Importance of spore mutants for fed-batch and continuous fermentation of Bacillus subtilis

To alleviate plasmid instability and to prolong the production phase of subtilisin, integrable plasmid and spore mutants are used. Compared with batch-type shake flask cultures, spore mutants' ability to produce subtilisin can be well pronounced in fed-batch and continuous cultures. Hence, the two culture methods make it possible to identify the peculiar characteristics of the spore mutants unobtainable in batch culture. Spore mutants can enhance subtilisin productivity and prolong subtilisin production time in fed-batch culture as well as enable us to use very low dilution rates (<0.1 h(-1)) without losing productivity in continuous culture, thereby improving the conversion yield of the nitrogen source. At 0.05 h(-1) the spollG mutant of Bacillus subtilis DB104 (Deltanpr Deltaapr) (Em(r)) spollG (Bim(r)):: pMK101 (Cm(r)) showed a subtilisin yield about ten times higher than that from wild-type DB104 (Deltanpr Deltaapr)::pMK101 (Cm(r)). (c) 1995 John Wiley & Sons, Inc.     

Conformational changes in subdomain 2 of G-actin: fluorescence probing by dansyl ethylenediamine attached to Gln-41.

Gln-41 on G-actin was specifically labeled with a fluorescent probe, dansyl ethylenediamine (DED), via transglutaminase reaction to explore the conformational changes in subdomain 2 of actin. Replacement of Ca2+ with Mg2+ and ATP with ADP on G-actin produced large changes in the emission properties of DED. These substitutions resulted in blue shifts in the wavelength of maximum emission and increases in DED fluorescence. Excitation of labeled actin at 295 nm revealed energy transfer from tryptophans to DED. Structure considerations and Cu2+ quenching experiments suggested that Trp-79 and/or Trp-86 serves as energy donors to DED. Energy transfer from these residues to DED on Gln-41 increased with the replacement of Ca2+ with Mg2+ and ATP with ADP. Polymerization of Mg-G-actin with MgCl2 resulted in much smaller changes in DED fluorescence than divalent cation substitution. This suggests that the conformation of loop 38-52 on actin is primed for the polymerization reaction by the substitution of Ca2+ with Mg2+ on G-actin.     

A scanning electron microscope study of Craspedella sp. from the branchial chamber of redclaw crayfish,Cherax quadricarinatus,from Queensland,Australia

Epidermal topography was examined, including papillate ridges, grooves and ciliated sensory papillae of Craspedella sp. from the branchial chamber of redclaw crayfish, Cherax quadricarinatus, from Queensland, Australia. Rhandites were observed to discharge from ducts opening mainly in a small distal region of the ventral epidermis of the three central (of five) tentacles. These regions, devoid of ciliated sensory papillae, serve to adhere the anterior end of the worms during locomotion. Secretions from glands associated with the posterior attachment organ were observed to discharge from pores on the outside region of the ventral surface of the disc.A comparison of various scanning electron microscopy (SEM) fixation techniques showed that (1) hot fixatives at 90 °C provide most information on the largest number of epidermal structures and (2) different fixation regimes highlight different epidermal features.     

The cytoplasmic tyrosine kinase FER is associated with the catenin-like substrate pp120 and is activated by growth factors.

The FER gene encodes a cytoplasmic tyrosine kinase with a single SH2 domain and an extensive amino terminus. In order to understand the cellular function of the FER kinase, we analyzed the effect of growth factor stimulation on the phosphorylation and activity of FER. Stimulation of A431 cells and 3T3 fibroblasts with epidermal growth factor or platelet-derived growth factor results in the phosphorylation of FER and two associated polypeptides. The associated polypeptides were shown to be the epidermal growth factor receptor or the platelet-derived growth factor receptor and a previously identified target, pp120. Since pp120 had previously been shown to interact with components of the cadherin-catenin complex, these results implicate FER in the regulation of cell-cell interactions. The physical association of FER with pp120 was found to be constitutive and was mediated by a 400-amino-acid sequence in the amino terminus of FER. Analyses of that sequence revealed that it has the ability to form coiled coils and that it oligomerizes in vitro. The identification of a coiled coil sequence in the FER kinase and the demonstration that the sequence mediates association with a potential substrate suggest a novel mechanism for signal transduction by cytoplasmic tyrosine kinases.     

Independent changes in type I and type II receptors for transforming growth factor beta induced by bone morphogenetic protein 2 parallel expression of the osteoblast phenotype.

Transforming growth factor beta (TGF-beta), a potent regulator of bone formation, has bifunctional effects on osteoblast replication and biochemical activity that appear differentiation dependent. We now show that cell surface binding sites for TGF-beta vary markedly among fibroblasts, bone-derived cells, and highly differentiated osteosarcoma cultures from fetal rats. Expression of betaglycan and type II receptors decline relative to type I receptor expression in parallel with an increase in osteoblast-like activity, predicting that the ratio among various TGF-beta binding sites could influence how its signals are perceived. Bone morphogenetic protein 2 (BMP-2), which induces osteoblast function, does not alter TGF-beta binding or biochemical activity in fibroblasts and has only small effects in less differentiated bone cells. In contrast, BMP-2 rapidly reduces TGF-beta binding to betaglycan and type II receptors in osteoblast-enriched primary cell cultures and increases its relative binding to type I receptors in these cells and in ROS 17/2.8 cultures. Pretreatment with BMP-2 diminishes TGF-beta-induced DNA synthesis in osteoblast-enriched cultures but synergistically enhances its stimulatory effects on either collagen synthesis or alkaline phosphatase activity, depending on the present state of bone cell differentiation. Therefore, BMP-2 shifts the TGF-beta binding profile on bone cells in ways that are consistent with progressive expression of osteoblast phenotype, and these changes distinguish the biochemical effects mediated by each receptor. Our observations indicate specific stepwise actions by TGF-beta family members during osteoblast differentiation, developing in part from changes imprinted by BMP-2 on TGF-beta receptor stoichiometry.     

Plasmid replication and partition in Escherichiacoli: is the cell membrane the key?

The DNA–membrane complex has been the subject of intensive investigation for over 35 years as the possible site for DNA replication in the prokaryotic cell and the site through which newly synthesized chromosomes are segregated into daughter cells. However, the molecular mechanisms which control these phenomena are, for the most part, poorly understood despite genetic, biochemical, and morphologic evidence in favour of their existence. This is probably due to the transient nature and non-covalent interactions that occur between DNA and the membrane. In addition, there is a paucity of knowledge concerning the nature of the membrane receptors for DNA and whether the membrane plays simply a structural or metabolic role in the two processes. Plasmids can provide important insights into the role of the membrane in replication and partitioning because the plasmid life cycle is relatively simple, with replication occurring during the cell cycle and partitioning during cell division. The replicon model of Jacob et al. (1963, Cold Spring Harbor Symp Quant Biol 28: 329–348) still represents a good conceptual framework (with modifications) to explain how plasmid replication and partitioning are linked by the membrane. In its simplest form, the model focuses on specific membrane binding sites (possibly along the equator of the cell) for plasmid (or bacterial) replication, with the membrane acting as a motive force to separate the newly synthesized replicons and their attached sites into daughter cells. Indeed, proteins involved in both plasmid replication and partitioning have been found in membrane fractions and some plasmids require membrane binding for initiation and an active partitioning. We propose that several factors are critical for both plasmid DNA replication and partitioning. One factor is the extent of negative supercoiling (brought about by an interplay of various topoisomerases, but most importantly by DNA gyrase). Supercoiling is known to be critical for initiation of DNA replication but may also be important for the formation of a partition complex in contact with the cell membrane. Another factor is the presence of specific subdomains of the membrane which can interact specifically with origin DNA and possibly other regions involved in partitioning. Such domains may be induced transiently or be present at all times during the cell cycle.