Homeoviscous adaptation, growth rate, and morphogenesis in bacteria.

Fluorescence polarization, P, of 1,6-diphenyl-1,3,5-hexatriene was studied in Escherichia coli B/r. Modification of nutritional conditions was not compensated by homeoviscous adaptation, demonstrated to exist for temperature variations. Cell diameter, which is known also to vary with nutrition but not with temperature, was found to be positively correlated with 1/P, and may therefore be regulated by membrane lipid order and fluidity.     

Replication forks of Escherichia coli are not the preferred sites for lysogenic integration of bacteriophage Mu.

The question of whether bacteriophage Mu prefers replication forks for lysogenic integration into Escherichia coli chromosomes was tested by using two different systems. In the first, inactivation of genes was scored in synchronized cultures infected by Mu at various times. No increase in the mutation frequency of a gene was found after infection at the time of its replication. In the second, the composition of colonies formed by bacteria lysogenized by Mu was determined; the newly formed lysogens should give rise to mixed colonies (containing lysogenized as well as nonlysogenized bacteria), uniform colonies, or both, depending on the mode of integration. Both types of colonies were found, and the fraction of uniform colonies was proportional to the relative length of the unreplicated segment of an average chromosome in the culture. The results in both systems clearly preclude the possibility that a lysogenizing Mu integrates with high preference at the chromosome replication forks.     

Mosquito larvicidal activity of Escherichia coli with combinations of genes from Bacillus thuringiensis subsp. israelensis.

The genes cryIVA and cryIVD, encoding 134- and 72-kDa proteins, respectively, and the gene for a regulatory 20-kDa polypeptide of Bacillus thuringiensis subsp. israelensis (serovar H14) were cloned in all seven possible combinations by the Escherichia coli expression vectors pT7 and pUHE. The four combinations containing cryIVA (cryIVA alone, with cryIVD, with the 20-kDa-protein gene, and with both) displayed high levels of mosquito larvicidal activity in pUHE. The toxicity of the combination of cryIVA and cryIVD, with or without the 20-kDa-protein gene, was higher than has ever been achieved with delta-endotoxin genes in recombinant E. coli. Fifty percent lethal concentrations against third-instar Aedes aegypti larvae for these clones decreased (i.e., toxicity increased) continuously to about 3 x 10(5) cells ml-1 after 4 h of induction. Larvicidal activities, obtained after 30 min of induction, were lower for clones in pT7 and decreased for an additional 3.5 h. Induction of either cryIVD or the 20-kDa-protein gene alone resulted in no larvicidal activity in either pT7 or pUHE20. Cloned together, these genes were slightly toxic in pT7 but not in pUHE20. Five minutes of induction of this combination (cryIVD with the 20-kDa-protein gene) in pT7 yielded a maximal mortality of about 40%, which decreased rapidly and disappeared completely after 50 min. CryIVD is thus apparently degraded in E. coli and partially stabilized by the 20-kDa regulatory protein. Larvicidal activity of the combination of cryIVA and cryIVD was sevenfold higher than that of cryIVA alone, probably because of the cross-stabilization of the polypeptides or the synergism between their activities.     

Rate maintenance of cell division in Escherichia coli B/r: analysis of a simple nutritional shift-down.

A competitive (nonmetabolizable) inhibitor of glucose uptake, alpha-methylglucoside, was used to limit the growth of Escherichia coli. Cell division during such a nutritional shift-down was studied in batch cultures and with the "baby-machine" technique. Following a brief delay, the rate of division was maintained for 60 to 70 min in batch cultures and for an extended period in the baby machine. Decreases in cell size were due, in part, to a possible reduction in the mass per chromosome origin at the time of replication initiation and a shorter time interval between initiation and the subsequent division. These unusual findings suggest that this method for abrupt change in growth rate without modifying repression patterns is useful for studying the control of various aspects of the bacterial cell.     

Deviation from homeoviscous adaptation in Escherichia coli membranes.

The process by which an organism changes the composition of its membranal fatty acids in response to growth temperature, so as to maintain optimal membrane functioning, is known as homeoviscous adaptation (HA). One expression of HA is the constancy of the fluorescence polarization (P) of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene (DPH) in membranes of cells grown at various temperatures. The P of DPH in the membranes of Escherichia coli was shown by us to be inversely proportional to bacterial growth rate on different carbon sources. This result, implying failure of HA, is now complemented by measurements of DPH lifetimes, which indicate that the dominant variables contributing to the drop in P are (a) the order parameter of the membrane, which goes down, and (b) the fluidity, which may slightly increase. These are then the changes induced by enhanced growth rate. Two additional effects, cell membrane permeability and sensitivity to thermal shock, determined by the diffusion of o-nitrophenylgalactoside (ONPG) and by exposure to 52 degrees C, respectively, are reported to increase with growth rate. We can now conclude that there is a deviation from the principle of HA in E. coli grown at various rates, brought about by controlling the growth media at constant temperatures.     

Measurement of membrane potential inBacillus subtilis: A comparison of lipophilic cations,rubidium ion,and a cyanine dye as probes

Summary Two of the commonly used probes for measuring membrane potential—lipophilic cations and the cyanine dye diS-C₃(5)—indicated nominally opposite results when tetraphenylarsonium ion was added as a drug to suspensions of metabolizingBacillus subtilis cells. [³H]-Triphenylmethylphosphonium uptake was enhanced by the addition, indicating hyperpolarization, yet fluorescence of diS-C₃(5) was also enhanced, indicating depolarization. Evidence is presented that both effects are artifactual, and can occur without any change in membrane potential, as estimated by⁸⁶Rb⁺ uptake in the presence of valinomycin. The fluorescence studies suggest that tetraphenylarsonium ion displaces the cyanine dye from the cell envelope, or other binding site, into the aqueous phase.The uptake characteristics of the radiolabeled lipophilic cations were quite unusual: At low concentrations (e.g., less than 10 m for triphenylmethylphosphonium) there was potential-dependent uptake of the label to a stable level, but subsequent addition of nonradioactive lipophilic cation caused further uptake of label to a new stable level. Labeled triphenylmethylphosphonium ion taken up to the first stable level could be displaced by 10mm magnesium ion, whereas⁸⁶Rb⁺ uptake was unperturbed. Association of the lipophilic cations with the surface of de-energized cells was concentration-dependent, but there was no evidence for cooperative binding. This phenomenon of stimulated uptake inB. subtilis (which was not seen inEscherichia coli cells or vesicles) is consistent with a two-compartment model with access to the second compartment only being possible above a critical cation concentration. We tentatively propose such a model, in which these compartments are the cell surface and the cytoplasm, respectively.Triphenylmethylphosphonium up to 0.5mm exhibited linear binding to de-energized cells; binding of tetraphenylphosphonium and tetraphenylarsonium was nonlinear but was not saturated at the highest concentration tested (1mm). The usual assumption, that association of the cation with cell surfaces is saturated and so can be estimated on de-energized cells, therefore leads to undercorrected estimates of cytoplasmic uptake inB. subtilis, and hence to overestimates of membrane potential. We describe a more realistic procedure, in which the estimate of extent of binding is based on a mean aqueous concentration related both to the external concentration and to the much higher internal concentration that exists in energized cells. Using this procedure we estimate the membrane potential inB. subtilis to be 120 mV, inside-negative. The procedure is of general applicability, and should yield more accurate estimates of membrane potential in any system where there is significant potential-dependent binding.Work performed while on sabbatical leave from Department of Biology, Ben-Gurion University of the Negev, Beer-Sheva, Israel.     

DNA synthesis in Escherichia coli during a nutritional shift-up

Summary DNA synthesis in a thymine-requiring Escherichia coli K12 strain was studied by exploiting deoxyguanosine, so simulating the behaviour of Thy⁺ strains. DNA synthesis is inhibited during the first 25 min after a nutritional shift-up. The new DNA/mass is lower than that predicted by current models for initiation control.Dedicated to the memory of Shmuel Zabrovitz, whose high spirits and good humor enabled him to complete the work while struggling with his lethal diseaseDeceased     

Crystal structure studies and inhibition kinetics of tripeptide chloromethyl ketone inhibitors with Streptomyces griseus protease B

The bacterial serine protease, SGPB, was inhibited by two specific tripeptide chloromethyl ketones, N-t-butyloxycarbonyl-l-alanylglycyl-l-phenylalanine chloromethyl ketone (BocAGFCK) and N-t-butyloxycarbonyl-glycyl-l-leucyl-l-phenylalanine chloromethyl ketone (BocGLFCK). Crystals of the inhibited complexes were grown and examined by X-ray crystallographic methods. The peptide backbone of each inhibitor is bound by three hydrogen bonds to the main chain of residues Ser214 to Gly216. There are two well-characterized hydrophobic pockets, S₁ and S₂, on the surface of SGPB which accommodate the P₁ and P₂ side-chains of the BocGLFCK inhibitor. A conformational change of Tyr171 is induced by the binding of this inhibitor. Both inhibitors make two covalent bonds to the SGPB enzyme. The imidazole ring of His57 is alkylated at the N^?2 atom and O^γ of Ser195 forms a hemiketal bond with the carbonyl-carbon atom of the inhibitor. Comparison of the binding modes of the two tripeptides in conjunction with the differences in their inhibition constants (K_I) allows one to estimate the binding energy of the leucyl side-chain as ?2.6 kcal mol^?1. The importance of an electrophilic component in the serine protease mechanism, which involves the polarization of the susceptible carbonyl bond of a substrate or inhibitor by the peptide NH groups of Gly193 and Ser195 is discussed.     

162 Dominant forces in protein folding

In the first part of this talk, I will discuss the need for a paradigm shift from hydrophobic (HφO) to a hydrophilic ((HφI) based theory of protein folding. Next, I will discuss the various types of solvent-induced forces that are exerted on various groups on the protein. It is argued, both theoretically and by simulations, that the HφI–HφI solvent-induced forces are likely to be the strongest. Therefore, it is suggested that these forces are also the forces that force the protein to fold, in a short time, along a narrow range of pathways. This paradigm shift also answers Levinthal’s question about the factors that “speed” and “guide” the folding of proteins.