The protein capsid of filamentous bacteriophage PH75 from Thermus thermophilus.

The PH75 strain of filamentous bacteriophage (Inovirus) grows in the thermophilic bacterium Thermus thermophilus at 70 degrees C. We have characterized the viral DNA and determined the amino acid sequence of the major coat protein, p8. The p8 protein is synthesized without a leader sequence, like that of bacteriophage Pf3 but unlike that of bacteriophage Pf1, both of which grow in the mesophile Pseudomonas aeruginosa. X-ray diffraction patterns from ordered fibres of the PH75 virion are similar to those from bacteriophages Pf1 and Pf3, indicating that the protein capsid of the PH75 virion has the same helix symmetry and subunit shape, even though the primary structures of the major coat proteins are quite different and the virions assemble at very different temperatures. We have used this information to build a molecular model of the PH75 protein capsid based on that of Pf1, and refined the model by simulated annealing, using fibre diffraction data extending to 2.4 A resolution in the meridional direction and to 3.1 A resolution in the equatorial direction. The common design may reflect a fundamental motif of alpha-helix packing, although differences exist in the DNA packaging and in the means of insertion of the major coat protein of these filamentous bacteriophages into the membrane of the host bacterial cell. These may reflect differences in the assembly mechanisms of the virions.

A novel covalent modification of nitrogenase in a cyanobacterium.

In extracts of the unicellular cyanobacterium Gloeothece, the Fe-protein of nitrogenase can be separated by SDS-PAGE into two antigenically identifiable components. Unlike the situation in photosynthetic bacteria such as Rhodospirillum rubrum, these two forms do not arise from covalent modification of the protein by ADP-ribosylation. Rather, the Fe-protein of Gloeothece nitrogenase is subjected to modification by palmitoylation.

The use of nickel to probe the role of hydrogen metabolism in cyanobacterial nitrogen fixation.

The hydrogenase activities of the heterocystous cyanobacteria Anabaena cylindrica and Mastigocladus laminosus are nickel dependent, based on their inability to consume hydrogen with various electron acceptors or produce hydrogen with dithionite-reduced methyl viologen, after growth in nickel-depleted medium. Upon addition of nickel ions to nickel-deficient cultures of A. cylindrica, the hydrogenase activity recovered in a manner which was protein synthesis-dependent, the recovery being inhibited by chloramphenicol. We have used the nickel dependence of the hydrogenase as a probe of the possible roles of H2 consumption in enhancing nitrogen fixation, and particularly for protecting nitrogenase against oxygen inhibition. Although at the usual growth temperatures (25 degrees for A. cylindrica and 40 degrees for M. laminosus), the cells consume H2 vigorously in an oxyhydrogen reaction after growth in the presence of nickel ions, we have not found that the reaction confers any significant additional protection of nitrogenase, either at aerobic pO2 (for both organisms) or at elevated pO2 (for A. cylindrica). However, at elevated temperatures (e.g., 40 degrees for A. cylindrica and 48 degrees for M. laminosus) a definite protective effect was observed. At these temperatures both organisms rapidly lost acetylene reduction activity under aerobic conditions. When hydrogen gas (10%) was present, the cells retained approximately 50% of the nitrogenase activity observed under anaerobic conditions (argon gas phase). No such protection by hydrogen gas was observed with nickel-deficient cells. Studies with cell-free extracts of A. cylindrica showed that the predominant effect of temperature was not due to thermal inactivation of nitrogenase.

Isozymes of alpha-galactosidase from Bacillus stearothermophilus.

Two molecular forms of alpha-galactosidase (EC 3.2.1.22) synthesized constitutively by Bacillus stearothermophilus, strain AT-7, have been purified. alpha-Galactosidase I (with the substrate p-nitrophenyl alpha-D-galactopyranoside (PNPG)) has a pH optimum of 6 and half-life at 65 degrees C of > 2 h at low protein concentration. alpha-Galactosidase II has a pH optimum of 7 with PNPG and a half-life at 65 degrees C of about 3 min. The isozymes also differ with respect to their Km with PNPG and melibiose. Both enzymes are inhibited competitively by D-galactose, melibiose, and Tris. With the beta-glycosides cellobiose and lactose either noncompetitive or mixed-type inhibition is observed, with the pattern dependent on both the pH and the isozyme. The two isozymes have similar Arrhenius activation energies (about 20 kcal/mol, 1 kcal = 4.184 kJ). Their molecular weights, estimated by disc gel electrophoresis, are alpha-galactosidase I, 280 000 +/- 30 000 and alpha-galactosidase II, 325 000 +/- 15 000. Dodecyl sulfate gel electrophoresis gave a single band for each enzyme. The respective molecular weights, 81 000 +/- 500 for alpha-galactosidase I and 84 000 +/- 500 for alpha-galactosidase II, suggest that both enzymes consist of four subunits.

beta-Galactosidase from Bacillus stearothermophilus.

Several strains of thermophilic aerobic spore-forming bacilli synthesize beta-galactosidase (EC 3.2.1.23) constitutively. The constitutivity is apparently not the result of a temperature-sensitive repressor. The beta-galactosidase from one strain, investigated in cell-free extracts, has a pH optimum between 6.0 and 6.4 and a very sharp pH dependence on the acid side of its optimum. The optimum temperature for this enzyme is 65 degrees C and the Arrhenius activation energy is about 24 kcal/mol below 47 degrees C and 16 kcal/mol above that temperature. At 55 degrees C the Km is 0.11 M for lactose and 9.8 X 10(-3) M for 9-nitrophenyl-beta-D-galactopyranoside. The enzyme is strongly product-inhibited by galactose (Ki equals 2.5 X 10(-3) M). It is relatively stable at 50 degrees C, losing only half of its activity after 20 days at this temperature. At 60 degrees C more than 60% of the activity is lost in 10 min. However, the enzyme is protected somewhat against thermal inactivation by protein, and in the presence of 4 mg/ml of bovine serum albumin the enzyme is only 18% inactivated in 10 min at 60 degrees C. Its molecular weight, estimated by disc gel electrophoresis, is 215 000.