Purification of Poly-3-Hydroxybutyrate by Density Gradient Centrifugation in Sodium Bromide

Fractionation of fully sporulated cultures of Bacillus thuringiensis by density gradient centrifugation in NaBr produced two bands which were identified as poly- β -hydroxybutyrate. This technique generated high yields of membranebound and unbound granules of exceptional purity and degree of polymerization

Sodium Dodecyl Sulfate Hypersensitivity of \u3ci\u3eclpP\u3c/i\u3e and \u3ci\u3eclpB\u3c/i\u3e Mutants of \u3ci\u3eEscherichia coli\u3c/i\u3e

We studied the hypersensitivity of clpP and clpB mutants of Escherichia coli to sodium dodecyl sulfate (SDS). Both wild-type E. coli MC4100 and lon mutants grew in the presence of 10% SDS, whereas isogenic clpP and clpB single mutants could not grow above 0.5% SDS and clpA and clpX single mutants could not grow above 5.0% SDS. For wild-type E. coli, cellular ClpP levels as determined by Western immunoblot analysis increased ca. sixfold as the levels of added SDS increased from 0 to 2%. Capsular colanic acid, measured as uronic acid, increased ca. sixfold as the levels of added SDS increased from 2 to 10%. Based on these findings, 3 of the 19 previously identified SDS shock proteins (M. Adamowicz, P. M. Kelley, and K. W. Nickerson, J. Bacteriol. 173:229-233, 1991) are tentatively identified as ClpP, ClpX, and ClpB

A Circular Dichroic Study of Cu (II) -Ribonuclease Complexes

The visible and ultraviolet circular dichroic (CD) spectra resulting from the interaction of ribonuclease with successive Cu(I1) ions have been recorded under a variety of conditions. At pH 7 in the presence of 0.16 M KC1 a broad, negative band was found in the visible region. This band increased in intensity and changed in shape as successive coppers were added. The circular dichroic spectra could be analyzed in terms of two kinds of binding sites: a single strong site with CD minimum at about 710 nm, and four weaker sites with CD minimum at about 600 nm. The binding constants observed are close to those obtained by more conventional means. Carboxymethylation of one histidine results in loss of one of the weaker sites. In 0.01 M salt, only the 600-nm band is seen. Binding at pH 9.6 differed in that saturation did not occur until about 33 sites had been filled. The presence of tetra coordination at this pH was indicated by the shift of the primary d-d transition down to 530 nm. Additional structure in the visible and near ultraviolet CD was now present in the form of a negative band at 355 nm and, for the first two Cu(II)‘s added, a positive one at 480 nm. Strong positive bands were observed at 251 and 305 nm for all pH values ≥7. These are tentatively ascribed to charge transfer complexes between Cu(I1) and the peptide backbone. The relationship of the Cu(II)-ribonuclease CD spectra to those of natural, copper-containing metalloproteins, both “blue” and “non-blue”, is discussed, with special emphasis on the oxyhemocyanins

Quorum sensing activity and control of yeast-mycelium dimorphism in \u3ci\u3eOphiostoma floccosum\u3c/i\u3e

Quorum sensing (QS) activity in Ophiostoma fungi has not been described. We have examined the growth conditions on the control of dimorphism in Ophiostoma floccosum, an attractive biocontrol agent against blue-stain fungi, and its relationship with QS activity. In a defined culture medium with l-proline as the N source, a high inoculum size (107 c.f.u. ml-1) was the principal factor that promoted yeast-like growth. Inoculum size effect can be explained by the secretion of a QS molecule(s) (QSMs) responsible for inducing yeast morphology. QSM candidates were extracted from spent medium and their structure was determined by GC–MS. Three cyclic sesquiterpenes were found. The most abundant molecule, and therefore the principal candidate to be the QSM responsible for yeast growth of O. floccosum, was 1,1,4a-trimethyl-5,6-dimethylene-decalin (C15H24). Other two compounds were also detected

Quorum sensing activity and control of yeast-mycelium dimorphism in \u3ci\u3eOphiostoma floccosum\u3c/i\u3e

Quorum sensing (QS) activity in Ophiostoma fungi has not been described. We have examined the growth conditions on the control of dimorphism in Ophiostoma floccosum, an attractive biocontrol agent against blue-stain fungi, and its relationship with QS activity. In a defined culture medium with l-proline as the N source, a high inoculum size (107 c.f.u. ml-1) was the principal factor that promoted yeast-like growth. Inoculum size effect can be explained by the secretion of a QS molecule(s) (QSMs) responsible for inducing yeast morphology. QSM candidates were extracted from spent medium and their structure was determined by GC–MS. Three cyclic sesquiterpenes were found. The most abundant molecule, and therefore the principal candidate to be the QSM responsible for yeast growth of O. floccosum, was 1,1,4a-trimethyl-5,6-dimethylene-decalin (C15H24). Other two compounds were also detected

Structural Disulfide Bonds in the \u3ci\u3eBacillus thuringiensis\u3c/i\u3e subsp. \u3ci\u3eisraelensis\u3c/i\u3e Protein Crystal

We examined disulfide bonds in mosquito larvicidal crystals produced by Bacillus thuringiensis subsp. israelensis. Intact crystals contained 2.01 x 10-8 mol of free sulfhydryls and 3.24 x 10--8 mol of disulfides per mg of protein. Reduced samples of alkali-solubilized crystals resolved into several proteins, the most prominent having apparent molecular sizes of 28, 70, 135, and 140 kilodaltons (kDa). Non-reduced samples contained two new proteins of 52 and 26 kDa. When reduced, both the 52- and 26-kDa proteins were converted to 28-kDa proteins. Furthermore, both bands reacted with antiserum prepared against reduced 28-kDa protein. Approximately 50% of the crystal proteins could be solubilized without disulfide cleavage. These proteins were 70 kDa or smaller. Solubilization of the 135- and 140-kDa proteins required disulfide cleavage. Incubation of crystals at pH 12.0 for 2 h cleaved 40% of the disulfide bonds and solubilized 83% of the crystal protein. Alkali-stable disulfides were present in both the soluble and insoluble portions. The insoluble pellet contained 12 to 14 disulfides per 100 kDa of protein and was devoid of sulfhydryl groups. Alkali-solubilized proteins contained both intrachain and interchain disulfide bonds. Despite their structural significance, it is unlikely that disulfide bonds are involved in the formation or release of the larvicidal toxin

Structural Disulfide Bonds in the \u3ci\u3eBacillus thuringiensis\u3c/i\u3e subsp. \u3ci\u3eisraelensis\u3c/i\u3e Protein Crystal

We examined disulfide bonds in mosquito larvicidal crystals produced by Bacillus thuringiensis subsp. israelensis. Intact crystals contained 2.01 x 10-8 mol of free sulfhydryls and 3.24 x 10--8 mol of disulfides per mg of protein. Reduced samples of alkali-solubilized crystals resolved into several proteins, the most prominent having apparent molecular sizes of 28, 70, 135, and 140 kilodaltons (kDa). Non-reduced samples contained two new proteins of 52 and 26 kDa. When reduced, both the 52- and 26-kDa proteins were converted to 28-kDa proteins. Furthermore, both bands reacted with antiserum prepared against reduced 28-kDa protein. Approximately 50% of the crystal proteins could be solubilized without disulfide cleavage. These proteins were 70 kDa or smaller. Solubilization of the 135- and 140-kDa proteins required disulfide cleavage. Incubation of crystals at pH 12.0 for 2 h cleaved 40% of the disulfide bonds and solubilized 83% of the crystal protein. Alkali-stable disulfides were present in both the soluble and insoluble portions. The insoluble pellet contained 12 to 14 disulfides per 100 kDa of protein and was devoid of sulfhydryl groups. Alkali-solubilized proteins contained both intrachain and interchain disulfide bonds. Despite their structural significance, it is unlikely that disulfide bonds are involved in the formation or release of the larvicidal toxin

Proteomic Adaptations to Starvation Prepare \u3ci\u3eEscherichia coli\u3c/i\u3e for Disinfection Tolerance

Despite the low nutrient level and constant presence of secondary disinfectants, bacterial re-growth still occurs in drinking water distribution systems. The molecular mechanisms that starved bacteria use to survive low-level chlorine-based disinfectants are not well understood. The objective of this study is to investigate these molecular mechanisms at the protein level that prepare starved cells for disinfection tolerance. Two commonly used secondary disinfectants chlorine and monochloramine, both at 1 mg/L, were used in this study. The proteomes of normal and starved Escherichia coli (K12 MG1655) cells were studied using quantitative proteomics. Over 60-min disinfection, starved cells showed significantly higher disinfection tolerance than normal cells based on the inactivation curves for both chlorine and monochloramine. Proteomic analyses suggest that starvation may prepare cells for the oxidative stress that chlorine-based disinfection will cause by affecting glutathione metabolism. In addition, proteins involved in stress regulation and stress responses were among the ones up-regulated under both starvation and chlorine/monochloramine disinfection. By comparing the fold changes under different conditions, it is suggested that starvation prepares E. coli for disinfection tolerance by increasing the expression of enzymes that can help cells survive chlorine/monochloramine disinfection. Protein co-expression analyses show that proteins in glycolysis and pentose phosphate pathway that were up-regulated under starvation are also involved in disinfection tolerance. Finally, the production and detoxification of methylglyoxal may be involved in the chlorine-based disinfection and cell defense mechanisms

Bioassay of Solubilized \u3ci\u3eBacillus thuringiensis\u3c/i\u3e var. \u3ci\u3eisraelensis\u3c/i\u3e Crystals by Attachment to Latex Beads

Solubilized crystals of Bacillus thuringiensis var. israelensis were 7,000 times less toxic to Aedes aegypti larvae than intact crystals, presumably because mosquito larvae are filter feeders and selectively concentrate particles while excluding water and soluble molecules. A procedure is described whereby soluble toxins are adsorbed to 0.8- micrometer latex beads, with retention of toxicity. The latex bead assay should make it possible to analyze the structure and mode of action of the mosquito toxin

Lipid Metabolism During Bacterial Growth, Sporulation, and Germination: Differential Synthesis of Individual Branched and Normal-Chain Fatty Acids During Spore Germination and Outgrowth of \u3ci\u3eBacillus thuringiensis\u3c/i\u3e

The biosynthesis of individual branched- and normal-chain fatty acids during Bacillus thuringiensis spore germination and outgrowth was studied by comparing pulsed and continuous labeling of these fatty acids with [U- 14C ]acetate. The relative specific activity of each fatty acid varies with time as the cell progresses through outgrowth. However, fatty acid synthesis does occur in two distinct phases. Upon germination, acetate is incorporated only into the iso-isomers i-C13, i-C14, and i-C16; no normal or anteiso synthesis occurs. Subsequent to T30, the full complement of branched- and normal-chain homologues is formed and there is a dramatic enhancement in the overall rate of fatty acid synthesis. Significantly, this rate increase coincides with a marked shift from the synthesis of short-chain to long-chain fatty acids. These findings illustrate a dichotomy in synthesis that may result from initial fatty acid formation by preexisting spore fatty acid biosynthetic enzymes in the absence of de novo protein synthesis. Elucidation of the timing and kinetics of individual fatty acid formation provides a biochemical profile of activities directly related to membrane differentiation and cellular development