Physiological and genomic features of a novel violacein-producing bacterium isolated from surface seawater
Physiological and genomic features of a novel violacein-producing bacterium isolated from surface seawater
Yue-Hong Wu 0 1
Hong Cheng 0 1
Lin Xu 0 1
Xiong-Bin Jin 0 1
Chun-Sheng Wang 0 1
Xue-Wei Xu 0 1
0 Key Laboratory of Marine Ecosystem and Biogeochemistry, Second Institute of Oceanography, State Oceanic Administration , Hangzhou , P. R. China
1 Editor: Beom Seok Kim, Korea University , REPUBLIC OF KOREA
Strains JW1T and JW3, isolated from surface seawater of the Arabian Sea, were subjected to polyphasic taxonomic analysis. Cells of both strains were Gram-stain-negative, aerobic, and rod-shaped. They formed violet pigment and produced violacein. On the basis of 16S rRNA gene sequence analysis, strains JW1T and JW3 showed high 16S rRNA gene sequence similarity with Pseudoalteromonas byunsanensis JCM12483T (98.2%), P. shioyasakiensis SE3T (97.8%), P. arabiensis JCM 17292T (97.3%), and P. gelatinilytica NH153T (97.1%). The 16S rRNA gene sequence similarity between JW1T and JW3 was 100%. Phylogenetic analyses revealed that both strains fell within the cluster of the genus Pseudoalteromonas and represented an independent lineage. The average nucleotide identity and in silico DNA-DNA hybridization values between JW1T and type strains of the closely related Pseudoalteromonas species were 70.9±83.3% and 20.0±26.4%, respectively. The sole respiratory quinone in both strains is ubiquinone 8 (Q-8). The principal fatty acids are summed feature 3 (C16:1ω7c and/or iso-C15:0 2OH), C18:1ω7c, and C16:0. The major polar lipids are phosphatidylethanolamine, phosphatidylglycerol, one unidentified glycolipid, one unidentified aminolipid, and one unidentified phospholipid. The DNA G+C content was 43.3 mol%. Differential phylogenetic distinctiveness, chemotaxonomic differences, and phenotypic properties indicated that strains JW1T and JW3 could be differentiated from the Pseudoalteromonas species with validly published names. Therefore, it is proposed that strains JW1T and JW3 represent a novel species of the genus Pseudoalteromonas, for which the name Pseudoalteromonas amylolytica sp. nov. (type strain, JW1T = CGMCC 1.15681T = KCTC 52406T = MCCC 1K02162T) is proposed.
Data Availability Statement: The GenBank/EMBL/
DDBJ accession numbers for the 16S rRNA gene
sequence of strains JW1T and JW3 are KU535632
and KU535631. The GenBank accession numbers
for the whole genome sequences of strains JW1T,
JW3 and P. byunsanensis JCM12483T are
MKJU00000000, MKJT00000000 and
MNAN00000000, respectively. Other relevant data
are within the paper and its Supporting Information
The genus Pseudoalteromonas, the type genus of the family Pseudoalteromonadacea e [
proposed by Gauthier et al. (1995) [
]. Initially, the genus Pseudoalteromonas was
differentiated from the genus Alteromonas based on the phylogenetic analysis of 16S rRNA gene
]. Currently, the genus Pseudoalteromonas consists of 43 species with validly
Funding: This work was supported by the National
Natural Science Foundation of China granted to YW
(no. 41406174, http://www.nsfc.gov.cn/publish/
portal1/); the National Key Basic Research Program
of China granted to XX (2014CB441503, http://
most.gov.cn/); the Natural Science Foundation of
Zhejiang Province granted to XX (LR17D060001,
http://www.zjnsf.gov.cn/). The funders had no role
in study design, data collection and analysis,
decision to publish, or preparation of the
Competing interests: The authors have declared
that no competing interests exist.
published names (http://www.bacterio.net/p/pseudoalteromonas.html). Members of the genus
Pseudoalteromonas are widespread in nature and have a great adaptability to marine
environments, such as coastal, open, and deep seawaters, sediments, marine invertebrates, fish, and
]. The genus Pseudoalteromonas is Gram-negative, aerobic or facultatively anaerobic,
and rod-shaped, it requires Na+ ions for growth, usually does not denitrify, and possesses
ubiquinone-8 (Q8) as major respiratory quinone [
Some Pseudoalteromonas species produce a variety of primary and secondary metabolites,
including antibiotics [
], exopolymers [
], hydrolytic enzymes [
], and pigments [
Violacein is a natural indolocarbazole compound formed by condensation of two molecules of
tryptophan . It is a potential pharmaceutical agent owing to its extensive biological
properties, such as antibacterial, antiviral, antioxidant, and antitumor activities [
Pseudoalteromonas luteoviolacea has been reported to produce violacein [
]. Here, we present a polyphasic
study describing two novel violacein-producing strains, both of which were isolated from
surface water of the Arabian Sea.
Materials and methods
Organisms and culture conditions
Strains JW1T and JW3 were isolated from the surface seawater collected from the Arabian Sea
(E67Ê N24Ê). The seawater samples were stored at 4ÊC until use. Natural seawater agar (pH
7.2±7.4) supplemented with 0.05% peptone (w/v; BD, Sparks, MD, USA) and 0.01% yeast
extract (w/v; BD) was used for isolation. The seawater samples were diluted using the standard
ten-fold dilution plating technique and spread on natural seawater agar. After ten days of
aerobic incubation at 30ÊC, two violet colonies, designated as JW1T and JW3, were picked from
different samples and purified by repeated restreaking. The purity was confirmed by the
uniformity of cell morphology. The reference strains P. byunsanensis JCM 12483T, P.
shioyasakiensis JCM 18891T, and P. arabiensis JCM 17292T were obtained from the JCM (Japan
Collection of Microorganisms). The reference strain P. gelatinilytica NH153T was available in
our lab [
]. Unless otherwise stated, the two strains were routinely cultured in marine broth
2216 (MB; BD) or on marine agar 2216 (MA; BD) at 30ÊC and stored at ±80ÊC with 30% (v/v)
16S rRNA gene and genome sequence determination
The 16S rRNA gene was amplified and analyzed as described previously [
]. PCR products
were cloned into the vector pMD 19-T (TaKaRa, Dalian, China) and then sequenced to
determine the almost-complete sequence of the 16S rRNA gene. High-quality genomic DNA was
extracted with the AxyPrep™ Bacterial Genomic DNA Miniprep Kit (Axygen Scientific, Inc.,
Union City, CA, USA). The genomes of strains JW1T, JW3, and P. byunsanensis JCM 12483T
were sequenced using the Solexa paired-end sequencing technology with the Illumina HiSeq
2000 platform (Anoroad Gene Technology Co. Ltd, Beijing, China). One paired-end library
was constructed with 500-bp insert size. The sequencing generated approx. 1 Gb of clean data
(approx. 500-fold genome coverage). De novo assembly of the reads was carried out using
SOAPdenovo (version 2.0.1) [
]. Assembly k-mer was tested from 57 to 64 for seeking the
optimal value, using the abyss-pe script. Assembly quality was estimated using MUMmer [
Completeness of the genome sequence was addressed using the bioinformatics tool CheckM
]. The complete sequence of the 16S rRNA gene
was annotated via the RNAmmer 1.2 Server [
] and was compared with related sequences of
reference organisms using the EzTaxon-e service [
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Phylogenetic analysis was carried out using ARB (release 6.0.2) [
] and the All-Species Living
Tree Project database (LTPs123, September 2015) [
]. The 16S rRNA gene sequences of
strains JW1T and JW3 were aligned with SILVA Incremental Aligner (SINA, version 1.2.11)
]. The alignment sequences were imported into the LTPs
database and implemented in ARB. On the basis of the obtained All-Species Living Tree and the
EzTaxon-e results, 24 species were selected and sequence data were aligned with ClustalW
]. Phylogenetic trees were reconstructed using the MEGA 5 program package [
Algicola sagamiensis B-10-31T as the outgroup, by neighbor-joining [
], and maximum-likelihood methods [
]. Tree topology was evaluated by bootstrap
analysis using 1000 resample datasets. Kimura two-parameter model [
] was used to calculate
evolutionary distances and reconstruct phylogeny (neighbor-joining and maximum-likelihood
The genomes of 20 type strains of Pseudoalteromonas species were retrieved from the
GenBank database (S1 Fig). Six housekeeping genes, atpD (beta subunit for ATP synthase), gyrB
(DNA gyrase beta subunit), mreB (rod shape-determining protein), recA (RNA recombinase
alpha subunit), rpoD (RNA polymerase), and topA (DNA topoisomerase I) were used for
multilocus sequence analysis (MLSA). The concatenated sequence of six single genes was obtained
from the genome and subjected to maximum-likelihood phylogenetic analyses [
Cell morphology, size, and motility were examined using confocal laser scanning microscopy
(TCS SP5; Leica) and transmission electron microscopy (JEM-1230; JEOL). The hanging-drop
method was used for motility testing. Cell morphology and ultrastructure were observed using
transmission electron micrographs.
The growth at various temperatures (4, 15, 20, 28, 30, 37, 45, and 50ÊC) was tested in MB.
The pH range for growth was determined in the range of 5.0±10.5 with interval of 0.5 in MB
by adding MES (pH 5.0±6.0), PIPES (pH 6.5±7.0), Tricine (pH 7.5±8.5), and CAPSO (pH 9.0±
10.5) at a final concentration of 50 mM. pH values changed only minimally after autoclaving.
Growth at different concentrations of NaCl (0, 0.5, 1.0, 3.0, 5.0, 7.5, 10.0, and 15.0%, w/v) was
investigated using NaCl-free MB (prepared according to the MB formula, but without NaCl).
Sea-salt requirement for growth was measured in the PY medium (peptone 5.0 g, yeast extract
1.0 g and distilled water 1 L, pH 7.6) supplemented with sea salts (Sigma) at various
concentrations (0, 0.5, 1.0, 2.0, 3.0, 4.0, 4.5, and 5.0%, w/v). Growth was measured at 590 nm (OD590)
with a UV/visible spectrophotometer (Ultrospec 6300 pro; Amersham Biosciences). Upper
and lower limits for growth were confirmed when no growth was observed after cultivation for
one month. For anaerobic growth, strains were incubated in the AnaeroPack-MicroAero
anaerobic system (Mitsubishi). Sodium nitrate (20 mM) or sodium nitrite (20 mM) was used
as a potential electron acceptor.
Gram reaction, oxidase and catalase activities, and hydrolysis of starch and Tween-20, -40,
and -80 were tested according to [
]. Violacein was extracted according to [
] and the
absorbance of the violet pigment was monitored from 350 nm to 1000 nm using a UV/visible
spectrophotometer (DU800, Beckman Coulter). The molecular weight of violacein was deduced by
LC-MS analysis according to [
]. Chromatography was carried out on a Agilent 1200.
Compound separation was achieved on an analytical column (Extend-C18, 3.5 μm, 2.1 × 100 mm;
Agilent). Analysis of the pigment by eletrospray ionization mass spectrometry was conducted
with Finnigan LCQ DECA XP MAX mass spectrometer (Thermo Electron Corp., USA). The
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flow rate of the pigment solution was 15 μL/min. The utilization of carbon substrates as sole
carbon and energy sources was tested in BM [
] supplemented with filter-sterilized complex
nutrients (yeast extract, peptone and tryptone, 0.2%, w/v), sugars (0.2%, w/v), alcohols (0.2%,
w/v), organic acids (0.1%, w/v), or amino acids (0.1%, w/v). Yeast extract (0.01%, w/v) was
added as a growth factor. Acid production was evaluated using marine oxidation-fermentation
medium supplemented with 1% filter-sterilized sugars [
]. API 20NE and API 20E tests
(bioMeÂrieux) were used according to the manufacturer's instructions to determine additional
physiological and biochemical characteristics. Strips were inoculated with a heavy bacterial
suspension (MacFarland 5 standard) in AUX medium supplemented with 2% (w/v) sea salts
]. API 20 NE and API 20 E strips were read after 48 h. Sensitivity to antimicrobial
agents was determined with a two-layer plate method according to Wu et al. (2015)[
reference strains, P. byunsanensis JCM 12483T, P. shioyasakiensis JCM 18891T, P. arabiensis
JCM 17292T, and P. gelatinilytica NH153T were used as controls in the above tests.
The cellular fatty acids of strains JW1T, JW3, and the reference strains were determined under
identical conditions in parallel. The quadrant streak method was used for inoculation and
cellular fatty-acid methyl esters were obtained from cells grown on MA at 30ÊC for 16 h from
quadrant 3 (late exponential phase). Whole cell fatty acids were analyzed using the Microbial
Identification System (MIDI Inc.) according to the manufacturer's instructions. Isoprenoid
quinones were extracted and purified by two-dimensional thin-layer chromatography (TLC)
and then analyzed by LC-MS (Agilent 1200 and Thermo Finnigan LCQ DECA XP MAX mass
]. Total lipids were extracted and separated by two-dimensional TLC [
silica gel 60 F254 plates (Merck). Four types of spray reagent were used to detect the
corresponding lipids, including molybdophosphoric acid for total lipids, α-naphthol reagent for
glycolipids, ninhydrin reagent for lipids containing free aminolipids, and molybdenum blue for
phosphorus-containing lipids [
Average nucleotide identities and genome analysis
The average nucleotide identity (ANI) was calculated using the OrthoANIu algorithm by
ChunLab's online Average Nucleotide Identity calculator [
]. In silico DNA-DNA
hybridization (DDH) values were calculated by GGDC [
rRNA genes were identified using the RNAmmer 1.2 Server [
] and tRNA genes were
searched with the tRNAscan-SE 2.0 online server [
]. Gene prediction and functional
annotation were carried out using the Rapid Annotation using Subsystem Technology (RAST) server
]. Selected predicted genes were classified using RPSBLAST against the COG
database . Translated genes were assigned to KEGG pathway using KEGG Automatic
Annotation Server (KAAS) with the BBH method [
]. Orthologous cluster analyses were carried
out using OrthoMCL . Shared and unique orthologous clusters were established with
inhouse shell scripts. CRISPR structures in the genomes were predicted with the CRISPRFinder
program online (http://crispr.i2bc.paris-saclay.fr/Server/).
Results and discussion
Strains JW1T and JW3 were Gram-stain-negative, aerobic and rod-shaped (0.7±1.2 μm in
width and 1.8±3.0 μm in length) (S2 Fig). Colonies were violet, circular, convex, smooth, and
1±2 mm in diameter after one day of incubation at 30ÊC on MA. Optimal growth occurred at
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30ÊC and pH 7.5. UV-visible absorption spectra and LC-MS analysis of the pigment isolated
from strains JW1T, JW3, and P. byunsanensis JCM 12483T showed the presence of violacein.
Maximal absorption of UV-visible light was at 575 nm (S3A Fig). Mass spectrometry of the
pigment revealed a parent ion [M-H]−at m/z 342.1, which was identical to that of violacein
(S3B Fig) [
]. Both strains were positive for catalase, oxidase, tryptophan deaminase, and
Voges±Proskauer reaction, able to hydrolyze esculin, gelatin, starch, Tween-20, -40 and -80,
susceptible to (μg per disc unless otherwise stated) chloramphenicol (30), erythromycin (10),
gentamicin (10), kanamycin (30), mefoxin (30), neomycin (30), nitrofurantoin (300),
norfloxacin (10), polymyxin B (300 IU), rifampicin (5), streptomycin (10), tetracycline (30), and
vancomycin (30), and resistant to ampicillin (10), cefalexin (30), nystatin (100), and penicillin G (10
IU). Detailed phenotypic characteristics are given in the species description and Table 1.
16S rRNA gene sequence similarities and phylogenetic analysis
The 16S rRNA gene sequences of strains JW1T and JW3 (1527 nt) were obtained. According
to EzTaxon as well as ClustalW results of 16S rRNA gene sequence comparison to
representative bacteria with validly published names, strains JW1T and JW3 showed high 16S rRNA gene
sequence similarity to P. byunsanensis JCM12483T (98.2%), P. shioyasakiensis SE3T (97.8%), P.
arabiensis JCM 17292T (97.3%), and P. gelatinilytica NH153T (97.1%), and exhibited less than
97.0% 16S rRNA gene sequence similarity with the type strains of other Pseudoalteromonas
species. The 16S rRNA gene sequence similarity between strains JW1T and JW3 was 100%.
The All-Species Living Tree indicated that the genus Pseudoalteromonas forms a
monophyletic clade and strains JW1T and JW3 fall within the cluster comprising the Pseudoalteromonas
species. The topologies of neighbor-joining, maximum-likelihood, and maximum-parsimony
phylogenetic trees based on the 16S rRNA gene also supported the notion that strains JW1T
and JW3 formed a stable lineage, with a high bootstrap value of 100%, and a distinct lineage
from P. byunsanensis (bootstrap value 91%) (Fig 1). Phylogenetic trees based on concatenated
sequences of the six housekeeping genes atpD, gyrB, mreB, recA, rpoD, and topA confirmed
that the two strains formed a clade with P. byunsanensis as well as P. citrea and could not be
associated with any of the recognized species in the genus Pseudoalteromonas (S1 Fig).
Phylogenetic analysis indicated JW1T and JW3 form another, independent lineage and might
represent a novel member of the genus Pseudoalteromonas.
Chemotaxonomic data supported the results of the phylogenetic analysis. The sole respiratory
quinone found in strains JW1T and JW3 was Q8, in line with all members of the genus
]. Fatty-acid analysis revealed that summed feature 3, C18:1ω7c, and C16:0
were the major fatty acids in JW1T and JW3 and the references strains (Table 2). JW1T and
JW3 possessed phosphatidylethanolamine and phosphatidylglycerol as the major polar lipids,
similar to the reference strains. In addition, JW1T and JW3 possessed three unidentified
glycolipids (GL2±GL4), one unidentified aminolipid (AL2), and one unidentified phospholipid
(PL1) as moderate-to-minor polar lipids, which were similar to those of P. byunsanensis JCM
12483T (S4 Fig). Moreover, JW1T and JW3 possessed aminolipid, glycolipid, and phospholipid,
all of which were detected in the four reference strains (S4 Fig) [
Chemotaxonomic data of JW1T, JW3, and their relatives also showed some clear differences
in fatty acid composition and polar lipid profile. The percentage of C16:0 of strains JW1T and
JW3 (18.4% and 16.5%, respectively) was lower than that of the reference strains (21.0±27.1%).
JW1T and JW3 contained C18:1ω6c (4.1% and 4.6%, respectively), which were not detected in
the reference strains (Table 2). One unidentified aminolipid (AL1) was present in both strain
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JW1T and JW3, but was not detected in the P. byunsanensis JCM 12483T. P. byunsanensis JCM
1248 possessed three unidentified aminolipids (AL3±AL5) and three unidentified lipids (L3±
L5), while strains JW1T and JW3 did not. Diphosphatidylglycerol was present in P.
shioyasakiensis JCM 18891T, P. arabiensis JCM 17292T, and P. gelatinilytica NH153T [
], but not in
JW1T and JW3 (S4 Fig).
In silico DNA-DNA relatedness
The DNA G+C content of strains JW1T and JW3 calculated from the genome sequence was
43.3 mol%, a value in the range reported for members of the genus Pseudoalteromonas, i.e. 38±
48 mol% [
]. JW1T and the reference strains exhibited ANI values of 70.9±83.3% (Table 1).
These ANI values were far below the threshold of species boundary (94±96%) [
low taxonomic relatedness between JW1T and the reference strains. The recommended results
(formula 2) of the in silico DDH analysis revealed that JW1T and the reference strains shared
20.0±26.4% DNA relatedness (Table 1). The values were below 70%, indicating that the strains
should be assigned to different genomic species . In addition, the ANI and in silico DDH
values between JW1T and JW3 were 99.9%. These values were above the species boundary
(94±96% for ANI values and 70% for in silico DDH values), suggesting that JW1T and JW3
represent an identical genospecies.
General features of JW1T and JW3 are displayed in Table 3 and S1 Table. The bioinformatics
tool CheckM indicated that the genome completeness was 100% for both JW1T and JW3, with
a contamination percentage of 0.4% and 0.5%, respectively. Genome sequence completeness
95%, with 5% contamination, is considered to indicate an excellent reference genome for
deeper analyses [
]. The genome size of the two strains and their related Pseudoalteromonas
species varied from 4.5 Mb to 4.8 Mb. This variation can be attributed partially to the draft
nature of the sequence. COG assignments were similar for all genomes. Twenty-two COG
classes were annotated in the genomes of JW1T, JW3, P. byunsanensis JCM 12483T, and P.
arabiensis JCM 17292T, and 23 COG classes (with an extra Cytoskeleton class) were detected in the
genomes of P. shioyasakiensis JCM 18891T and P. gelatinilytica NH153T (S1 Table).
Furthermore, 13 and 6 unique orthologous clusters were found in the genomes of JW1T and JW3,
respectively (Table 3). The two strains shared 640 unique orthologous clusters.
Genome analysis of JW1T, JW3, and the reference strains indicated the presence of genes
encoding clustered regularly interspaced short palindromic repeats (CRISPRs; Table 3).
CRISPRs, in association with CRISPR-associated (Cas) proteins, make up the immune system
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Fig 1. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic
relationships of JW1T, JW3, and related taxa. Bootstrap values (>60%) based on 1,000 replications are shown at branch
nodes. Filled circles indicate that the corresponding nodes were also recovered in the trees generated with the
maximumlikelihood and maximum-parsimony algorithms. Bar, 0.005 substitutions per nucleotide position.
that confers resistance to foreign genetic elements, such as plasmids and phages [
CRISPRs/Cas system is being used for gene editing. Colonies of JW1T and JW3 were violet
and the two strains produced violacein. The biosynthesis of violacein begins with L-tryptophan
and is successively catalyzed by enzymes VioA, B, E, D, and C, all of which are encoded by the
vioABCDE operon [
]. Strains JW1T, JW3, and P. byunsanensis JCM 12483T produced
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*Summed features represent groups of two fatty acids that could not be separated by GLC with the MIDI system. Summed feature 2 contained C14:0 3OH
and/or iso-C16:1 I; Summed feature 3 contained C16:1ω7c and/or iso-C15:0 2OH.
violacein and possessed vioABCDE operon, while P. arabiensis JCM 17292T, P. shioyasakiensis
JCM 18891T and P. gelatinilytica NH153T did not (Table 3 and S3 Fig).
The genomes of strains JW1T and JW3 were annotated and analyzed to identify the major
metabolic pathways of carbon, nitrogen, sulfur, and phosphorus based on key genes. JW1T
and JW3 can use organic carbon sources (refer to the species description). They harbor key
genes of the Entener±Doudoroff pathway, glycolysis pathway, pentose phosphate pathway,
and tricarboxylic acid cycle. The genomes of JW1T and JW3 possess an ammonium
transporter gene, but they lack genes involved in nitrate reduction, nitrite reduction, nitrogen
fixation, nitrification, or anaerobic ammonium oxidation. Thus, both strains can utilize only
reduced nitrogen. Genes encoding urease and urea transporter were not detected, suggesting
that JW1T and JW3 are incapable of utilizing urea as a C or N source. Both genomes possess a
variety of sulfate permease genes involved in assimilatory SO42+ reduction. Sulfate can be
reduced to sulfide and is subsequently incorporated into amino acids (e.g. cysteine). The
genomes of JW1T and JW3 harbor genes encoding low-affinity inorganic phosphate
transporter and sodium-dependent phosphate transporter. The presence of alkaline phosphatase
genes in both genomes indicates that JW1T and JW3 are capable of utilizing both inorganic
and organic forms of phosphorus.
Strains JW1T and JW3 possess some properties, particularly chemotaxonomic characteristics,
that species of the genus Pseudoalteromonas all share. On the other hand, JW1T and JW3 could
be distinguished from the type strains of their closely related species on the basis of phenotypic
differences (e.g., color, NaCl, pH and temperature ranges and optima, arginine dihydrolase,
citrate utilization, nitrate reduction, tryptophan deaminase, urease, Voges±Proskauer reaction,
carbohydrate utilization, and acid production, Table 1). In addition, strains JW1T and JW3
produce violacein, a potential pharmaceutical agent. On the basis of phylogenetic, genome,
and chemotaxonomic data, as well as phenotypic characteristics, strains JW1T and JW3
represent a novel species of the genus Pseudoalteromonas, for which the name Pseudoalteromonas
amylolytica sp. nov. is proposed.
Description of Pseudoalteromonas amylolytica sp. nov
Pseudoalteromonas amylolytica (a.my.lo.ly'ti.ca. Gr. n. amylon, starch; N.L. adj. lyticus -a -um
(from Gr. adj. lytikos -ê -on), able to loosen, able to dissolve; N.L. fem. adj. amylolytica, starch
Cells are Gram-stain-negative, rod-shaped, 0.7±1.2 μm in width and 1.8±3.0 μm in length.
Colonies are violet, circular, convex, smooth and 1±2 mm in diameter after one day of
incubation at 30ÊC on MA. Grow on NaCl-free MB supplemented with 0.5±10% (w/v) NaCl
(optimum 1.0±3.0%). pH and temperature ranges for growth are pH 6±10.5 and 20±40ÊC
(optimum at pH 7.5 and 30ÊC). Require sea salts for growth. No anaerobic growth occurs on
MA supplemented with sodium nitrate or sodium nitrite. Produce violacein. Positive for
catalase, oxidase, tryptophan deaminase, and Voges±Proskauer reaction. Negative for arginine
dihydrolase, citrate utilization, β-galactosidase, glucose fermentation, nitrate reduction, lysine
and ornithine decarboxylases, indole formation, H2S production, and urease. Esculin, gelatin,
starch, Tween-20, -40, and -80 are hydrolyzed. The following compounds are utilized as sole
carbon and energy sources: N-acetyl-glucosamine, L -alanine, L-arginine, D-glucose, L
-histidine, L -isoleucine, D-maltose, sodium acetate, sodium propionate, sodium pyruvate, sucrose
and D-trehalose. Acid is produced from D-glucose, D-maltose, sucrose, and D-trehalose.
Principal fatty acids (> 10%) are summed feature 3 (C16:1ω7c and/or iso-C15:0 2OH), C18:1ω7c, and
C16:0. Sole respiratory quinone is Q-8. Major polar lipids are phosphatidylethanolamine,
phosphatidylglycerol, one unidentified glycolipid, one unidentified aminolipid, and one
unidentified phospholipid. In addition, moderate to minor amounts of three unidentified glycolipids,
one unidentified aminolipid, one unidentified phospholipid, and two unidentified lipids are
present. DNA G+C content is 43.3 mol%.
The type strain, JW1T (= CGMCC 1.15681T = KCTC 52406T = MCCC 1K02162T), and
additional strain JW3 were isolated from surface seawater.
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S1 Table. COG annotations of JW1T, JW3, and related Pseudoalteromonas species.
S1 Fig. Maximum-likelihood phylogenetic tree based on concatenated sequences of the six
housekeeping genes atpD, gyrB, mreB, recA, rpoD, and topA showing the phylogenetic
relationships of JW1T, JW3, and related taxa. The gene sequences were obtained from the
genomes, the accession numbers of which are indicated in parentheses. Bootstrap values
(>90%) based on 1,000 replications are shown at branch nodes. Bar, 0.05 substitutions per
S2 Fig. Transmission electron micrographs showing the cell ultrastructure of strains
JW1T (a) and JW3 (b). Bar, 0.5 μm.
S3 Fig. Absorption profile (a) and mass spectrum (b) of violacein.
S4 Fig. Thin-layer chromatograms after staining with molybdatophosphoric acid, α-naph
thol reagent, ninhydrin reagent, and molybdenum blue showing the total polar lipid
profiles of strains JW1T (a1-a4), JW3 (b1-b4), and P. byunsanensis JCM 12483T (c1-c4). PE,
Phosphatidylethanolamine; PG, phosphatidylglycerol; AL, aminolipid; GL, glycolipid; PL,
phospholipid; L, other lipid.
We would like to thank Prof. Aharon Oren for help with etymology.
Conceptualization: YW XX.
Data curation: HC LX.
Formal analysis: HC LX.
Funding acquisition: YW XX.
Investigation: YW XJ.
Methodology: YW XX.
Project administration: XX.
Software: HC LX.
Writing ± original draft: YW XX.
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Writing ± review & editing: XX.
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