Molecular cloning and nucleotide sequence of a gene for alkaline cellulase from Bacillus sp. KSM-635

A gene for alkaline cellulase from the alkalophilic Bacillus sp. KSM-635 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. Although the recombinant plasmid contained two HindIII inserts of 2.6 kb and 4.0 kb, the inserts were found to be contiguous in the Bacillus genome by hybridization analysis. Nucleotide sequences of a 2.4 kb region which was indispensable for the production of cellulase, and the flanking, 1.1 kb region, were determined. There was an open reading frame (ORF) of 2823 bp in the 3498 bp sequence determined, which encoded 941 amino acid residues. Two putative ribosome-binding sites and a sigma 43-type, promoter-like sequence were found upstream from an initiation codon in the ORF. The deduced amino-terminal sequence resembles the signal peptide of extracellular proteins. A region of amino acids, 249 to 568, of the deduced amino acid sequence of the cellulase from this organism is homologous with those of alkaline and neutral enzymes of other micro-organisms, but nine amino acid residues were found to be conserved only in the alkaline enzymes.     

Synthetic inositol trisphosphate analogs and their effects on phosphatase, kinase, and the release of Ca2+

A series of inositol 1,4,5-trisphosphate (IP3) analogs and positional isomers was examined to explore the structure-activity relationships among IP3 5-phosphatase, IP3 3-kinase, and the release of Ca2+. All analogs with additional groups on the 2nd position of IP3 inhibited the hydrolysis of [5-32P]IP3 catalyzed by erythrocyte ghosts, with a lower Ki value than seen with IP3. IP3 dehydroxylated at the 2nd position also had a lower Ki, while 2,4,5-IP3 or cyclic(1:2), 4,5-IP3 had higher Ki values. Among these compounds 2-deoxy-IP3 was as potent as IP3 in inhibiting the phosphorylation by [3H] IP3-3-kinase in rat brain cytosol. The other compounds, except for 2,4,5-IP3 inhibited the phosphorylation, however, 2-30 times higher concentrations were required. By lowering free Ca2+, the concentrations required for half-maximal inhibition were low, while those of IP3, 2-deoxy-IP3, and positional isomers remained unchanged. These compounds acted as full agonists in releasing Ca2+ from permeabilized macrophages, although 1.6-50-fold higher concentrations than IP3 were required. These compounds also inhibited the binding of [3H]IP3 to rat cerebellum and bovine adrenal cortex microsomes, but the potencies were 2.9-33 times less than that of IP3. Thus, the 2nd position of IP3 can be modified with only a slight loss of biological activity.     

Functional mapping of amino acid residues responsible for the antibacterial action of apidaecin.

Functional mapping was carried out to address the amino acid residues responsible for the activity of the antibacterial peptide apidaecin from the honeybee by an in vivo assay system developed previously. The C-terminal region and many of the proline and arginine residues which are present at high frequency in apidaecin were found to play an important role in its antibacterial activity.     

Comparative studies of genes encoding thermostable L-2-halo acid dehalogenase from Pseudomonas sp. strain YL, other dehalogenases, and two related hypothetical proteins from Escherichia coli.

We have determined the nucleotide sequence of the gene encoding thermostable L-2-halo acid dehalogenase (L-DEX) from the 2-chloroacrylate-utilizable bacterium Pseudomonas sp. strain YL. The open reading frame consists of 696 nucleotides corresponding to 232 amino acid residues. The protein molecular weight was estimated to be 26,179, which was in good agreement with the subunit molecular weight of the enzyme. The gene was efficiently expressed in the recombinant Escherichia coli cells: the amount of L-DEX corresponds to about 49% of the total soluble proteins. The predicted amino acid sequence showed a high level of similarity to those of L-DEXs from other bacterial strains and haloacetate dehalogenase H-2 from Moraxella sp. strain B (38 to 57% identity) but a very low level of similarity to those of haloacetate dehalogenase H-1 from Moraxella sp. strain B (10%) and haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (12%). By searching the protein amino acid sequence database, we found two E. coli hypothetical proteins similar to the Pseudomonas sp. strain YL L-DEX (21 to 22%).     

Identification and inter-relationship analysis of Bradyrhizobium japonicum strains by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD)

Genomic DNA of 13 Bradyrhizobium japonicum strains was prepared and analysed by restriction fragment length polymorphism (RFLP) with nif and nod probes, and by random amplified polymorphic DNA (RAPD) with 11 primers of arbitrary nucleotide sequence. Polymorphism was observed in both analyses. The RFLP and RAPD banding patterns of different strains were used to calculate genetic divergence and to construct phylogenetic trees, allowing studies on the relationships between the strains. RFLP with nif and nod probes permitted the separation of the strains into two divergent groups, whereas RAPD separated them into four main groups. RAPD allowed closely related strains to be distinguished.     

Phenylalanine production by metabolically engineered Corynebacterium glutamicum with the pheA gene of Escherichia coli

The bifunctional enzyme chorismate mutase (CM)-prephenate dehydratase (PD), which is encoded by the pheA gene of Escherichia coli, catalyses the two consecutive key steps in phenylalanine biosynthesis. To utilize the enzyme for metabolic engineering of phenylalanine-producing Corynebacterium glutamicum KY10694, the intact gene was cloned on a multicopy vector to yield pEA11. C. glutamicum cells transformed with pEA11 exhibited a more than tenfold increase in CM and PD activities relative to the host cells. Moreover, the level of pheA expression was further elevated a fewfold when cells were starved of phenylalanine, suggesting that the attenuation regulation of pheA expression functions in heterogeneous C. glutanicum. Plasmid pEA11 encoding the wild-type enzyme was mutated to yield pEA22, which specified CM-PD exhibiting almost complete resistance to end-product inhibition. When pEA22 was introduced into KY10694, both the activities of CM and PD were highly maintained throughout the cultivation, thus leading to a 35% increased production (23 g/l) of phenylalanine.     

Analysis of feeding mechanism in a pitcher ofNepenthes hybrida

The process of digestion of captured feeds in a pitcher, an insect-trapping organ, ofNepenthes was studied. Changes in bacterial population, pH and NH₄ ⁺ concentrations in pitcher juice were examined. Strong activities of both acid- and alkaline phosphatase, phosphoamidase, esterase C4 and esterase C8 were found in the pitcher juice. Optimum pH of proteases in the juice and those secreted from bacteria showed pH 3.0 and pH 8.0–9.0, respectively. Twenty six strains of bacteria were isolated from 4 pitchers: 10 strains were gram positive, 16 strains were gram negative (10 strains had casein hydrolase activity). A proton excretion was induced by NH₄ ⁺ released from the added solutions, and accordingly, the pH of the solutions fell. As a simulation model of the digestion process of feeds in pitcher juice and polypeptone solution was added into the washed pitcher. A good correlation was found among the NH₄ ⁺ concentration, pH and bacterial cell titer.