Hiroo Yasukawa

Genetic and biochemical characterization of glutamyl endopeptidase of Staphylococcus warneri M

A Staphylococcus warneri strain M, newly isolated from processed seafood (smoked Watasenia scintillans), produced an extracellular protease. The protease, designated to as m-PROM (the mature form of PROM), selectively cleaved the carbonyl side of glutamic acid residues in beta-casein. Sequence of N-terminal 27 amino acids of m-PROM, RANVILPNNDRHQINDTTLGHYAPVTF, was found to be similar to those of other glutamyl endopeptidases, V8 protease (Staphylococcus aureus strain V8) and SPase (S. aureus ATCC 12600). To determine the complete primary structure and precursor of PROM, its gene (proM) was cloned and sequenced. The gene proM was found to encode for a protein of 316 amino acids. The amino acid residues from 64 to 90 completely coincided with the N-terminal 27 amino acids of the m-PROM, suggesting that the N-terminal 63 amino acids region of p-PROM (the precursor form of PROM) might be processed posttranslationally. Moreover, the whole amino acid sequence deduced from the primary structure of proM shows significant similarity to those of other glutamyl endopeptidases, V8 protease and SPase. These results suggested that PROM belongs to the glutamyl endopeptidase class. PROM, however, differs from V8 and SPase proteases in the processing site and the C-terminal region.

Two cell-counting factors regulate the aggregate size of the cellular slime mold Dictyostelium discoideum

Countin, a cell‐counting factor in Dictyostelium discoideum, is considered to limit the maximum size of the multicellular structure, because a countin null strain forms a huge fruiting body compared to that of the wild‐type. A novel gene, countin2, that is highly homologous to countin (40% identity in amino acid sequence) was identified in the D. discoideum genome. The countin2 null strain formed a 1.7‐fold higher number of the aggregates, resulting in smaller fruiting bodies compared with those of wild‐type cells. Thus, the Countin2 protein is thought to limit the minimum size of the multicellular structure. The size and number of aggregates formed by a mixture of countin null and countin2 null strains were the same as those of the wild‐type. These findings demonstrate that a combination of Countin and Countin2 proteins determines the appropriate size of the multicellular structure of D. discoideum.

Promoter/repressor system of Lactobacillus plantarum phage øg1e: characterization of the promoters pR49–pR–pL and overproduction of the Cro-like protein Cng in Escherichia coli

The Lactobacillus plantarum phage og1e (42259bp) has two repressor-like genes cng and cpg oriented oppositely, accompanied by three potential promoters pR, pL and pR49, and seven operator-like sequences (GATAC-boxes) (Kodaira et al., 1997). In this study, the og1e putative promoters were introduced into the Escherichia coli promoter-detecting plasmid pKK232-8. In E. coli CK111, pR (pKPR1), pL (pKPL1) and pR49 (pKPR49) exhibited distinct CAT activities. When pKPR1 or pKPL1 was coexistent with a compatible plasmid pACYC184 carrying pR-cng (pA4PRCN1), the CAT activity was decreased significantly. On the other hand, cng directed a protein (Cng) of 10.1 kDa in E. coli under the control of T7 promoter. Gel mobility-shift assays demonstrated that Cng binds specifically to a DNA region containing the GATAC-boxes. In addition, primer extension analyses demonstrated that the two sequences pR and pL act as a promoter in L. plantarum as well as in E. coli. These results suggested that the potential promoters pR and pL probably function for the lytic and lysogenic pathways, respectively, and Cng may act as a repressor presumably through the GATAC-boxes as operators.

Copper/zinc superoxide dismutases in Dictyostelium discoideum: amino acid sequences and expression kinetics.

Genes for Copper/Zinc superoxide dismutases, designated sodA and sodB, in the cellular slime mold Dictyostelium discoideum were analyzed. It was found that these gene products contain charged amino acid residues and hydrophobic stretches in their N-terminal regions, suggesting that they are extracellular Cu/Zn superoxide dismutases. The sodA and sodB are expressed in cells in the growth phase and throughout the developmental phases, suggesting that they are the housekeeping genes. The sodA is induced by H(2)O(2) exposure but not by UV irradiation, whereas sodB is induced both by H(2)O(2) exposure and UV irradiation. Such distinct kinetics of the expression patterns suggests that these enzymes play unique roles in D. discoideum.

Identification of the homolog of cell-counting factor in the cellular slime mold Dictyostelium discoideum

Genes for the cell-counting factors in Dictyostelium discoideum, countin and countin2, are considered to control the size of the multicellular structure of this organism. A novel gene, countin3, that is homologous to countin and countin2 genes (49 and 39% identity in amino acid sequence, respectively) was identified in the D. discoideum genome. The expression of countin3 was observed in the vegetatively growing cells, decreased in the aggregating stage, increased in the mid-developmental stage and decreased again in subsequent stages. This expression pattern is different from that of countin and countin2. The distinct expression kinetics of three genes suggests that they would have unique roles in size control of D. discoideum.

Endonuclease IV homolog from Dictyostelium discoideum: sequencing and functional expression in AP endonuclease-deficient Escherichia coli

We sequenced a gene encoding AP endonuclease DdAPN in Dictyostelium discoideum. The sequence predicts a protein of 542 amino acids, showing high homology to Escherichia coli Endonuclease IV (Endo IV). There is 45% identity to Endo IV using the C-terminal 282 amino acids of the Dictyostelium protein. The DdAPN conserves nine residues for the metal-binding identified in Endo IV. The truncated DdAPN protein containing these sites partially complemented E. coli RPC501 (xth(-), nfo(-)).

Myb‐binding site regulates the expression of glucosamine‐6‐phosphate isomerase in Dictyostelium discoideum

A homolog of the glucosamine‐6‐phosphate isomerase in the cellular slime mold Dictyostelium discoideum has been analyzed. The gene disruption mutant was arrested at the mound stage, demonstrating that the gene is important for development. The gene was expressed in vegetatively growing cells, silenced on starvation and expressed again in prestalk cells during the multicellular stages. The upstream region of the gene (1376 bp relative to ATG) was cloned and sequenced to study the transcription control mechanisms. Analysis of deletion mutants and a site‐directed mutant indicated that the Myb‐binding sequence (5′‐AACTG‐3′) localized in the upstream region is important for gene expression. The results of gel‐shift assays showed the presence of an Myb‐related protein binding to the sequence at the growing phase and another protein binding to the sequence at developmental stages.

FebA: a gene for eukaryotic translation initiation factor 4E-binding protein (4E-BP) in Dictyostelium discoideum.

We have identified a gene encoding a eukaryotic initiation factor 4E-binding protein (4E-BP) in the EST database of the Dictyostelium cDNA project. The Dictyostelium 4E-BP, designated febA (four e-binding), showed significant similarity to mammalian 4E-BPs. Northern blot analysis revealed that febA was expressed at a high level in the vegetative growth phase but the level of expression decreased during late development. The gene was shown to be non-essential since disruption of the gene had no severe effect; the null mutant proliferated normally and formed normal fruiting bodies. However, strains overexpressing the gene could not be established, suggesting that an excess of FebA protein may have a lethal effect on the cells.