Subcellular localization of Bacillus subtilis SMC, a protein involved in chromosome condensation and segregation

We have investigated the subcellular localization of the SMC protein in the gram-positive bacterium Bacillus subtilis. Recent work has shown that SMC is required for chromosome condensation and faithful chromosome segregation during the B. subtilis cell cycle. Using antibodies against SMC and fluorescence microscopy, we have shown that SMC is associated with the chromosome but is also present in discrete foci near the poles of the cell. DNase treatment of permeabilized cells disrupted the association of SMC with the chromosome but not with the polar foci. The use of a truncated smc gene demonstrated that the C-terminal domain of the protein is required for chromosomal binding but not for the formation of polar foci. Regular arrays of SMC-containing foci were still present between nucleoids along the length of aseptate filaments generated by depleting cells of the cell division protein FtsZ, indicating that the formation of polar foci does not require the formation of septal structures. In slowly growing cells, which have only one or two chromosomes, SMC foci were principally observed early in the cell cycle, prior to or coincident with chromosome segregation. Cell cycle-dependent release of stored SMC from polar foci may mediate segregation by condensation of chromosomes.     

Subcellular localization of the heparin-neutralizing factor in blood platelets

1. The distribution of the heparin-neutralizing factor (platelet factor 4, PF4) in subcellular organelles of blood platelets of rabbits and man was investigated. 2. In both species the organelles storing 5-hydroxytryptamine (5-HT storage organelles) contained only trivial amounts of PF4. 3. In contrast, the content of PF4 was highest in the subcellular fractions rich in alpha-granules. 4. In conclusion, PF4 is probably localized in the alpha-granules and therefore the platelets contain at least two types of organelles (5-HT organelles and alpha-granules) capable of releasing their contents in response to the same stimuli, such as exposure to collagen, thrombin, etc.     

Solution structure of SpoIIAA, a phosphorylatable component of the system that regulates transcription factor sigmaF of Bacillus subtilis

The establishment of differential gene expression in sporulating Bacillus subtilis involves four protein components, one of which, SpoIIAA, undergoes phosphorylation and dephosphorylation. We have used NMR spectroscopy to determine the solution structure of the nonphosphorylated form of SpoIIAA. The structure shows a fold consisting of a four-stranded beta-sheet and four alpha-helices. Knowledge of the structure helps to account for the phenotype of several strains of B. subtilis that carry known spoIIAA mutations and should facilitate investigations of the conformational consequences of phosphorylation.     

Cell surface localization and processing of the ComG proteins, required for DNA binding during transformation of Bacillus subtilis

The comG operon of Bacillus subtilis encodes seven proteins essential for the binding of transforming DNA to the competent cell surface. We have explored the processing of the ComG proteins and the cellular localization of six of them. All of the proteins were found to be membrane associated. The four proteins with N-terminal sequence motifs typical of type 4 pre-pilins (ComGC, GD, GE and GG) are processed by a pathway that requires the product of comC, also an essential competence gene. The unprocessed forms of ComGC and GD behave like integral membrane proteins. Pre-ComGG differs from pre-ComGC and pre-ComGD, in that it is accessible to proteolysis only from the cytoplasmic face of the membrane and at least a portion of it behaves like a peripheral membrane protein. The mature forms of these proteins are translocated to the outer face of the membrane and are liberated when peptidoglycan is hydrolysed by lysozyme or mutanolysin. ComGG exists in part as a disulphide-cross-linked homodimer in vivo. ComGC was found to possess an intramolecular disulphide bond. The previously identified homodimer form of this protein is not stabilized by disulphide bond formation. ComGF behaves as an integral membrane protein, while ComGA, a putative ATPase, is located on the inner face of the membrane as a peripheral membrane protein. Possible roles of the ComG proteins in DNA binding to the competent cell surface are discussed in the light of these and other results.     

SinI modulates the activity of SinR, a developmental switch protein of Bacillus subtilis, by protein-protein interaction

SinR, a 111-amino-acid DNA-binding protein, is a pleiotropic regulator of several late growth processes in Bacillus subtilis. It acts as a developmental switch, positively regulating genes for competence and motility and repressing aprE and stage II sporulation genes. It is encoded by the second gene in a two gene operon, but previous results have also indicated that these two genes are differently regulated. We show in this discussion that the product of sinI, the first open reading frame (ORF) of this operon, interferes with the function of SinR. In vivo experiments have demonstrated that overexpression of sinI results in phenotypes that are observed in cells with a null mutation of sinR. A chromosomal in-frame deletion of sinI gives rise to a phenotype associated with higher levels of SinR. Thus, SinI acts as an antagonist to SinR. In vitro experiments have shown that the interaction between these two proteins is a direct one. SinI prevents SinR from binding to its target sequence on aprE, and the two proteins form a complex that can be immunoprecipitated with antibodies to either SinR or SinI.     

Subcellular localization of lead in synaptosomes

The neurotoxicology of lead is reported to involve effects on cholinergic and monoaminergic pathways. However, no information is available on the site of action at which lead exerts neurotoxic effects. The technique of x-ray elemental analysis coupled scanning-transmission mode electron microscopy has been applied to study the in vitro interactions of lead with synaptosomes. To minimize possible translocation effects on elemental distribution, isolated synaptosomes were air dried on EM grids; no fixing or staining procedures were used. In such preparations, synaptosomes and intrasynaptosomal mitochondria were distinguishable. Lead was found only in mitochondria in association with Ca ad P. Thus, lead appears to enter isolated nerve terminals and to be sequestered in mitochondria.