Isolation and characterization of the genes encoding two metalloproteases (MprI and MprII) from a marine bacterium, Alteromonas sp. strain O-7

An extracellular alkaline metalloprotease (MprI) from Alteromonas sp. strain O-7 was purified and characterized. The molecular mass of the purified enzyme was estimated to be 56 kDa by SDS-PAGE. The optimum pH and temperature were pH 10.0 and 60 degrees C, respectively. The gene (mprI) encoding MprI was cloned and its nucleotide sequence was analyzed. The deduced amino acid sequence of MprI showed significant similarity to metalloproteases classified into the thermolysin family. Furthermore, sequence analysis showed that another metalloprotease (MprII)-encoding gene was located downstream from mprI. The deduced amino acid sequence of MprII showed high similarity to metalloproteases of the aminopeptidase family. Similar repeated C-terminal extensions were found in both MprI and MprII.     

Characterization of Chitinase Genes from an Alkaliphilic Actinomycete, Nocardiopsis prasina OPC-131

An alkaliphilic actinomycete, Nocardiopsis prasina OPC-131, secretes chitinases, ChiA, ChiB, and ChiBΔ, in the presence of chitin. The genes encoding ChiA and ChiB were cloned and sequenced. The open reading frame (ORF) of chiA encoded a protein of 336 amino acids with a calculated molecular mass of 35,257 Da. ChiA consisted of only a catalytic domain and showed a significant homology with family 18 chitinases. The chiB ORF encoded a protein of 296 amino acids with a calculated molecular mass of 31,500 Da. ChiB is a modular enzyme consisting of a chitin-binding domain type 3 (ChtBD type 3) and a catalytic domain. The catalytic domain of ChiB showed significant similarity to Streptomyces family 19 chitinases. ChiBΔ was the truncated form of ChiB lacking ChtBD type 3. Expression plasmids coding for ChiA, ChiB, and ChiBΔ were constructed to investigate the biochemical properties of these recombinant proteins. These enzymes showed pHs and temperature optima similar to those of native enzymes. ChiB showed more efficient hydrolysis of chitin and stronger antifungal activity than ChiBΔ, indicating that the ChtBD type 3 of ChiB plays an important role in the efficient hydrolysis of chitin and in antifungal activity. Furthermore, the finding of family 19 chitinase in N. prasina OPC-131 suggests that family 19 chitinases are distributed widely in actinomycetes other than the genus Streptomyces.     

Structure-activity relationship and biological property of cortistatins, anti-angiogenic spongean steroidal alkaloids

Previously, bioassay-guided separation led us to isolate eleven novel steroidal alkaloids named cortistatins from the marine sponge Corticium simplex. These cortistatins were classified into three types based on the chemical structure of the side chain part, that is, isoquinoline, N-methyl piperidine or 3-methylpyridine units. From the structure-activity relationship study, the isoquinoline unit in the side chain was found to be crucial for the anti-angiogenic activity of cortistatins. Cortistatin A (1) showed cytostatic growth-inhibitory activity against human umbilical vein endothelial cells (HUVECs). Cortistatin A (1) also inhibited VEGF-induced migration of HUVECs and bFGF-induced tubular formation. Although cortistatin A (1) showed no effect on VEGF-induced phosphorylation of ERK1/2 and p38, which are one of the signaling pathways for migration and tubular formation, the phosphorylation of the unidentified 110kDa protein in HUVECs was inhibited by the treatment with cortistatin A.     

Construction of a new cloning vector utilizing a cryptic plasmid and the highly expressed melanin-synthesizing gene operon from Streptomyces castaneoglobisporus

Streptomyces castaneoglobisporus HUT6202 overproduces a diffusible melanin pigment and harbors a cryptic 7.4-kb plasmid, pHY6202. We constructed a Streptomyces cloning vector, pSY10CMM, consisting of the HUT6202 rep gene, the thiostrepton resistance gene and an operon encoding the synthesis of melanin pigment, abbreviated mel, from S. castaneoglobisporus. The vector, which has SphI and BamHI sites as cloning sites with insertional inactivation of the mel, is a more convenient cloning vector than commonly used pIJ702, because of its broad host range for antibiotic-producing Streptomyces strains and its much greater production of diffusible melanin pigment.     

Characterization of chitinase genes from an alkaliphilic actinomycete,Nocardiopsis prasina OPC-131

An alkaliphilic actinomycete, Nocardiopsis prasina OPC-131, secretes chitinases, ChiA, ChiB, and ChiB Delta, in the presence of chitin. The genes encoding ChiA and ChiB were cloned and sequenced. The open reading frame (ORF) of chiA encoded a protein of 336 amino acids with a calculated molecular mass of 35,257 Da. ChiA consisted of only a catalytic domain and showed a significant homology with family 18 chitinases. The chiB ORF encoded a protein of 296 amino acids with a calculated molecular mass of 31,500 Da. ChiB is a modular enzyme consisting of a chitin-binding domain type 3 (ChtBD type 3) and a catalytic domain. The catalytic domain of ChiB showed significant similarity to Streptomyces family 19 chitinases. ChiB Delta was the truncated form of ChiB lacking ChtBD type 3. Expression plasmids coding for ChiA, ChiB, and ChiB Delta were constructed to investigate the biochemical properties of these recombinant proteins. These enzymes showed pHs and temperature optima similar to those of native enzymes. ChiB showed more efficient hydrolysis of chitin and stronger antifungal activity than ChiB Delta, indicating that the ChtBD type 3 of ChiB plays an important role in the efficient hydrolysis of chitin and in antifungal activity. Furthermore, the finding of family 19 chitinase in N. prasina OPC-131 suggests that family 19 chitinases are distributed widely in actinomycetes other than the genus Streptomyces.     

IutB participates in the ferric-vulnibactin utilization system in <Emphasis Type="Italic">Vibrio vulnificus</Emphasis> M2799

Vibrio vulnificus, an opportunistic pathogen that causes a serious, often fatal, infection in humans, requires iron for its growth. This bacterium utilizes iron from the environment via the vulnibactin-mediated iron uptake system. The mechanisms of vulnibactin biosynthesis, vulnibactin export, and ferric-vulnibactin uptake systems have been reported, whereas the ferric-vulnibactin reduction mechanism in the cell remains unclear. The results of our previous study showed that VuuB, a member of the flavin adenine dinucleotide-containing siderophore-interacting protein family, is a ferric-vulnibactin reductase, but there are other reductases that can complement for the defective vuuB. The aim of this study was to identify these proteins that can complement the loss of function of VuuB. We constructed mutants of genes encoding putative reductases in V. vulnificus M2799, and analyzed their growth under low-iron conditions. Complementation analyses confirmed that IutB, which functions as a ferric-aerobactin reductase, participates in ferric-vulnibactin reduction in the absence of VuuB. This is the first genetic evidence that ferric-vulnibactin is reduced by a member of the ferric-siderophore reductase protein family. In the aerobactin-utilization system, IutB plays a major role in ferric-aerobactin reduction in V. vulnificus M2799, and VuuB and DesB can compensate for the defect of IutB. Furthermore, the expression of iutB and desB was found to be regulated by iron and a ferric uptake regulator.