RESUMEN
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.
Asunto(s)
Quitina/metabolismo , Quitinasas/genética , Regulación Bacteriana de la Expresión Génica , Pseudoalteromonas/genética , Pseudoalteromonas/metabolismo , Secuencia de Bases , Quitinasas/biosíntesis , Quitinasas/metabolismo , Huella de ADN , Proteínas de Unión al ADN/genética , Ensayo de Cambio de Movilidad Electroforética/métodos , Mutagénesis Sitio-Dirigida , Organofosfatos/química , Fosforilación , Filogenia , Pseudoalteromonas/enzimología , Transducción de SeñalRESUMEN
Alteromonas sp. strain O-7 secretes several proteins in addition to chitinolytic enzymes in response to chitin induction. In this paper, we report that one of these proteins, designated MprIII, is a metalloprotease involved in the chitin degradation system of the strain. The gene encoding MprIII was cloned in Escherichia coli. The open reading frame of mprIII encoded a protein of 1,225 amino acids with a calculated molecular mass of 137,016 Da. Analysis of the deduced amino acid sequence of MprIII revealed that the enzyme consisted of four domains: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The C-terminal extension (PkdDf) was characterized by four polycystic kidney disease domains and two domains of unknown function. Western and real-time quantitative PCR analyses demonstrated that mprIII was induced in the presence of insoluble polysaccharides, such as chitin and cellulose. Native MprIII was purified to homogeneity from the culture supernatant of Alteromonas sp. strain O-7 and characterized. The molecular mass of mature MprIII was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 115 kDa. The optimum pH and temperature of MprIII were 7.5 and 50 degrees C, respectively, when gelatin was used as a substrate. Pretreatment of native chitin with MprIII significantly promoted chitinase activity. Furthermore, the combination of MprIII and a novel chitin-binding protease (AprIV) remarkably promoted the chitin hydrolysis efficiency of chitinase.
Asunto(s)
Alteromonas/enzimología , Quitina/metabolismo , Metaloendopeptidasas/genética , Alteromonas/metabolismo , Secuencia de Aminoácidos , Western Blotting , Clonación Molecular , ADN Bacteriano/análisis , Inducción Enzimática , Expresión Génica , Biología Marina , Metaloendopeptidasas/química , Metaloendopeptidasas/aislamiento & purificación , Metaloendopeptidasas/metabolismo , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Microbiología del AguaRESUMEN
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.
Asunto(s)
Alteromonas/genética , Genes Bacterianos , Isoenzimas/genética , Metaloendopeptidasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Isoenzimas/química , Biología Marina , Metaloendopeptidasas/química , Datos de Secuencia Molecular , TemperaturaRESUMEN
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 alpha-chitin and beta-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 degrees C, respectively, and even at 10 degrees C the enzyme showed 25% of the maximum activity. Pretreatment of native chitin with AprIV significantly promoted chitinase activity.