Maintenance of the cellobiose utilization genes of Escherichia coli in a cryptic state

The genes for cellobiose utilization are normally cryptic in Escherichia coli. The cellobiose system was used as a model to understand the process by which silent genes are maintained in microbial populations. Previously reported was (1) the isolation of a mutant strain that expresses the cellobiose-utilization (Cel) genes and (2) that expression of those genes allows utilization of three beta- glucoside sugars: cellobiose, arbutin, and salicin. The Cel gene cluster has now been cloned from that mutant strain. In the course of locating the Cel genes within the cloned DNA segment, it was discovered that inactivation of the Cel-encoded hydrolase rendered the host strain sensitive to all three beta-glucosides as potent inhibitors. This sensitivity arises from the accumulation of the phosphorylated beta- glucosides. Because even the fully active genes conferred some degree of beta-glucoside sensitivity, the effects of cellobiose on a series of five Cel+ mutants of independent origin were investigated. Although each of those strains utilizes cellobiose as a sole carbon and energy source, cellobiose also acts as a potent inhibitor that reduces the growth rate on glycerol 2.5-16.5-fold. On the other hand, wild-type strains that cannot utilize cellobiose are not inhibited. The observation that the same compound can serve either as a nutrient or as an inhibitor suggests that, under most conditions in which cellobiose will be present together with other resources, there is a strong selective advantage to having the cryptic (Cel0) allele. In those environments in which cellobiose is the sole, or the best, resource, mutants that express the genes (Cel+) will have a strong selective advantage. It is suggested that temporal alternation between these two conditions is a major factor in the maintenance of these genes in E. coli populations. This alternation of environments and fitnesses was predicted by the model for cryptic-gene maintenance that was previously published.      

The effect of counterions on melittin aggregation.

Melittin, a surface-active polypeptide from bee venom, has an overall hydrophobic N-terminus, with basic residues clustered at the C-terminus. In aqueous solution melittin exists as a mixture of monomer and tetramer, the monomer adopting a predominantly random-coil configuration, whereas the tetramer is rich in alpha-helix. The tendency of melittin to aggregate is dependent on the counter-anions present in solution, the effect being most marked with phosphate, decreasing in the order HPO4(2-) greater than SO4(2-) greater than ClO4- greater than Cl-.     

Rho guanine nucleotide dissociation inhibitor protein (RhoGDI) inhibits exocytosis in mast cells.

Introducing non-hydrolysable analogues of GTP into the cytosolic compartment of mast cells results in exocytotic secretion through the activation of GTP binding proteins. The identity and mechanism of action of these proteins are not established. We have investigated the effects of Rho GDP dissociation inhibitor (RhoGDI) on exocytosis induced by guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) in rat mast cells, introducing the protein into cells by means of a patch pipette and recording the progress of exocytosis by monitoring cell capacitance. To allow time for the protein to enter the cells and find its correct location, stimulation was provided 5-10 min after patch rupture by photolysing caged GTP-gamma-S included in the pipette solution. When bovine RhoGDI was introduced into mast cells, exocytosis was inhibited at concentrations of 200-400 nM for native protein and 800 nM to 8 microM for the recombinant form. Protein denatured by heat or N-ethylmaleimide treatment did not inhibit. In permeabilized cells, recombinant RhoGDI increased the rate at which cells lose their ability to respond to GTP-gamma-S. These data demonstrate that one or more small GTP binding proteins of the Rho family has a central role in the exocytotic mechanism in mast cells.     

On alternatives to selection-induced mutation in the Bgl operon of Escherichia coli

Selection-induced mutations are nonrandom mutations that occur as specific and direct responses to environmental challenge. Examples of selection-induced mutations have been reported both in bacteria and in yeast. I previously showed (Hall 1988) that excisions of the mobile genetic element IS150 from within bglF are selection induced and argued that they occurred because they were potentially advantageous under the selective conditions employed. Mittler and Lenski (Mittler and Lenski 1992) have argued that such excisions are not selection induced but that they occur randomly in nondividing cells. Here I provide further evidence that IS150 excisions are induced by selection and that the excisions are immediately, rather than only potentially, advantageous to the cell. I also provide evidence that excisions, which Mittler and Lenski claim occur randomly in saturated broth cultures, actually occur after samples from those cultures are plated onto selective medium.      

SUMOylation stabilizes sister kinetochore biorientation to allow timely anaphase

During mitosis, sister chromatids attach to microtubules from opposite poles, called biorientation. Sister chromatid cohesion resists microtubule forces, generating tension, which provides the signal that biorientation has occurred. How tension silences the surveillance pathways that prevent cell cycle progression and correct erroneous kinetochore–microtubule attachments remains unclear. Here we show that SUMOylation dampens error correction to allow stable sister kinetochore biorientation and timely anaphase onset. The Siz1/Siz2 SUMO ligases modify the pericentromere-localized shugoshin (Sgo1) protein before its tension-dependent release from chromatin. Sgo1 SUMOylation reduces its binding to protein phosphatase 2A (PP2A), and weakening of this interaction is important for stable biorientation. Unstable biorientation in SUMO-deficient cells is associated with persistence of the chromosome passenger complex (CPC) at centromeres, and SUMOylation of CPC subunit Bir1 also contributes to timely anaphase onset. We propose that SUMOylation acts in a combinatorial manner to facilitate dismantling of the error correction machinery within pericentromeres and thereby sharpen the metaphase–anaphase transition.     

Wheat seed proteins exhibit a complex mechanism of protein elasticity

Elastomeric proteins are found in a number of animal tissues (elastin, abductin and resilin), where they have evolved to fulfil a range of biological functions. All exhibit rubber-like elasticity, undergoing deformation without rupture, storing the energy involved in deformation, and then recovering to their initial state when the stress is removed. The second part of the process is passive, entropy decreasing when the proteins are deformed, with the higher entropy of the relaxed state providing the driving force for recoil. In plants there is only one well-documented elastomeric protein system, the alcohol-soluble seed storage proteins (gluten) of wheat. The elastic properties of these proteins have no known biological role, the proteins acting as a store for the germinating seed. Here we show that the modulus of elasticity of a group of wheat gluten subunits, when cross-linked by gamma-radiation, is similar to that of the cross-linked polypentapeptide of elastin. However, thermoelasticity studies indicate that the mechanism of elastic recoil is different from elastin and other characterized protein elastomers. Elastomeric force, f, has two components, an internal energy component, f(e), and an entropic component, f(s). The ratio f(e)/f can be determined experimentally; if this ratio is less than 0.5 the elastomeric force is predominantly entropic in origin. The ratio was determined as 5.6 for the cross-linked high M(r) subunits of wheat glutenin and near zero for the cross-linked polypentapeptide of elastin. Tensile stress must be entropic or energetic in origin, the results would suggest that elastic recoil in the wheat gluten subunits, in part, may be associated with extensive hydrogen bonding within and between subunits and that entropic and energetic mechanisms contribute to the observed elasticity.     

Role of two residues proximal to the active site of Ubc9 in substrate recognition by the Ubc9.SUMO-1 thiolester complex

The small ubiquitin-like modifier SUMO-1 is covalently attached to lysine residues on target proteins by a specific conjugation pathway involving the E1 enzyme SAE1/SAE2 and the E2 enzyme Ubc9. In an ATP-dependent manner, the C-terminus of SUMO-1 forms consecutive thiolester bonds with cysteine residues in the SAE2 subunit and Ubc9, before the Ubc9.SUMO-1 thiolester complex catalyzes the formation of an isopeptide bond between SUMO-1 and the epsilon-amino group of the target lysine residue on the protein substrate. The SUMO-1 conjugation pathway bears many similarities with that of ubiquitin and other ubiquitin-like protein modifiers (Ubls), and because of its production of a singly conjugated substrate and the lack of absolute requirement in vitro for E3 enzymes, the SUMO-1/Ubc9 system is a good model for the analysis of protein conjugation pathways that share this basic chemistry. Here we describe methods of both steady-state and half-reaction kinetic analysis of Ubc9, and use these techniques to determine the role of two residues, Asp(100) and Lys(101) of Ubc9 which are not found in E2 enzymes from other protein conjugation pathways. These residues are found close to the active site Cys in the tertiary structure of Ubc9, and although they are shown to inhibit the transesterification reaction from SAE1/SAE2, they are important for substrate recognition in the context of the thiolester complex with SUMO-1.     

Structural characterization of a methionine-rich, emulsifying protein from sunflower seed

The 2 S seed storage protein, sunflower albumin 8, contains an unusually high proportion of hydrophobic residues including 16 methionines in a mature protein of 103 amino acids. A structural model, based on the known structure of a related protein, has been constructed as a four-helix bundle cross-linked by four disulphide bonds. This model structure is consistent with data from circular dichroism and nuclear magnetic resonance experiments. Analysis of the model's surface shows the presence of a large hydrophobic face that may be responsible for the highly stable emulsions this protein is known to form with oil/water mixtures.     

High-level expression of the Endo-beta-N-acetylglucosaminidase F2 gene in E.coli: one step purification to homogeneity

The Endo F2gene was overexpressed in E.coli as a fusion protein joined to the maltose-binding protein. MBP-Endo F2was found in a highly enriched state as insoluble, inactive inclusion bodies. Extraction of the inclusion bodies with 20% acetic acid followed by exhaustive dialysis rendered the fusion protein active and soluble. MBP-Endo F2was digested with Factor Xaand purified on Q-Sepharose. The enzyme was homogeneous by SDS-PAGE, and appeared as a single symmetrical peak on HPLC. Analysis of the amino-terminus demonstrated conclusively that recombinant Endo F2was homogeneous and identical to the native enzyme.