1506
S.-J. HA et al.
template. The PCR cycling conditions were as follows: initial
denaturation at 94 ꢀC for 5 min, followed by 30 cycles of denaturation
at 94 ꢀC for 30 s, annealing at 50 ꢀC for 30 s, and an extension at 72 ꢀC
for 1 min, with an additional extension at 72 ꢀC for 5 min during the
final cycle. After amplification, the PCR products in each reaction were
analyzed by agarose gel electrophoresis. The amplified PCR products
were then purified using the QIAquick gel extraction kit and were
cloned into the pGEM-T Easy vector. The nucleotide sequence of the
subcloned PCR products was determined using the BigDye Terminator
Cycle Sequencing Kit for an ABI377 PRISM (PE Applied Biosystems,
Boston, MA). The resulting DNA sequences were analyzed using the
Basic Local Alignment Search Tool (BLAST) server available at NCBI
rium isolated at a depth of 3,500 meters in the Urania
Basin, has been sequenced and made available at
the National Center for Biotechnology Information
(NCBI).15) A putative protein tagged MADE 00676,
which is significantly homologous to known ASases, is
located in the genome of the A. macleodii Deep ecotype.
Based on the fact that A. macleodii produces a xanthan-
like exopolysaccharide and ASase forms an ꢀ-1,4-linked
glucan polysaccharide, which has a similar backbone to
xanthan polymers, we hypothesized that the putative
ASase homolog of A. macleodii is a functional protein
with a role in the biosynthesis of exopolysaccharides.
In this study, we sought homologs of Neisseria and
Deinococcus ASase in various Alteromonas strains and
cloned the potential ASase gene from A. macleodii
(amas). We further expressed amas in E. coli and
characterized the enzymatic properties of the recombi-
nant protein.
Cloning and analysis of the amas gene. Southern blot analysis was
performed with A. macleodii KCTC 2957 genomic DNA. Genomic
DNA was isolated from A. macleodii KCTC 2957 and digested with
BamHI, EcoRI, HindIII, SphI, XbaI, or XhoI. The DNA digests were
separated on 0.6% agarose gels and then transferred by capillary-
blotting onto Hybond-Nþ nylon membranes (GE Healthcare, Buck-
inghamshire, UK). Membranes were hybridized with digoxigenin
(DIG)-labeled probe, which was generated by PCR with the AS-N1
and AS-C1 primer set. Detection was performed with a chemilumi-
nescent detection system according to the manufacturer’s protocols
(Roche Applied Science, Indianapolis, IN). Restriction endonuclease
mapping localized the amas gene within a 7.5 kb fragment spanning
two SphI sites. Colony hybridization was used to screen a mini-library
made from 7–8 kb SphI digests of A. macleodii KCTC 2957 genomic
DNA. A positive clone was identified with the DIG-labeled probe used
in Southern blot analysis. An isolated positive clone was designated
pGEM-Sph7.5. The insert was fully sequenced by the dideoxy chain
termination method, using T7 or SP6 sequencing primers and the
BigDye Terminator Cycle Sequencing Kit for the ABI 377 PRISM
(PE Applied Biosystems). The DNA and amino acid sequences were
subsequently analyzed with DNASIS version 2.1 (Hitachi Software,
Yokohama, Japan) and the BLAST server at NCBI.
Materials and Methods
Bacterial strains, media, and plasmids. Twelve strains belonging to
the genera Alteromonas and Pseudoalteromonas were obtained from
the Korean Collection for Type Cultures (KCTC). These strains were
Alteromonas addita KCTC 12195, Alteromonas macleodii KCTC
2957, Pseudoalteromonas byunsanensis KCTC 12274, Pseudoalter-
omonas issachenkonii KCTC 12958, Pseudoalteromonas maricaloris
KCTC 12960, Pseudoalteromonas marina KCTC 12241, Pseudoalter-
omonas phenolica KCTC 12086, Pseudoalteromonas ruthenica KCTC
12959, Pseudoalteromonas sp. KCTC 12273, Pseudoalteromonas sp.
KCTC 12275, Pseudoalteromonas ulvae KCTC 12940, and Pseudoal-
teromonas undina KCTC 12423. They were grown in Marine broth or
on Marine agar (Difco Laboratories, Detroit, MI) at 25–30 ꢀC for 48 h
and used as the source of chromosomal DNA. E. coli DH5ꢀ
þ
[Fꢁꢀ80lacZ ꢁM15 ꢁ(lacZYA-argF)U169 recA1 endA1 hsdR17(rk
,
Phylogenetic analysis. Known ASase sequences of N. polysaccharea,
D. geothermalis, and D. radiodurans and the complete amino acid
sequences of AAAS and AMAS were aligned with the default
parameters of the Clustal X (version 1.81) multiple sequence alignment
algorithm.16) A phylogenetic tree was constructed using the Neighbor-
joining (NJ) method, version 3.6b of PHYLIP software. The phyloge-
netic tree was evaluated by a bootstrap test on 1,000 replicates.17)
mkþ) phoA supE44 thi-1 gyrA96 relA1ꢂꢁ] and E. coli BL21 [FꢁompT
hsdSB (rBꢁ, mBꢁ) gal dcm ꢂ(DE3) pLysS T1R] were used as hosts in
DNA manipulation and for gene expression respectively. The selection
of recombinant E. coli clones was performed on LB agar plates
supplemented with 100 mg/ml ampicillin, 0.5 mM of isopropyl-ꢁ-D-
thiogalactopyranoside (IPTG), and 40 mg/ml of 5-bromo-4-chloro-
3-indolyl-ꢁ-D-galactopyranoside (Xgal). pGEM-T Easy plasmid
(Promega, Madison, WI) and the pGEX-4T-1 vector (Amersham
Biosciences, Buckinghamshire, UK) were utilized in the cloning
of PCR products and the construction of the expression vector
respectively.
Nucleotide sequence accession numbers. The AAAS and AMAS
nucleotide sequences were submitted to GenBank (accession
nos. AB469415 and AB469558 respectively).
Expression and purification of AMAS. The putative AMAS coding
region was amplified by PCR from pGEM-Sph7.5 with primers
AMASNBamHI (50-GGA TCC ATG AGC TAT GCT GCT GAC-30)
and AMASCXhoI (50-CTC GAG TTA ATC AGC AAG AAG CCA-
30). The introduced BamHI and XhoI restriction sites of each primer are
underlined. The amplified products were cloned into pGEX-4T-1
vector (Amersham Biosciences, Piscataway, NJ, USA) to create
pGEX-AMAS, in which the glutathione S-transferase (GST) was fused
to AMAS. Cultures of recombinant E. coli BL21(DE3)/pGEX-AMAS
were grown in 1 liter of LB medium supplemented with 0.1 mg/ml of
ampicillin at 37 ꢀC with agitation. When the optical density at 600 nm
reached 0.5 to 0.6, IPTG was added to a final concentration of 1 mM,
and the cells were grown for 3 h to induce expression of the amas gene.
After induction, the cells were harvested by centrifugation at 5;000 ꢂ g
for 20 min at 4 ꢀC. The pellet was resuspended in 5 ml of phosphate-
buffered saline (PBS) buffer (140 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, and 1.8 mM KH2PO4, pH 7.3) per g (wet weight) of cells.
The resuspended cells were incubated on ice for 30 min after adding
1 mg/ml lysozyme and were disrupted by sonication (Sonifier 450,
Branson, Danbury, CT; output 4, 6 times for 10 s, constant duty) in an
ice bath. The cell lysate was centrifuged at 10;000 ꢂ g for 10 min at
4 ꢀC. The clarified supernatant was applied directly onto a Glutathione-
Sepharoseꢀ high performance affinity column (Amersham Bioscien-
ces) that was pre-equilibrated with PBS. The column was washed with
Enzymes and chemicals. Restriction endonucleases and other DNA
modifying enzymes, such as T4 DNA ligase and Pfu DNA polymerase,
were purchased from New England Biolabs (Beverly, MA) and
Stratagene (La Jolla, CA). The genomic DNAs of Alteromonas and
Pseudoalteromonas strains was isolated and purified with the Genomic
DNA Prep Kit for Bacteria (Solgent, Seoul, Korea). The purification of
PCR products and DNA restriction fragments was performed using the
QIAquick gel extraction kit from Qiagen (Valencia, CA). All other
chemicals used were of reagent-grade quality and were obtained from
Sigma Chemical (St. Louis, MO).
Design of degenerate primers specific to the ASase gene and
detection of it. To detect potential ASase genes from the Alteromonas
and Pseudoalteromonas strains, a degenerate ASase primer set was
developed (Fig. 1). The primer set AS-N1 and AS-C1, specific to the
ASase gene, was designed based on conserved regions of known
ASases of N. polysaccharea, D. radiodurans, and D. geothermalis
(Fig. 2). The presence of an ASase was detected by PCR amplification
using Taq polymerase (Promega). Standard PCR reactions were carried
out in a 20 ml volume at the following final concentrations: 50 mM KCl,
10 mM Tris–HCl (pH 9.0 at 25 ꢀC), 0.1% Triton X-100, 1.5 mM MgCl2,
100 mM of each dNTP, 0.5 mM of each primer, and 0.5 U Taq DNA
polymerase. Thirty nanograms of genomic DNA was used as a