but not carbohydrate (fructose, arabinose, galactose, glycerol, and gluconate) as C sources for growth was used to identify the car (carbohydrate regulation) locus by complementation analysis. The 2.8-kb genomic fragment that complemented the Car- defect of CR17 and overlapped the fru operon (S. Chattopadhyay, A. Mukherjee, and S. Ghosh, J. Bacteriol. 175:3240-3243, 1993) has now been completely sequenced. The sequence contains an operon, carRS, coding for two proteins, CARR and CARS, having 236 and 352 amino acid residues, respectively. The 3'-flanking region of the carRS operon showed sequence homology with the 5' terminus of the fruB gene of a related bacterium, Rhodobacter capsulatus. A complementation study with carRS deletion clones showed that only the carR+ gene was required to complement the Car- defect of CR17, signifying that the carbohydrate pleiotropy was due to a lesion within this gene. Although the 2.8-kb DNA containing the carRS operon when introduced by conjugation into CR17 also complemented the Car- defect, the complemented transconjugant was unable to utilize succinate as a C source. The reason for this is not clear. A sequence analysis of the two protein products strongly suggests that the protein pair may constitute a novel two-component regulatory system for global expression of carbohydrate catabolic pathways in A. brasilense.     

Structural insights into thermostable direct hemolysin of Vibrio parahaemolyticus using single-particle cryo-EM

Thermostable direct hemolysin (TDH) is a ~19 kDa, hemolytic pore-forming toxin from the gram-negative marine bacterium Vibrio parahaemolyticus, one of the causative agents of seafood-borne acute gastroenteritis and septicemia. Previous studies have established that TDH exists as a tetrameric assembly in physiological state; however, there is limited knowledge regarding the molecular arrangement of its disordered N-terminal region (NTR)—the absence of which has been shown to compromise TDH's hemolytic and cytotoxic abilities. In our current study, we have employed single-particle cryo-electron microscopy to resolve the solution-state structures of wild-type TDH and a TDH construct with deletion of the NTR (NTD), in order to investigate structural aspects of NTR on the overall tetrameric architecture. We observed that both TDH and NTD electron density maps, resolved at global resolutions of 4.5 and 4.2 Å, respectively, showed good correlation in their respective oligomeric architecture. Additionally, we were able to locate extra densities near the pore opening of TDH which might correspond to the disordered NTR. Surprisingly, under cryogenic conditions, we were also able to observe novel supramolecular assemblies of TDH tetramers, which we were able to resolve to 4.3 Å. We further investigated the tetrameric and inter-tetrameric interaction interfaces to elaborate upon the key residues involved in both TDH tetramers and TDH super assemblies. Our current structural study will aid in understanding the mechanistic aspects of this pore-forming toxin and the role of its disordered NTR in membrane interaction.     

Ter-Seq: A high-throughput method to stabilize transient ternary complexes and measure associated kinetics

Regulation of biological processes by proteins often involves the formation of transient, multimeric complexes whose characterization is mechanistically important but challenging. The bacterial toxin CcdB binds and poisons DNA Gyrase. The corresponding antitoxin CcdA extracts CcdB from its complex with Gyrase through the formation of a transient ternary complex, thus rejuvenating Gyrase. We describe a high throughput methodology called Ter-Seq to stabilize probable ternary complexes and measure associated kinetics using the CcdA-CcdB-GyrA14 ternary complex as a model system. The method involves screening a yeast surface display (YSD) saturation mutagenesis library of one partner (CcdB) for mutants that show enhanced ternary complex formation. We also isolated CcdB mutants that were either resistant or sensitive to rejuvenation, and used surface plasmon resonance (SPR) with purified proteins to validate the kinetics measured using the surface display. Positions, where CcdB mutations lead to slower rejuvenation rates, are largely involved in CcdA-binding, though there were several notable exceptions suggesting allostery. Mutations at these positions reduce the affinity towards CcdA, thereby slowing down the rejuvenation process. Mutations at GyrA14-interacting positions significantly enhanced rejuvenation rates, either due to reduced affinity or complete loss of CcdB binding to GyrA14. We examined the effect of different parameters (CcdA affinity, GyrA14 affinity, surface accessibilities, evolutionary conservation) on the rate of rejuvenation. Finally, we further validated the Ter-Seq results by monitoring the kinetics of ternary complex formation for individual CcdB mutants in solution by fluorescence resonance energy transfer (FRET) studies.     

PPL catalyzed monoesterification of α,ω-dicarboxylic acids

Summary A regioselective strategy for PPL catalysed monoesterification of aliphatic ,-dicarboxylic acids with n-butanol have been developed. In addition to high regioselectivity, the method also ensures chemoselective esterification of a saturated acid moiety in presence of a conjugated acid function.