Construction of Mutant Genes for a Non-Toxic Verotoxin 2 Variant (VT2vp1) of Escherichia coli and Characterization of Purified Mutant Toxins

The gene encoding a Verotoxin 2 variant, VTvp1, was mutated by oligonucleotide-directed site-specific mutagenesis. Among 6 mutant toxins encoded by the mutated genes, E167Q-R170L (glutamic acid at position 167 and arginine at position 170 from N-terminus of the A subunit were replaced by glutamine and leucine, respectively) was found to have markedly decreased activities; inhibition of protein synthesis, Vero cell cytotoxicity and mouse lethality of the purified E167Q-R170L were 1/1,900, 1/125,000 and 1/2,000, respectively, of those of the purified wild-type VT2vp1. Since the antigenic property of the E167Q-R170L was demonstrated to be similar to that of the wild-type VT2vp1 by Ouchterlony double gel diffusion test and by neutralization test of Vero cell cytotoxicity of the VT2vp1, a possibility to use the mutant VT2vp1, E167Q-R170L, as a toxoid is discussed.     

Complementation of an Enterococcus hirae (Streptococcus faecalis) mutant in the alpha subunit of the H+-ATPase by cloned genes from the same and different species

We isolated an Enterococcus hirae (formerly Streptococcus faecalis) mutant, designated MS117, in which ‘G’ at position 301 of the alpha-subunit gene of the F₁F₀ type of H⁺-ATPase was deleted. MS117 had low H⁺-ATPase activity, was deficient in the regulatory system of cytoplasmic pH, and was unable to grow at pH6.0. When the alpha-subunit gene of E. hirae H⁺-ATPase was ligated with the shuttle vector pHY300PLK at the downstream region of the tet gene of the vector, it was expressed without its own promoter in MS117, and the mutation of MS117 was complemented; the mutant harbouring the plasmid had the ability to maintain a neutral cytoplasm and grew at pH6.0. We next transformed MS117 with pHY300PLK containing the alpha-subunit gene of Bacillus megaterium F₁F₀-ATPase constructed in the same way. The transformant grew at pH 6.0, and the ATP hydrolysis activity was recovered. These results suggested that an active hybrid H⁺-ATPase containing the B. megaterium alpha subunit was produced, and that the hybrid enzyme regulated the enterococcal cytoplasmic pH, although the function of the B. megaterium enzyme did not include pH regulation. Thus, our present results support the previous proposal that the enterococcal cytoplasmic pH is regulated by the F₁F₀ type of H⁺-ATPase.     

Effect of charge density of cationic polyelectrolytes on complex formation with DNA

The interaction between DNA and ionen polymers, -[N⁺(CH₃)₂(CH₂)_mN⁺(CH₃)₂(CH₂)_n], with m-n of 3–3, 6–6, and 6–10 were examined in order to know how the binding behavior of cationic polymers with DNA depends on the charge density of polycation. The ionen polymer has no bulky side chain and the binding forces with DNA would be attributed mainly to electrostatic interaction. When 3–3 ionen polymers were added to DNA solution, precipitable complexes with the ratio of cationic residue to DNA phosphate (+/?) of 1/1 and the free DNA molecules were segregated, while 6–6 and 6–10 ionen polymers formed soluble complexes with DNA molecules up to (+/?) = 0.5. This suggests that 3–3 ionen polymers bind cooperatively with DNA while 6–6 and 6–10 ionen polymers bind noncooperatively. The cooperative binding of 3–3 ionen polymer and the noncooperative binding of 6–6 ionen polymer were also supported by the thermal melting and recooling profiles from the midpoint between first and second meltings. It was concluded that the charge density of DNA phosphate is a critical value determining whether the ionen polymers bind to DNA by a cooperative or by a noncooperative binding, since the distance between successive cationic charges of 3–3 ionen polymer is shorter than that between successive phosphate charges on DNA double helix and those of 6–6 and 6–10 ionen polymers are longer.     

Kinetics of chemical modification of arginine and lysine residues in calf thymus histone H1

The arginine and lysine residues of calf thymus histone H1 were modified with large molar excesses of 2,3-butanedione and O-methylisourea, respectively. Kinetic study of the modification reaction of the arginine residue revealed that the reaction is divided into the two pseudo-first-order processes. About a third (1 Arg) of the total arginine residues of the H1 molecule was rapidly modified without causing any detectable structural change of the molecule, and the slow modification of the remaining arginine residues (2 Arg) led to a loss of the folded structure of H1. In the case of lysine residue modification, 93% (56 Lys) of the total lysine residues of the H1 was modified with the same rate constant, while 7% (4 Lys) of lysine residue remained unmodified. When the reaction was performed in the presence of 6M guanidine-HCl, all of lysine residues were modified. It is concluded that the 2 arginine and 4 lysine residues resistant to modification are buried in interior regions of the H1 molecule and play an important role in the formation of the H1 globular structure, while the other 1 arginine and 56 lysine residues are exposed to solvent.     

Influence of steric factors on oxygen binding. I. Studies on 2,4-diisopropyldeuteroheme-myoglobin.

Sperm whale apomyoglobin was recombined with 2,4-diisopropyldeuterohemin to form 2,4-diisopropyldeuteroheme-myoglobin and its various physico-chemical properties were investigated to get an insight into the structural and functional role of the peripheral vinyl groups. 2,4-Diisopropyldeuteroheme-myoglobin showed a four times lower oxygen affinity at 25 degrees C and larger enthalpy and entropy changes of oxygenation than the corresponding values of native myoglobin. 2,4-Diisopropyldeuteroheme-metmyoglobin shows a pKa value of 9.68 which is higher than those of native metmyoglobin and mesoheme-metmyoglobin. The rate of autooxidation of oxy-form was about seven times larger in 2,4-diisopropyldeuteroheme-myoglobin than in native myoglobin. The electron-donating effect of isopropyl groups does not give straightforward explanation for these anomalous properties of 2,4-diisopropyldeuteroheme-myoglobin. It is proposed that site and stereospecific van der Waals' interaction between the polypeptide side chains and the peripheral 2,4-diisopropyl groups may weaken the interaction between the bound oxygen molecule and the distal His, resulting in the decrease in the stability of oxyform.     

Structure and function of polyamine-amino acid antiporters CadB and PotE in Escherichia coli

The structure and function of a cadaverine-lysine antiporter CadB and a putrescine-ornithine antiporter PotE in Escherichia coli were evaluated using model structures based on the crystal structure of AdiC, an agmatine-arginine antiporter, and the activities of various CadB and PotE mutants. The central cavity of CadB, containing the substrate binding site, was wider than that of PotE, mirroring the different sizes of cadaverine and putrescine. The size of the central cavity of CadB and PotE was dependent on the angle of transmembrane helix 6 (TM6) against the periplasm. Tyr(73), Tyr(89), Tyr(90), Glu(204), Tyr(235), Asp(303), and Tyr(423) of CadB, and Cys(62), Trp(201), Glu(207), Trp(292), and Tyr(425) of PotE were strongly involved in the antiport activities. In addition, Trp(43), Tyr(57), Tyr(107), Tyr(366), and Tyr(368) of CadB were involved preferentially in cadaverine uptake at neutral pH, while only Tyr(90) of PotE was involved preferentially in putrescine uptake. The results indicate that the central cavity of CadB consists of TMs 2, 3, 6, 7, 8, and 10, and that of PotE consists of TMs 2, 3, 6, and 8. These results also suggest that several amino acid residues are necessary for recognition of cadaverine in the periplasm because the level of cadaverine is much lower than that of putrescine in the periplasm at neutral pH. All the amino acid residues identified as being strongly involved in both the antiport and uptake activities were located on the surface of the transport path consisting of the central cavity and TM12.