Analysis by fluorescence microscopy of the development of compartment-specific gene expression during sporulation of Bacillus subtilis.

The use of a fluorogenic substrate, 5-octanoylaminofluorescein-di-beta-D-galactopyranoside, for beta-galactosidase has made it possible to visualize enzyme activity in individual cells of sporulating populations of Bacillus subtilis by fluorescence microscopy. lacZ fusions to different sporulation-associated genes have been used to investigate the cell compartmentalization of gene expression during sporulation. A strain with a lacZ fusion to sspA, a gene which is transcribed by E-sigma G at a late stage of sporulation, displayed predominantly compartment-specific fluorescence. Expression of the early-expressed spoIIA locus, which includes the structural gene for sigma F, was seen not to be compartmentalized. Populations of strains with lacZ fusions to gpr and dacF, genes which are transcribed by E-sigma F at intermediate stages of sporulation, included some organisms showing uncompartmentalized fluorescence and others showing compartment-specific fluorescence; the proportion showing compartment-specific fluorescence increased in samples taken later in sporulation. Several possible explanations of the results obtained with gpr and dacF are considered. A plausible interpretation is that sigma F activity is initially not compartmentalized and becomes compartmentalized as sporulation progresses. The progression to compartmentalization does not require the activities of the sporulation-specific factor sigma E or sigma G but may require some product of sigma F activity.     

The division during bacterial sporulation is symmetrically located in Sporosarcina ureae

Immunofluorescence microscopy was used to visualize the FtsZ band that marks the site of septation in Sporosarcina ureae . Image analysis indicated that the vegetative division was symmetrically located with respect to the ends of the cells. Fusions of lacZ to the sporulation loci, spoIIA and cotE , of Bacillus subtilis were introduced into S . ureae by mobilization of plasmids containing the fusions from Escherichia coli . The fusions showed similar patterns of sporulation-associated expression in S . ureae to those observed in B . subtilis . Formation of β-galactosidase encoded by the spoIIA–lacZ fusion made it possible to identify early sporulating cells by immunofluorescence microscopy. Analysis of the position of FtsZ bands in cells expressing spoIIA–lacZ indicated that the location of sporulation division was symmetrical with respect to the ends of the cells, in sharp contrast to the asymmetrical location of septation in sporulating Bacilli . It is inferred that asymmetry of location of the sporulation division is not essential for the compartmentalization of gene expression that follows the division.     

Use of immunofluorescence to visualize cell-specific gene expression during sporulation in Bacillus subtilis.

We have adapted immunofluorescence microscopy for use in Bacillus subtilis and have employed this procedure for visualizing cell-specific gene expression at early to intermediate stages of sporulation. Sporangia were doubly stained with propidium iodide to visualize the forespore and mother cell nucleoids and with fluorescein-conjugated antibodies to visualize the location of beta-galactosidase produced under the control of the sporulation RNA polymerase sigma factors sigma E and sigma F. In confirmation and extension of earlier reports, we found that expression of a lacZ fusion under the control of sigma E was confined to the mother cell compartment of sporangia at the septation (II) and engulfment (III) stages of morphogenesis. Conversely, sigma F-directed gene expression was confined to the forespore compartment of sporangia at postseptation stages of development. Little indication was found for sigma E- or sigma F-directed gene expression prior to septation or in both compartments of postseptation sporangia. Gene expression under the control of the forespore sigma factor sigma G also exhibited a high level of compartmentalization. A high proportion of sporangia exhibited fluorescence in our immunostaining protocol, which should be suitable for the subcellular localization of sporulation proteins for which specific antibodies are available.     

Directed evolution of biocatalysts

Directed evolution is being used increasingly in academic and industrial laboratories to modify and improve important biocatalysts. Significant advances during this period of review include compartmentalization of genes and the in vitro translation apparatus in emulsions, as well as several impressive demonstrations of catalyst improvement. Shuffling of homologous genes offers a new way to utilize natural diversity in the evolution of novel catalysts.     

Supramolecular aggregation and organization in peripheral nerve myelin

Under certain preparative conditions the lipid bilayers of glutaraldehyde-fixed, PNS myelin demonstrate a marked compartmentalization, which can be augmented by lipid extraction following sectioning. The results are interpreted as indicating a supramolecular domain pattern of arrangement centered upon the transmembrane protein (P0) molecules. The latter are thought to be surrounded by annuli of substantially immobilized phospholipids. In the lamellar planes particular lipids are considered to have a nonrandom distribution. The visualization of bilayer compartmentalization was seen only in negatively stained sections obtained from unembedded or glutaraldehyde-urea-embedded myelin. Lipids were unextracted in the basic preparations except in so far as some unfixed, amphipathic molecules escaped at the trough-fluid interface at the time of sectioning, an observed phenomenon which probably aided in the visualization of the compartmentalization. Visualization was also augmented by surface tension expanding section fragments as they floated on the trough fluid. All stages of transition between well-ordered myelin and dispersed globular units were commonly to be found. Deliberately delipidated myelin exposed more sharply defined and smaller globular units in bilayer regions, but even these are regarded as being supramolecular aggregates including residual lipid annuli around the transmembrane proteins. The addition of cadmium ions as a "fixative" for lecithin seemed to improve the preservation of glutaraldehyde-urea-embedded myelin but was not strictly necessary to reveal its domain structure. A secondary tannic acid fixation was required to process unembedded myelin so as to reveal the fundamental compartmentalization of its lipid bilayers.     

The effect of sublethal lead exposure on the ultrastructure and on the distribution of acid phosphatase activity in chloragocytes of earthworms (Annelida, Oligochaeta)

Summary Laboratory experiments were conducted to study the effects of the exposure to a sublethal concentration (500 p.p.m.) of lead on the ultrastructure and acid phosphatase compartmentalization of the chloragogenous tissue of earthworms,Eisenia foetida. For the cytochemical demonstration of acid phosphatase activity, lead and cerium were used as capturing agents. In both cases there was a change in the compartmentalization of acid phosphatase, the enzyme activity being localized within the chloragosomes in controls, but distributed throughout the cytosol in treated animals. In addition, acid phosphatase activity increased following lead exposure. At the ultrastructural level, disruption of the chloragosomal membranes, an increase in chloragosomal fusion processes and vesiculation of the cytoplasm were evident. Moreover, an enhanced release of chloragosomes to the extracellular space was found in lead-exposed worms.     

Promotion of sporulation by caffeine pretreatment in saccharomyces cerevisiae

Cells cultured in the presence of caffeine had high sporulation ability. The sporulation-promotive effect of caffeine was studied, special attention being paid upon changes in nucleic acid metabolism. When transferred to a sporulation medium, the breakdown of RNA, the synthesis of protein, RNA and DNA, commitment to sporulation and the appearance of mature asci took place in caffeine-treated cells significantly earlier than in control cells. Commitment to sporulation occurred before the completion of premeiotic DNA synthesis in both caffeine-treated and control cells.     

Bacilysin production and sporulation inBacillus subtilis

Bacilysin-negative(bac^–) strain NG79 was found to be oligosporogenous. When compared with the parental strain, it was 200–300 times less resistant to heat, chloroform, and lysozyme treatments, and the spores contained considerably less dipicolinate. When NG79 was transduced, the oligosporogenous phenotype was found to be cured in all the transductants tested. External addition of bacilysin to the cultures of this strain markedly improved each measure of spore quality. The time of its addition determined the extent of acquired resistance, the optimum being 4–7 h after inoculation. This suggested that bacilysin might influence sporulation prior to stage I. Bacilysin activity completely disappeared from the extracellular fluid of aged cultures. It was demonstrated that the dipeptide can be cleaved by the alkaline serine protease that is produced byBacillus subtilis.     

Dynamic compartmentalization of the voltage-gated sodium channels in axons

One of the major physiological roles of the neuronal voltage-gated sodium channel is to generate action potentials at the axon hillock/initial segment and to ensure propagation along myelinated or unmyelinated fibers to nerve terminal. These processes require a precise distribution of sodium channels accumulated at high density in discrete subdomains of the nerve membrane. In neurons, information relevant to ion channel trafficking and compartmentalization into sub-domains of the plasma membrane is far from being elucidated. Besides, whereas information on dendritic targeting is beginning to emerge, less is known about the mechanisms leading to the polarized distribution of proteins in axon. To obtain a better understanding of how neurons selectively target sodium channels to discrete subdomains of the nerve, we addressed the question as to whether any of the large intracellular regions of Nav1.2 contain axonal sorting and/or clustering signals. We first obtained evidence showing that addition of the cytoplasmic carboxy-terminal region of Nav1.2 restricted the distribution of a dendritic-axonal reporter protein to axons of hippocampal neurons. The analysis of mutants revealed that a di-leucine-based motif mediates chimera compartmentalization in axons and its elimination in soma and dendrites by endocytosis. The analysis of the others generated chimeras showed that the determinant conferring sodium channel clustering at the axonal initial segment is contained within the cytoplasmic loop connecting domains II-III of Nav1.2. Expression of a soluble Nav1.2 II-III linker protein led to the disorganization of endogenous sodium channels. The motif was sufficient to redirect a somatodendritic potassium channel to the axonal initial segment, a process involving association with ankyrin G. Thus, it is conceivable that concerted action of the two determinants is required for sodium channel compartmentalization in axons.     

Nucleotide sequence of sporulation locus spoIIA in Bacillus subtilis

We have determined a sequence of 2073 bp from two recombinant plasmids carrying the whole spoIIA locus from Bacillus subtilis, the expression of which is required for spore formation. The sequence contains three long open reading frames (ORFs), each of them being preceded by a ribosome binding site. These three putative proteins (mol. wts 13100, 16300 and 22200) are likely to be expressed and are probably encoded on the same mRNA. The stop codon of ORF1 overlaps with the start codon of ORF2 suggesting that there might be translational coupling between the two ORFs. Although some known promoter sequences were found, the only one upstream from the first open reading frame is about 260 bp from it.     

PIWI-interacting small RNAs: the vanguard of genome defence

PIWI-interacting RNAs (piRNAs) are a distinct class of small non-coding RNAs that form the piRNA-induced silencing complex (piRISC) in the germ line of many animal species. The piRISC protects the integrity of the genome from invasion by 'genomic parasites'--transposable elements--by silencing them. Owing to their limited expression in gonads and their sequence diversity, piRNAs have been the most mysterious class of small non-coding RNAs regulating RNA silencing. Now, much progress is being made into our understanding of their biogenesis and molecular functions, including the specific subcellular compartmentalization of the piRNA pathway in granular cytoplasmic bodies.