The feasibility of obtaining useful interspecific hybrids with high cellulolytic activities by breeding was investigated by a protoplast fusion technique using the citric acid hyper-producer, Aspergillus kawachii, and the cellulase hyper-producer, Aspergillus niger. Interspecific hybridization between two auxotrophic mutants of Aspergillus kawachii (his) and A. niger (met) was achieved by protoplast fusion in the presence of 35% polyethylene glycol as fusogen. Stable diploid strains were obtained from a heterokaryon strain by treatment with 0.1% d-camphor. The selected strains were specified by their conidial size and DNA content as well as the conidial germination. One of them showed high cellulase productivity besides the same level of amylase production and sporulation as that of A. kawachii, a higher level of citric acid and the lowering of mash viscosity than that of A. kawachii. From these results it is suggested that this diploid strain would be very useful for the degradation of cellulosic substances in the mash from shochu fermentation, consequently for its ready disposal thereafter.
A Bacillus subtilis germination gene gerK that is essential for spore's response to glucose has been cloned and sequenced. An ORF was found encoding a polypeptide comprising 407 amino acids that is considerably homologous with the product of gerAC, the third gene of the gerA operon. Neither promoter-like nor terminator-like sequence was found suggesting that the gerK also may be a constituent of an operon.
The primer protein (PP) and DNA polymerase (Pol) of bacteriophage M2, both of which are essential for the protein-primed replication of the genome, were purified from Escherichia coil cells that harbored the recombinant plasmid pIKM23, on which the genes for PP and Pol are under the control of the tac promoter. The purified PP and Pol formed a heterodimeric complex at a molar ratio of 1 to 1. Maximal activity of Pol for M2 DNA replication was attained when an equimolecular amount of PP, relative to that of Pol, was added to the replication system in vitro. When excess amount of PP was added, however, the replication activity was reduced. The activity of Pol for the priming reaction in M2 DNA replication was not reduced even by the presence of a five-fold molar excess of PP over Pol. By contrast, the activities for DNA chain elongation with templates of primed M2 DNA and with poly(dA): oligo (dT) were inhibited by 70% and by 80%, respectively, on the addition of PP in an equimolecular amount to that of Pol. Therefore, the reduction in M2 DNA replication appears to be caused by an inhibitory effect of PP on Pol in the elongation process.
Fourteen strains belonging to the genera Rhizobium, Bradyrhizobium and Agrobacterium were investigated for susceptibility to infection by a lysogenic phage (phage φU) from Rhizobium leguminosarum biovar trifolii. All strains tested were resistant to phage φU infection; however, the phage was able to adsorb to R. leguminosarum biovar viciae strain K5. Plasmid pCI6 carrying the attachment site (attP) of phage φU was introduced into all these strains by conjugation. The tetracycline resistance (Tcr) gene on pCI6 was used as a selectable marker. When R. leguminosarum biovar viciae strain K5 was used as a recipient, Tcr transconjugants appeared at high frequency (4.41×10-3). Southern hybridization with the attP fragment of phage φU indicates that pCI6 is integrated into the chromosome of 10 transconjugants. Furthermore, the restriction map of pCI6 integration region on the chromosome of strain K5 was shown to be identical to that of R. leguminosarum biovar trifolii strain 4S which was the original host strain of phage φU.
The possible participation of cyclic AMP (cAMP) in osmoregulation during the initial stages of salt stress was examined in cells of a salt-tolerant yeast, Zygosaccharomyces rouxii. When the cells were exposed to a medium with 1M NaCl, levels of cAMP reached a maximum within 10 min and then declined. The maximum level was about 4.6 times higher than the initial intracellular level, measured when the cells were inoculated into the medium. Adenylate cyclase (AC) activity, which catalyzes the synthesis of cAMP from ATP, increased immediately after exposure of cells to medium with 1M NaCl, reached a maximum of 4.43 pmol cAMP produced/min•mg protein at 15min and then decreased. The activity of phosphodiesterase (PDE), which catalyzes the conversion of cAMP to 5′-AMP, increased slowly after salt stress and reached a maximum at 25min. The increase in PDE activity resulted in a decrease in the cAMP content of cells. These observations indicate that the levels of cAMP during salt stress are controlled by the activation and inactivation of the two enzymes. When GTPγS was added at 10μM to medium with 1M NaCl, AC activity increased to about 2.5 times that in the case of medium with 1M NaCl but without GTPγS. This result suggests that AC activity may be regulated by a GTP-binding regulatory protein (G-protein) during salt stress. When 2-deoxy-D-glucose (DG), an inhibitor of AC, was added at 8mM to the medium with 1M NaCl, synthesis of cAMP and AC activity were completely inhibited. Both the accumulation of glycerol and activation of the plasma membrane ATPase, induced by salt stress, were simultaneously inhibited in the presence of DG. This result indicates that the increase in the cAMP content of cells was related to the intracellular accumulation of glycerol and to activation of a plasma membrane ATPase during salt stress. These results together suggest that cAMP synthesized under regulatory control exerted by a G-protein may function as a second messenger in osmoregulation in response to salt stress.