Journal of Environmental Biotechnology
Vol. 7, No. 2, 99–102, 2007

Original paper (regular paper)

Cloning and Characterization of A Gene Encoding Algicidal Serine
Protease from Pseudoalteromonas sp. Strain A28
DAIKI KOHNO1, YUICHI SAKIYAMA1, SUN-OG LEE2, NOBORU TAKIGUCHI1, ATSUSHI MITSUTANI3,
HIROTAKA KITAGUCHI3 and JUNICHI KATO1*1
2

Department of Molecular Biotechnology, Hiroshima University, Higashi-Hiroshima, Hiroshima 739–8530, Japan

Bio-Control Research Team, Korea Research Institute of Chemical Technology, Yusong PO Box 107, Taejon 305–600,
Republic of Korea
3

Department of Marine Biotechnology, Fukuyama University, Fukuyama, Hiroshima 729-0292, Japan* TEL: +81–(0)82–424–7757 FAX: +81–(0)82–424–7047* E-mail: jun@hiroshima-u.ac.jp(Received; 25 October, 2007/Accepted; 2 November, 2007)

The gene (espI) encoding an algicidal extracellular serine protease was isolated from Pseudoalteromonas sp. strain A28.
DNA sequence analysis revealed that EspI is synthesized as a 711-amino-acid prepropeptide and is further processed to the50-kDa matured protease after cleavage of a 148-amino-acid N-terminal domain and 6.7-kDa C-terminal domain. TheConserved Domain Search program found that EspI belongs to the subtilisin-like serine protease family. The program also
found two PPC domains in the C-terminal region. The PPC domain is normally found at the C-terminus of secreted bacterial
peptidases and they are not present in the active peptidase. However, the molecular size of purified EspI predicts that it
contains one and half PPC domains in the matured EspI protease.
Key words: red tide algae, serine protease, alga-lysing bacterium, Pseudoalteromonas

Skeletonema costatum is the marine diatom whichoccasionally produces blooms in the Ariake sea, Fukuoka,
Japan6). Its undesirable blooms cause severe damage toaquacultured laver. Pseudoalteromonas sp. strain A28,
which was isolated from the Ariake Sea, is capable of lysing
S. costatum2). The culture supernatant of strain A28 shows
algicidal activity, suggesting that strain A28 produces
an extracellular algicidal compound(s)3). Genetic analysis
revealed an involvement of extracellular proteases inalgicidal activity of strain A28. We identified two serine
proteases, designated serine protease I (EspI) and serine
protease II (EspII), as the major extracellular proteases.
EspI is a monomeric protease with molecular weight of
50 kDa, whereas EspII is a heterodimer of 48-kDa and33-kDa subunits. We demonstrated that EspI was responsible
for the algicidal activity but not EspII3). Interestingly, somecommercially available proteases, including trypsin, pepsin,subtilisin, and pronase, did not show algicidal activity. These
results suggest that some properties of EspI, for example,
substrate specificity or aﬃnity to algal cells, are required for
its algicidal activity. To further investigate molecular mechanism of algicidal activity of EspI, we cloned and characterized the espI gene encoding EspI from strain A28 in thisstudy.
In the previous study, the N-terminal amino acid sequenceof purified EspI was determined to be Ala-Thr-Pro-Asn-Asp-Pro3). To design an oligonucleotide probe, we determined the N-terminal amino acid sequence again. Aminoacid sequencing enabled determination of the first 15 aminoacid residues (Ala-Thr-Pro-Asn-Asp-Pro-Arg-Tyr-Asp-GlnTrp-His-Tyr-Tyr-Glu). A degenerated 23-mer oligonucleotide (5’-TA(C/T)GA(C/T)CA(A/G)TGGCA(C/T)TA(C/T)TA(C/T)GA-3’) derived from the N-terminal sequence,residue 8–15 was synthesized as a hybridization probe.
Southern blot analysis7) detected a 2.1-kb EcoRI fragmentof the Pseudoalteromonas sp. strain A28 genome. A sizefractionated library of EcoRI fragments of strain A28
genomic DNA (1.5–2.5 kb in size) was created with apUC118 vector13) and Escherichia coli MV118413). Plasmids
DNAs, isolated from recombinant colonies, were screened
with the oligonucleotide probe. One positive plasmid, designated as pPA28-1, was obtained. Restriction endonucleaseanalysis showed that pPA28-1 contained a 2.1-kb EcoRI
fragment of the strain A28 genomic DNA. Nucleotide
sequence analysis of the 2.1-kb EcoRI fragment found partof an open reading frame (espI). To obtain the 5’ region ofespI, Southern blot analysis with a 0.8-kb EcoRI-HindIII
fragment as probe (Fig. 1) was performed and the 2.0-kb
HindIII fragment hybridized to the 0.8-kb probe was clonedinto pUC118. DNA sequencing found that the 2.0-kb HindIII
fragment contained the 5’ region of espI. The espI gene of2133 bp could code for a protein of 711 amino acid residues

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having a calculated molecular weight of 73.2 kDa. The
N-terminal amino acid sequence of purified EspI was locatedwithin the 711-amino acid polypeptide starting at Ala-149
and is in perfect agreement with all 15 sequence amino acids(Fig. 1). To confirm that the cloned espI gene really encodes
EspI, an espI mutant of strain A28 was constructed by inactivating the chromosomal espI gene. The espI gene on
pPA28-1 was disrupted by inserting a kanamycin resistantgene cassette12) into the PstI site within the espI gene (Fig.
1). The resulting plasmid was introduced into strain A28 byelectroporation2) and kanamycin resistant transformants

were selected. The insertion mutation of the chromosomal
espI gene was confirmed by Southern blot analysis. The
culture supernatant of the espI- mutant was examined forproteolytic and algicidal activities as described previously3).
It showed ca. 70% decreased proteolytic and algicidal activities compared to that of the parent strain (Fig. 2), confirming that the cloned espI gene encodes algicidal serine protease I.
A computer-assisted sequence similarity search revealed
that EspI showed 74% identity with Pseudolateromonas
piscicida strain O-7 (formerly named Alteromonas sp.
strain O-7) serine protease AprI11). P. piscicida stain O-7
was isolated from a sediment sample at the Sagami Bay ofJapan as a chitin-degrading bacterium9). There is no information about algicidal activity of P. piscicida strain O-7
and AprI. It is predicted that the AprI protein is synthesized
as a preproprotein with a 40-amino-acid signal peptide, aFig. 1. Restriction map of the espI region of Pseudoalteromonas sp. strain A28 (A) and the nucleotide sequence of the cloned espI gene andthe deduced amino acid sequence of EspI (B).
(A) The location and orientation of espI are indicated by a horizontal arrow. The gray bar indicates a DNA fragment used as a probe forthe gene walking experiment. Plasmid pPA28-2 was constructed by inserting a kanamycin resistant gene cassette into the PstI sited indicated by the vertical arrow. Restriction sites: E, EcoRI; H, HindIII; P, PstI. (B) The 15-amino acid N-terminal amino acid residues of purified EspI are underlined. PPC1 and PPC2 are illustrated by solid boxes. Potential catalytic triad residues, 217-Asp, 249-His, and430-Ser, are indicated by gray boxes. Nucleotide sequence of espI has been deposited in the GSDB, EMBL, DDBJ, and NCBI nucleotide
sequence databases under the accession number AB365799.

Algicidal serine protease gene110-amino-acid N-terminal propeptide, a 346-mino-acidmature protease, and a 219-amino-acid C-terminal propeptide which is processed into the 32.4-kDa mature protease11).
In the case of Pseudoalteromonas sp. strain 28 EspI, theN-terminal domain with a similar size (148 amino acidresidues) to that of strain O-7 AprI is removed during thematuration. However, since the purified EspI protease is50 kDa3), only a 6.7-kDa C-terminal domain (ca. 60 aminoacid residues) is expected to be removed during the processing of EspI.
The Conserved Domain Search program5) of the NationalCenter for Biotechnology Information found a subtilase(subtilisin-like serine protease) family motif in EspI. Potential
catalytic triad residues, Asp, His, and Ser, which are common
in subtilisin-like serine proteases8), are conserved at thepositions corresponding to 217-Asp, 249-His, and 430-Ser(Fig. 1). The Conserved Domain Search program also444
found two PPC (bacterial pre-peptidase C-terminal) domains,designated PPC1 (residues 513–597) and PPC2 (residues624–708) in the C-terminal region of EspI (Fig. 1). The
PPC domain (Conserved Domain Database accession
number: pfam04151 [the National Center for BiotechnologyInformation]) is normally found at the C-terminus of many
secreted bacterial peptidases and they are removed during thematuration of the active peptidases14). Another P. piscicida
strain O-7 extracellular serine protease AprII10) and theMixococcus xanthus metal protease1) as well as EspI possess
two PPC domains in their C-terminal regions, whereas there
is only one PPC domain in many other bacterial proteases.
In P. piscicida strain O-7 AprI, two PPC domains arecleaved oﬀ during maturation, while the molecular weightFig. 2. Protease activity (A) and algicidal activity (B) in culture supernatants of strain A28 and its espI- mutant.
(A) Protease activity was measured by using azocasein as a
substrate as described by Lee, et al3). One unit protease activity was defined as the amount of enzyme that caused incremental change of one absorbance unit per hour. Vertical bars represent the standard deviations of measurements done in four
separate experiments. (B) Algicidal activity was detected as described previously3). Zones of clearing around paper disks indicate the lysis of the diatom S. costatum strain NIES-324.
Plates were photographed after 48 h of incubation at 20°C.

101(50 kDa) of the matured EspI protease predicts that matured
EspI still has whole PPC1 and half PPC2 in its C-terminal
region. EmpI is an algicidal metal protease produced bystrain A284). Expectedly, the EspI serine protease did notshow significant similarity to 38-kDa matured EmpI metal
protease, however, the C-terminal region (residues 476–711)
of EspI was 50.4% identical to that of EmpI. Sequence
analysis revealed that EmpI also possesses two PPC domains
in the C-terminal region and that these PPC domains areremoved from the matured EmpI protease. It was shownthat EmpI was 6-fold less algicidal to S. costatum than EspI
on the basis of the mass of proteins4). Thus, the PPC
domains in matured EspI might have a role in algicidal
activity. We are now investigating this possibility using
obtained espI gene.

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