Control of Enterococcus faecalis sex pheromone cAD1 elaboration: Effects of culture aeration and pAD1 plasmid-encoded determinants

Aeration of plasmid-free Enterococcus faecalis strains resulted in an 8- to 16-fold decrease in sex pheromone cAD1 activity in culture filtrates. Levels of two unrelated pheromones, cPD1 and cAM373, were unaffected by culture aeration. Aeration also resulted in a decrease in the expression of conjugative transfer functions observed in cells containing pAD1 traB mutations, verifying a link between traB function and pheromone “shutdown.” Tests with a series of pAD1 mini-plasmids indicated that the product of the traB gene was involved in, but not sufficient for, pheromone shutdown; the cooperation of one or more other gene products encoded within the pheromone response control region was required.     

Purification of ribulose 1,5-bisphosphate carboxylase/oxygenase and of carboxysomes from Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa (Montagu)

The bacterial symbionts of many marine invertebrates contain ribulose 1,5-bisphosphate (RuBP) carboxylase but apparently no carboxysomes, polyhedral bodies containing RuBP carboxylase. In the few cases where polyhedral bodies have been observed they have not been characterised enzymatically. Polyhedral bodies, 50–90 nm in diameter, were observed in thin cell sections of Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa and RuBP carboxylase activity was detected in both soluble and particulate fractions after centrifugation of cell-free extracts. RuBP carboxylase purified 90-fold from the soluble fraction was of high molecular weight and consisted of large and small subunits, with molecular weights of 53,110 and 11,100 respectively. Particulate RuBP carboxylase activity was associated with polyhedral bodies 50–100 nm in diameter, as revealed by density gradient centrifugation and electron microscopy. Therefore, the polyhedral bodies were inferred to be carboxysomes. Native electrophoresis of isolated carboxysomes demonstrated a major band which comigrated with the purified RuBP carboxylase and three minor bands of lower molecular weight. Sodium dodecyl-sulphate (SDS) gel electrophoresis of SDS-dissociated carboxysomes demonstrated nine major polypeptides two of which were the large and small subunits of RuBP carboxylase. The RuBP carboxylase subunits represented 21% of the total carboxysomal protein. The most abundant polypeptide had a molecular weight of 40,500. Knowledge of carboxysome composition is necessary to provide an understanding of carboxysome function.Abbreviations FPLC fast performance liquid chromatography - IB isolation buffer - PAGE polyacrylamide gel electrophoresis - RuBP carboxylase - ribulose 1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl-sulphate     

The bacteriophage Mu transposase protein can form high-affinity protein-DNA complexes with the ends of transposable elements of the Tn 3 family

The 37 kb transposable bacteriophage Mu genome encodes a transposase protein which can recognize and bind to a consensus sequence repeated three times at each extremity of its genome. A subset of this consensus sequence (5'-PuCGAAA(A)-3') is found in the ends of many class II prokaryotic transposable elements. These elements, like phage Mu, cause 5 bp duplications at the site of element insertion, and transpose by a cointegrate mechanism. Using the band retardation assay, we have found that crude protein extracts containing overexpressed Mu transposase can form high-affinity protein-DNA complexes with Mu att R and the ends of the class II elements Tn 3 (right) and IS101. No significant protein-DNA complex formation was observed with DNA fragments containing the right end of the element IS102, or a non-specific pBR322 fragment of similar size. These results suggest that the Mu transposase protein can specifically recognize the ends of other class II transposable elements and that these elements may be evolutionarily related.     

Purification and characterization of C1, the catalytic subunit of Saccharomyces cerevisiae cAMP-dependent protein kinase encoded by TPK1

In the yeast Saccharomyces cerevisiae, three genes TPK1, TPK2, and TPK3 encode catalytic subunits of cAMP-dependent protein kinase. We have purified and characterized the catalytic subunit, C1, encoded by the TPK1 gene. In order to purify C1 completely free of C2 and C3, a strain was constructed that contained only the TPK1 gene and genetic disruptions of the other two TPK genes. The cellular level of C1 was increased by expressing the genes for C1 (TPK1) and yeast regulatory subunit (BCY1) on multiple copy plasmids within this strain. Purification was accomplished by a two-column procedure in which holoenzyme was chromatographed on Sephacryl-200, then bound to an anti-regulatory subunit immunoaffinity column. Pure C1 was released from the antibody column by addition of cAMP. The protein migrated on a sodium dodecyl sulfate-polyacrylamide gel with an Mr of 52,000. Kinetic analysis showed that the apparent Km for ATP and Leu-Arg-Arg-Ala-Ser-Leu-Gly was 33 and 101 microM, respectively. The kcat was determined to be 640 min-1. The protein weakly autophosphorylated, incorporating less than 0.1 mol of phosphate/mol of catalytic subunit. NH2-terminal sequencing revealed that the protein was blocked.     

Prolyl isomerization: How significant for in vivo protein folding?

Suggestive but not decisive evidence indicates that in vivo peptide chain folding is completed in a time not much longer than that required for covalent peptide synthesis. Extrapolation of model peptide rates of the cis–trans prolyl isomerization leads to the prediction tht protein folding should be much slower than the apparent in vivo rates. On the assumption that rapid protein folding in vivo is the rule, three routes are suggested by which a protein undergoing biosynthesis can avoid a strongly slowed folding rate: (1) by a peptide chain-elongation process that adds only trans peptide bonds, follwed by a rapid folding process that incorporates them into a three-dimensional structure, raising the energy barrier to isomerization; (2) by folding to produce three dimensional structures that position prolyl residues largely in chain turns on the protein surface, where the residue may be either cis or trans without large effects on the protein structure and function; (3) prolyl cis–trans isomerization may be speeded by the formation of peptide loops.     

Accuracy and direction of error in the sexing of the skeleton: implications for paleodemography

Determinations of sex by subjective assessment of the skulls from a skeletal series of known sex were compared to fully independent assessments based on pelves of the same specimens. Within-sex correlations of cranial and pelvic morphologies measured on an android-gynecoid scale were smaller than expected. Subjective assessment by means of the skull compared favorably to that of the linear discriminant functions of Giles and Elliot; however, the direction of error was similar for both procedures. Of course, estimations based on the pelves were generally superior to both in terms of frequency and overall bias of error. The bias of sex estimation for paleodemographic purposes is contingent upon completeness of skeletal remains.     

Genetic locus (stmF) associated with cyclic GMP phosphodiesterase activity in Dictyostelium discoideum maps in linkage group II.

Previous attempts to map the stmF locus in Dictyostelium discoideum, by using only clone morphology as a marker, have led to equivocal results. Since strains carrying mutations at the stmF locus possess very low cyclic GMP phosphodiesterase activity, we have remapped this locus using both morphological and biochemical markers. Our results indicate that mutations producing a stable "streamer" phenotype and reduced cyclic GMP phosphodiesterase activity are located in linkage group II, probably centromere distal to acrA.     

Genetic and physical map of the 2,4-dichlorophenoxyacetic acid-degradative plasmid pJP4.

Plasmid pJP4 is an 80-kilobase, IncP1, broad-host-range conjugative plasmid of Alcaligenes eutrophus encoding resistance to mercuric chloride and phenyl mercury acetate and degradation of 2,4-dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid, and 3-chlorobenzoate. By the use of cloning, transposon mutagenesis, and restriction endonuclease analysis, a biophysical and genetic map of pJP4 was generated.     

The response of stream macroinvertebrates to substrate size and heterogeneity

Manipulations of substrate size and components of heterogeneity were designed to test their independent effects and interactions on the abundance and species richness of stream macroinvertebrates. Two components of substrate heterogeneity, variation in size class proportions and number of size classes, had no independent effect on abundance or richness; and in general did not interact with median particle size. Median particle size, stream current, and detritus accounted for most of the significant variation in macroinvertebrates colonizing the experimental substrates. Rocks with high surface heterogeneity (roughness) were colonized by more individuals (but not taxa) than rocks with low surface heterogeneity.