Pretreatment Of Walls Research Articles

Autolysis and extension of isolated walls from growing cucumber hypocotyls.

Walls isolated from cucumber hypocotyls retain autolytic activities and the ability to extend when placed under the appropriate conditions. To test whether autolysis and extension are related, we treated the walls in various ways to enhance or inhibit long-term wall extension ('creep') and measured autolysis as release of various saccharides from the wall. Except for some non-specific inhibitors of enzymatic activity, we found no correlation between wall extension and wall autolysis. Most notably, autolysis and extension differed strongly in their pH dependence. We also found that exogenous cellulases and pectinases enhanced extension in native walls, but when applied to walls previously inactivated with heat or protease these enzymes caused breakage without sustained extension. In contrast, pretreatment of walls with pectinase or cellulase, followed by boiling in methanol to inactivate the enzymes, resulted in walls with much stronger expansin-mediated extension responses. Crude protein preparations from the digestive tracts of snails enhanced extension of both native and inactivated walls, and these preparations contained expansin-like proteins (assessed by Western blotting). Our results indicate that the extension of isolated cucumber walls does not depend directly on the activity of endogenous wall-bound autolytic enzymes. The results with exogenous enzymes suggest that the hydrolysis of matrix polysaccharides may not induce wall creep by itself, but may act synergistically with expansins to enhance wall extension.

Effects of NaCl, pH and Ca2+ on autolysis of isolated tomato fruit cell walls

Cell walls isolated from ripening tomato (Lycopersicon esculentum Mill. cv. Rutgers) fruit released pectic polymers when incubated under conditions that allow activity of wall‐bound polygalacturonase (EC 3.2.1.15). Autolysis was optimally stimulated by 150–300 mM NaCl at either pH 2.5 or 4.5. This stimulation was negated by exposure to pH 6.5 or higher and by pretreatment of walls with boiling 80% ethanol. Five mM CaCl2 did not affect autolysis at pH 2.5, but significantly inhibited at pH 4.5 or higher. Inclusion of 1 M NaCl at selected steps in the extraction scheme did not inhibit subsequent autolysis of isolated walls. Exposure of isolated walls to 1 M NaCl at pH 2.5–8.5 also did not inhibit autolytic activity compared to walls that received no ionic treatment. These data support the concept that cell wall hydrolysis during tomato fruit softening is regulated by pH, Ca2+ levels and ionic strength of the apoplast.

Binding of deoxyribonucleic acid by cell walls of transformable and nontransformable streptococci.

Cell walls isolated from competent streptococci (group H strain Challis) were shown to bind more homologous and heterologous deoxyribonucleic acid (DNA) than noncompetent walls. Heat- and alkali-denatured DNA was not bound by either wall preparation. Pretreatment of cell walls with cetyltrimethylammonium bromide sharply increased the binding of DNA but did not increase transformation of whole cells. Pretreatment of the walls with either sodium dodecylsulfate, deoxyribonuclease and ribonuclease, or with crude competence-provoking factor did not affect the binding of DNA. Antiserum prepared against whole competent cells completely blocked transformation and also inhibited DNA binding to competent cell walls. Adsorption of this antiserum with competent Challis cells removed its blocking action for both binding and transformation. Pretreatment of walls with trypsin and Pronase destroyed their ability to bind DNA. Trypsin treatment also blocked transformation in whole cells. The transforming activity of DNA bound to cell walls was found to be protected from deoxyribonuclease action. Significant differences were observed in the arginine, proline, and phenylalanine content of competent and noncompetent walls. With few exceptions, the amino acids released from competent cell walls by trypsin were several-fold greater than from noncompetent walls. The results indicate that (i) two binding sites exist, one in competent cells only and essential for subsequent transformation, and a second, present in all cells, which is not involved in transformation; (ii) both sites are protein in nature; (iii) the transformation site is blocked by antibody; and (iv) the competent cell wall possesses tryptic-sensitive protein not present in the noncompetent wall.

On the Clumping of Corynebacterium xerosis as Affected by Temperature

SUMMARY: Corynebacterium xerosis Strain NCIB 9956 (minimum temperature for growth about 20°) formed clumps in cultures or suspensions of bacteria from these cultures, when grown at 30° and rapidly cooled to 15° with fast stirring. Electron microscopy showed whole organisms to be connected by an adhesive material. Electron micrographs of thin sections showed that clumps appeared to be formed by single layers of bacteria collecting round gas bubbles. The extent of clumping was greatest around pH 3, and increased as the temperature was decreased from 30° to 15°. Substitution of nitrogen or oxygen for air as gas phase had little effect on the clumping. The clumping ability increased with the age of the culture from the mid-exponential to the late-exponential phase of growth, but thereafter declined. Salts were necessary for clump formation. The relative effects of different monovalent cations and anions depended on the positions of the ions in the Hofmeister series. Incubation of bacteria in buffered solutions of proteolytic enzymes decreased their clumping ability. The extent of clumping also decreased when bacteria were incubated in buffered solutions of guanidine HCl (5 M), urea (8 M) or in a solution of uranyl nitrate (10 mM) and NaCl (0.1 M). Isolated walls of the organism formed clumps when suspensions in phosphate buffer (pH 7.0) were incubated at temperatures between 35° and 5° with fast stirring. Pretreatment of walls with trypsin decreased clumping ability. The ability to form clumps when cultures were cooled to 5° with fast stirring was demonstrated with several but not all of the strains of C. xerosis tested.