The Haloprotease CPI Produced by the Moderately Halophilic Bacterium Pseudoalteromonas ruthenica Is Secreted by the Type II Secretion Pathway

Abstract

The gene (cpo) encoding the extracellular protease CPI produced by the moderately halophilic bacterium Pseudoalteromonas ruthenica CP76 was cloned, and its nucleotide sequence was analyzed. The cpo gene encodes a 733-residue protein showing sequence similarity to metalloproteases of the M4 family. The type II secretion apparatus was shown to be responsible for secretion of the haloprotease CPI.Moderately halophilic microorganisms include a broad variety of bacteria that are able to grow in media containing a wide range of elevated NaCl concentrations (3 to 15% NaCl) (56). Considerable attention has been focused on enzymes of moderately halophilic bacteria, since they have substantial biotechnological potential (29,56). While several proteases from extreme halophiles, members of the halophilic archaea, have been characterized (12,15,19,36,44,49,50,51,57), fewer proteases from moderately halophilic bacteria have been purified and studied in depth (9,14,18,20,21,33).The moderately halophilic bacterium Pseudoalteromonas ruthenica CP76 was isolated from a saltern located in Isla Cristina (Huelva, Spain). This strain was selected for its ability to produce an extracellular protease, haloprotease CPI, which has a molecular mass of 38.0 kDa (46, 47). The haloprotease CPI was purified and biochemically characterized. It showed optimal activity at 55°C and pH 8.5 and tolerated a wide range of NaCl concentrations (0 to 4 M NaCl). In this work, we describe the molecular characterization of the operon encoding the haloprotease CPI and a second protease, CPII. We also report the first study of the mechanism used for the secretion of haloprotease CPI to the extracellular medium.The N-terminal amino acid sequence of the haloprotease CPI (ADATGPGGNQKTGQYNY) (47) allowed us to identify several homologues of CPI in a BLAST search (1). We aligned (16, 53) metalloproteases from Vibrio, Aeromonas, or Pseudomonas species (3,6,23,30). On the basis of the conserved regions, degenerate PCR primers AminoProL (GGYC CWGGYGGYAAYCAAAAR) and ProR (CCNGNATRTC HGARAANGCTTC) were designed. We first amplified a 500-bp DNA fragment from the total DNA (27) of P. ruthenica CP76, which was then used in two rounds of inverse PCR (37) to obtain a 4,680-bp fragment. Two open reading frames (ORFs) were found in this fragment (Fig. 1A). The first of them, cpo (2,202 bp), starts at position 148 and ends at position 2,349, encoding a polypeptide of 733 amino acids with an estimated molecular mass of 78.8 kDa. The mature secreted CPI protease corresponded to residues Ala-212 to Lys-542 of the cpo gene product, on the basis of its N-terminal sequence and molecular mass (38.0 kDa) (47). A BLAST analysis of the protein sequence deduced from cpo against the NCBI CDD database (26) revealed that the gene encodes a modular enzyme consisting of four domains: the signal sequence (28 amino acids); the N-terminal proregion (183 amino acids); the mature protease region (331 amino acids) of the M4 superfamily, with two distinct N-terminal and C-terminal domains; and the C...

Autocatalytic Maturation of the Tat-Dependent Halophilic Subtilase Nep Produced by the Archaeon Natrialba magadii

“…Recently, it was reported that the haloprotease CPI from the moderately halophilic bacterium Pseudoalternomonas ruthenica is exported by the Sec-dependent type II secretion system (27). Thus, it seems apparent that the mechanisms used by halophilic prokaryotes to deliver extracellular proteases are not conserved.…”

“…In this context, the maturation mechanism of the Sec-dependent subtilisin-like proteases secreted by the hyperthermophilic archaeon Thermococcus kodaraensis have been extensively characterized (8,33). Many halophilic bacteria and archaea produce extracellular proteases, however, there is limited information on the pathways used by halophiles to deliver secretory proteases (27,28), and the mechanism that leads to enzyme activation has not been examined.…”

“…In this context, the maturation mechanism of the Sec-dependent subtilisin-like proteases secreted by the hyperthermophilic archaeon Thermococcus kodaraensis have been extensively characterized (8, 33). Many halophilic bacteria and archaea produce extracellular proteases, however, there is limited information on the pathways used by halophiles to deliver secretory proteases (27,28), and the mechanism that leads to enzyme activation has not been examined.Most extracellular and membrane anchored proteins are exported through the general secretory (Sec) pathway, which is conserved in all life forms (21). The Sec pathway translocates a wide variety of unfolded substrates, including proteolytic enzymes.…”

Draft Genome Sequence of the Moderately Halophilic Bacterium Pseudoalteromonas ruthenica Strain CP76

“…Strain CP76 was isolated from a saltern located in Isla Cristina (Huelva, Spain). This strain is moderately halophilic (7), and the biochemical and molecular features of the protease, designated haloprotease CPI, were studied in detail (5, 8). It has a molecular mass of 38.0 kDa and showed optimal activity at 55°C, pH 8.5 (range, 6 to 10), and 7.5% NaCl (range, 0 to 4 M) (5).…”

“…A detailed taxonomic study placed strain CP76 within the species Pseudoalteromonas ruthenica (9). The cpo gene encoding the extracellular haloprotease CPI was cloned and its nucleotide sequence was analyzed (8). It encodes a 733-residue protein showing sequence similarity to metalloproteases of the M4 family (zincins superfamily).…”