Alteromonas sp. strain O-7 secretes several proteins in response to chitin induction. We have found that one of these proteins, designated AprIV, is a novel chitin-binding protease involved in chitinolytic activity. The gene encoding AprIV (aprIV) was cloned in Escherichia coli. DNA sequencing analysis revealed that the open reading frame of aprIV encoded a protein of 547 amino acids with a calculated molecular mass of 57,104 Da. AprIV is a modular enzyme consisting of five domains: the signal sequence, the N-terminal proregion, the family A subtilase region, the polycystic kidney disease domain (PkdD), and the chitin-binding domain type 3 (ChtBD3). Expression plasmids coding for PkdD or both PkdD and ChtBD (PkdD-ChtBD) were constructed. The PkdD-ChtBD but not PkdD exhibited strong binding to α-chitin and β-chitin. Western and Northern analyses demonstrated that aprIV was induced in the presence of N-acetylglucosamine, N-acetylchitobiose, or chitin. Native AprIV was purified to homogeneity from Alteromonas sp. strain O-7 and characterized. The molecular mass of mature AprIV was estimated to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature of AprIV were pH 11.5 and 35°C, respectively, and even at 10°C the enzyme showed 25% of the maximum activity. Pretreatment of native chitin with AprIV significantly promoted chitinase activity
An extracellular alkaline metalloprotease (MprI) from Alteromonas sp. strain O-7 was puriˆed and characterized. The molecular mass of the puriˆed enzyme was estimated to be 56 kDa by SDS-PAGE. The optimum pH and temperature were pH 10.0 and 609 C, respectively. The gene (mprI ) encoding MprI was cloned and its nucleotide sequence was analyzed. The deduced amino acid sequence of MprI showed signiˆcant similarity to metalloproteases classiˆed 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.
Pseudoalteromonas piscicida strain O-7 (formerly Alteromonas sp. strain O-7) is an efficient degrader of chitin in the marine environment. The chitinolytic system of the strain consists of many enzymes induced by N-acetylglucosamine (GlcNAc). This paper reports that CdsR, which is a response regulator of CdsS/CdsR two-component signal transduction system, is bound to near the promoter region of GlcNAc-induced aprIV gene. The CdsR protein as a response regulator was transphosphorylated by the CdsS protein as a sensor kinase. Furthermore, the transphosphorylation from CdsS to CdsR was promoted by chitin degradation products and a metabolite. The CdsR protein was also phosphorylated by acetyl phosphate which is an indicator of nutritive conditions of cells. Gel mobility shift assays demonstrated that phosphorylated CdsR (CdsR-P) was bound to not only near the promoter region of aprIV gene but also those of chiA, chiB, chiC, chiD and cbp1 genes which are induced in the presence of GlcNAc. Footprinting analysis demonstrated that CdsR-P was bound to the sequences around the transcriptional start sites of aprIV and chiD genes. These results indicate that CdsR is one of the common regulators of these genes involved in chitin degradation of the strain.
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