Microseiramide from the freshwater cyanobacterium Microseira sp. UIC 10445

Article abstract of DOI:10.1016/j.phytol.2015.05.003

Abstract Microseiramide (1), a cyclic heptapeptide, was isolated from a sample of the freshwater cyanobacterium Microseira sp. UIC 10445 collected in a shallow lake in Northern Indiana. Taxonomic identification of UIC 10445 was performed by a combination of morphological and phylogenetic characterization. Phylogenetic analysis revealed that UIC 10445 was a member of the recently described genus Microseira, which is phylogenetically distinct from the morphologically similar genera, Moorea and Lyngbya. The planar structure of microseiramide (1) was determined by extensive 1D and 2D NMR experiments as well as HRESIMS analysis. The absolute configurations of amino acid residues were determined using acid hydrolysis followed by the advanced Marfey's analysis. Microseiramide (1) is the first cyclic peptide reported from a Microseira sp., and the structure of microseiramide (1) is distinct from the previously known metabolites from cyanobacteria of the genera Moorea and Lyngbya.

Full text of DOI:10.1016/j.phytol.2015.05.003

Phytochemistry Letters 13 (2015) 47–52
Contents lists available at ScienceDirect
Phytochemistry Letters
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Short communication
Microseiramide from the freshwater cyanobacterium Microseira sp. UIC
10445
*
Shangwen Luo, Aleksej Krunic, George E. Chlipala, Jimmy Orjala
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 S. Wood Street, Chicago, IL 60612, United
States
A R T I C L E I N F O
A B S T R A C T
Article history:
Microseiramide (1), a cyclic heptapeptide, was isolated from a sample of the freshwater cyanobacterium
Microseira sp. UIC 10445 collected in a shallow lake in Northern Indiana. Taxonomic identification of UIC
Received 22 February 2015
Received in revised form 5 May 2015
Accepted 7 May 2015
10445 was performed by
a combination of morphological and phylogenetic characterization.
Phylogenetic analysis revealed that UIC 10445 was a member of the recently described genus Microseira,
which is phylogenetically distinct from the morphologically similar genera, Moorea and Lyngbya. The
planar structure of microseiramide (1) was determined by extensive 1D and 2D NMR experiments as well
as HRESIMS analysis. The absolute configurations of amino acid residues were determined using acid
hydrolysis followed by the advanced Marfey’s analysis. Microseiramide (1) is the first cyclic peptide
reported from a Microseira sp., and the structure of microseiramide (1) is distinct from the previously
known metabolites from cyanobacteria of the genera Moorea and Lyngbya.
Available online 23 May 2015
Keywords:
Cyanobacteria
Microseira sp.
Cyclic hetapeptide
Microseiramide
ã2015 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
1. Introduction
reported from freshwater Lyngbya spp. (Engene et al., 2010). To our
knowledge, the only metabolites reported from freshwater
Cyanobacteria, in particular marine members of the genus
Lyngbya (now classified as Moorea), are known to be a rich source of
biologically active natural products. Over 260 novel secondary
metabolites had been discovered from marine strains of the genus
Lyngbya up to 2010 (Engene et al., 2011). These metabolites account
for over 40% of all the metabolites reported from marine
cyanobacteria. These metabolites exhibit a broad spectrum of
activities including antimicrobial, antiproliferative, anticancer,
antifeedant, antifungal, and anti-inflammatory activities (Burja
et al., 2001; Jones et al., 2011). The genus Lyngbya is considered a
polyphyletic genus and cyanobacteria belong to the marine
Lyngbya lineage have been reclassified and constituted a new
genus named Moorea, in recognition of the extraordinary
contributions of Dr. Richard E. Moore in the area of marine natural
products research (Engene et al., 2012). A SciFinder search of
publications from January of 2009 to March of 2014 revealed that
84 novel metabolites were reported from marine cyanobacteria
identified as Lyngbya and six novel metabolites were obtained from
cyanobacteria of the genus Moorea. Freshwater cyanobacteria
strains identified as Lyngbya spp. do not form a monophyletic clade
in phylogenetic analysis, and few secondary metabolites have been
Lyngbya strains are lyngbyazothrins A–D and lyngbyaureidamides
A and B, all obtained from Lyngbya sp. (SAG 36.91) (Zainuddin et al.,
2009; Zi et al., 2012). Compounds structurally related to
lyngbyazothrins A–D have also been reported from strains that
are morphologically similar to Lyngbya. These compounds include
schizotrin A isolated from Schizotrix sp. (TAU strain IL-89-2)
(Pergament and Carmeli, 1994), pahayokolides A and B isolated
from a marine Lyngbya sp. (An et al., 2007), tychonamides A and B
isolated from
a Tychonema sp. (Mehner et al., 2008), and
portoamides A–D isolated from Oscillatoria sp. LEGE 5292 (Leao
et al., 2010).
We recently collected a cyanobacteria strain (UIC 10445) from
the bottom of a shallow freshwater lake in Northern Indiana. This
strain grew as a thick mat in its natural environment. Morphologi-
cal and phylogenetic analysis revealed that UIC 10445 was closely
related to strains previously identified as Lyngbya wollei, a known
producer of the neurotoxic saxitoxins, as well as the odor
compounds geosmin and 2-methylisoborneol (Carmichael et al.,
1997; Foss et al., 2012; Schrader and Blevins, 1993; Watson et al.,
2008). Recently, strains belong to the group of L. wollei were
considered to form a separate genus described as Microseira.
(McGregor and Sendall, 2015) Thus, UIC 10445 was identified as a
Microseira sp. 1H NMR and LC–MS dereplication of the organic
extract indicated the presence of a potentially new peptide (Orjala
et al., 2011). Herein, we present the isolation, structure
* Corresponding author. Tel.: +1 312 996 5583; fax: +1 312 996 7107.
E-mail address: orjala@uic.edu (J. Orjala).
http://dx.doi.org/10.1016/j.phytol.2015.05.003
1874-3900/ã 2015 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.

48
S. Luo et al. / Phytochemistry Letters 13 (2015) 47–52
determination, and biological evaluation of the first new cyclic
peptide named microseiramide (1), from a Microseira sp. UIC
10445.
by reversed-phase HPLC yielded microseiramide (1, 1.9 mg, 0.01%
of dry biomass).
Microseiramide (1) was obtained as a white, amorphous
powder. The molecular formula of
C
1 was determined as
37H56N8O9 (m/z 757.4258 [M + H]+) by HRESIMS analysis. The
2. Results and discussion
signal distribution pattern observed in the 1H NMR and DEPTQ
spectra (Table 1) indicated the peptidic nature of 1. 1H NMR of 1
exhibited a group of exchangeable amide NH signals (dH 7.2–8.7) as
well as one primary amide moiety (dH 7.19; 7.91), amino acid
The biomass was manually cleaned to remove debris, followed
by lyophilization and extraction. The extract was subjected to
vacuum column chromatography using Diaion HP-20SS resin with
a gradient of isopropyl alcohol (IPA) in H2O to yield eight fractions.
LC–MS and 1H NMR dereplication of the fraction eluting with 40%
IPA indicated the presence of a potentially new peptide with a
molecular weight of 756 Da (Orjala et al., 2011). Final purification
a
-proton signals (dH 3.6–4.7), and aliphatic methylene and methyl
signals (dH 0.7–3.0). DEPTQ spectrum of 1 exhibited amide
carbonyl signals (dC 169–173), signals typical of -carbons (dC
a
50–64), and aliphatic methyl, methylene and methine signals (dC
12–39). 1H NMR of 1 revealed the presence of several small peaks,
Table 1
NMR Spectroscopic data of microseiramide (1) in DMSO-d6.
Position
dCa, mult
dHb, mult.
(J in Hz)
COSYd
HMBCd
ROESYd
Ile
1
2
3
170.0, C
59.7, CH
35.2, CH
14.6, CH3
25.7, CH2
3.70, d (6.1)
1.64, m
0.92, d (6.9)
1.17, m
NH, 3
2, 3-Me, 4
3
NH
4, 3-Me, NHSer
3-Me
4
2, 3, 4
3. 5
1.64, m
5
NH
1
2
3
11.4, CH3
0.89, t (7.2)
8.61, s
4
2
4
2, 1Asn
2, 2Asn
Asn
170.9, C
49.6, CH
34.2, CH2
4.67, m
3.03, dd (17.3, 3.5)
3.07, dd (17.3, 3.5)
NH, 3
2
3, NHIle
5Pro
4
4
172.6, C
NH
NH2
8.63, s
7.19, s
7.91, s
2
1Val1
3, 4
2Val1
3
Val1
Phe
1
2
3
171.4, C
57.5, CH
32.4, CH
19.3, CH3
18.7, CH3
4.36, overlapped
1.72, m
0.90, d (6.7)
0.84, d (6.7)
7.29, overlapped
NH, 3
2, 3-Me, 4
3
3
2
3, 3-Me, NH
NH
2, 3
2, 3
1Phe
4, NHAsn
3-Me
4
NH
1
2
2Phe
170.5, C
54.2, CH
37.9, CH2
4.33, m
2.89, t (13.2)
3.20, dd (13.2, 3.2)
NH, 3
2
3, NH
2, 4, 5/9
NHVal1
3
4
138.4, C
5/9
6/8
7
NH
1
126.2, CH
128.0, CH
129.0, CH
7.18, m
7.25, m
7.13, m
8.08, d (10.4)
6/8, 7
5/9, 7
5/9, 6/8
2
3, 5/9, 6/8, 7
5/9, 6/8, 7
1Pro
3
3, 2Pro
Pro
169.9, C
62.9, CH
28.8, CH2
2
3
3.91, dd (7.5, 2.6)
1.23, m
3
2, 4
4, NHPhe
1.95, m
4
5
25.3, CH2
47.6, CH2
1.65, m
1.77, m
3.33, m
3, 5
4
2, 3
3Asn, 2Val2, 3Val2
3.83, m
Val2
Ser
1
2
3
169.7, C
61.4, CH
31.8, CH
19.5, CH3
19.4, CH3
4.02, overlapped
1.70, m
0.85, d (6.7)
0.77, d (6.7)
8.24, d (10.3)
NH, 3
2, 3-Me, 4
3
3
2
3, 4, NH
4, 5Pro
5Pro
3-Me
4
NH
1
2
2, 3
2, 3
1Ser
2Ser
168.9, C
58.9, CH
60.0, CH2
3.62, m
NH, 3
2
3
2
NHVal2
3
3.82, overlapped
4.04, overlapped
ndc
OH
NH
8.65, s
2
1Ile
2Ile
a
Carbon chemical shifts were assigned from DEPTQ spectrum recorded at 226 MHz.
Recorded at 600 MHz.
nd: not detected.
b
c
d
Correlations without a subscript are within the amino acid residue.

S. Luo et al. / Phytochemistry Letters 13 (2015) 47–52
49
presumably due to the presence of a minor conformer, which is
commonly observed for cyclic peptides with proline residues
(Brennan et al., 2008; Tripathi et al., 2009; Zhang et al., 2010). The
structure elucidation of
1 was performed using the major
conformer. Combined analysis of the COSY and TOCSY spectra
identified the presence of seven amino acid residues: serine (Ser),
isoleucine (Ile), proline (Pro), asparagine (Asn), phenylalanine
(Phe) and two valines (Val1 and Val2).
The complete sequence of the seven amino acid residues in 1
was established by analysis of the HMBC and ROESY spectra (Fig. 1
and Table 1). HMBC correlations from Val2-NH (dH 8.24) to Ser C-1
(dC 168.9), from Ser-NH (dH 8.65) to Ile C-1 (dC 170.0), from Ile-NH
(dH 8.61) to Asn C-1 (dC 170.9), from Asn-NH (dH 8.63) to Val1 C-1
(dC 171.4), from Val1-NH (dH 7.29) to Phe C-1 (dC 170.5), and from
Phe-NH (dH 8.08) to Pro C-1 (dC 169.9) suggested a sequence of
Val2-Ser-Ile-Asn-Val1-Phe-Pro (Table 1). ROESY correlations con-
firmed this linear sequence. ROESY correlations between Pro H2-5
(dH 3.83) and Val2 H-2 (dH 4.02), and between Pro H2-5 (dH 3.83)
and Val2 H-3 (dH 1.70) closed the macrocycle and identified the
peptide sequence as cyclo[Val2-Ser-Ile-Asn-Val1-Phe-Pro-]. This
sequence assignment was confirmed by MS/MS analysis using a
quadrupole-time-of-flight (Q-TOF) tandem mass spectrometer
(Fig. 2). The parent ion [M + H]+ 757.5 was fragmented using a
35 eV collision energy and the macrocycle was opened between
Pro and Val2 residues. Continuous fragmentation generated
fragment ions as shown in Fig. 1, and were in complete agreement
with the sequence determined by NMR analysis.
Fig. 2. Q-TOF MS/MS fragmentation analysis of microseiramide (1).
through the posttranslational modifications of short precursor
proteins, or through a nonribosomal peptide synthetase (NRPS)
pathway.
The absolute configurations of the amino acids were assigned
using the advanced Marfey’s method after acid hydrolysis
Microseiramide (1) was evaluated in the brine shrimp lethality
(Supporting information), and assigned
the amino acid residues. (Fujii et al., 1997a,b) Thus, the final
structure of 1 was established as cyclo[ -Val2- -Ser- -Ile- -Asn-
Val1- -Phe- -Pro-].
Microseiramide (1) is
proteinogenic amino acids with
L configurations for all
assay (132
435 human cancer cell lines (33
and chymotrypsin) inhibition assays (13
inhibition assays against Mycobacterium tuberculosis, Mycobacteri-
um smegmatis, Staphylococcus aureus, Escherichia coli and Candida
m
M), cytotoxicity assays against HT-29 and MDA-MB-
M), protease (elastase, trypsin,
M), as well as growth
m
L
L
L
L
L-
m
L
L
a
cyclic peptide containing seven
configurations. The structural
L
albicans (50 mg/mL), however, no activity was observed in any of
features of 1 matched with the definition of cyanobactins, which
are structurally variant and ribosomally synthesized cyclic
peptides composed of seven to 20 amino acids, including a
conserved proline residue (Leikoski et al., 2010; Sivonen et al.,
2010). Recently, genome sequencing revealed the broad distribu-
tion of ribosome-dependent biosynthetic pathways across the
multiple genera in the phylum Cyanobacteria (Shih et al., 2013).
Further, investigations of UIC 10445 are necessary to determine
whether microseiramide (1) is biosynthesized by ribosome
the assays at the highest concentration tested.
Although, the ecological functions of most natural products
produced by cyanobacteria are still undetermined, it is broadly
accepted that these compounds are produced to affect the
surrounding environment and act as defense against predators
and competitors (Burja et al., 2001). Microseiramide (1) may
contribute to the extremely high population density of UIC 10445
observed in its wild habitat. However, the limited supply
prevented evaluation in further assays to explore its potential
ecological function.
The initial taxonomic designation of UIC 10445 was performed
using traditional morphological analysis, and indicated that this
strain was morphologically similar to cyanobacteria of the genus
Lyngbya (Supporting information) (Castenholz, 2001; Komárek
et al., 2003). A phylogenetic analysis, using a 1.2 kb partial
sequence of 16S rDNA, was performed to assist the taxonomic
identification and indicated that UIC 10445 was a member of the L.
wollei clade. A very recently published report revealed that
cyanobacteria previously identified as L. wollei were distinct from
the other freshwater species of the genus Lyngbya and that these
cyanobacteria should be considered a separate genus described as
Microseira (McGregor and Sendall, 2015). Our data also showed
that the clade containing UIC 10445 also contains Microseira wollei
references strains. That clade was phylogenetically distinct from
the other Lyngbya clades, as well as the Moorea clade (previously
known as marine Lyngbya clade) (Fig. 3). Thus, we identify UIC
10445 as a Microseira sp.
Cyanobacteria of the genus Lyngbya are thought to be some of
the most prolific producers of secondary metabolites (Burja et al.,
2001; Engene et al., 2011; Jones et al., 2011). However, the
Fig. 1. Key 2D NMR correlations of microseiramide (1).

50
S. Luo et al. / Phytochemistry Letters 13 (2015) 47–52
Fig. 3. Neighbor-joining (NJ) phylogenetic tree computed using 16S rRNA gene sequence data. Species and strain numbers are given with accession numbers in parentheses.
Reference strains obtained from Bergey’s Manual were denoted with an asterisk (*) (Castenholz, 2001). Reference strains characterized by Engene et al. (2012) were denoted
with two asterisks (**). Only bootstrap values greater than or equal to 75% are displayed.
traditional taxonomic identification of cyanobacteria has been
based on morphological characterization, and cyanobacteria
previously identified as members of the genus Lyngbya have been
In summary, we have isolated a new cyclic heptapeptide from a
field collected sample of the freshwater cyanobacterium Microseira
sp. UIC 10445. This is the first report of cyclic peptide produced by a
Microseira sp. (previously known as L. wollei). Microseiramide (1)
found to be phylogenetically distinct from each other.
A
comprehensive study of the phylogeny of the genus Lyngbya
revealed that Lyngbya is a polyphyletic genus containing at least
three distinct lineages: a marine lineage, a lineage closely related
to the genus Oscillatoria, and a halophilic/brackish/freshwater
lineage (Engene et al., 2010). Cyanobacteria belonging to the
marine lineage, recently reclassified to be members of the genus
Moorea, are responsible for more than 40% of all reported marine
cyanobacterial secondary metabolites (Engene et al., 2011, 2012).
Freshwater Lyngbya strains either fall into the clade of halophilic/
brackish/freshwater lineage, or belong to a larger polyphyletic
group (Engene et al., 2010). Although, the freshwater L. wollei
cluster was recently redefined to form a new genus Microseira,
other cyanobacteria identified as freshwater Lyngbya spp. are still
polyphyletic, and the taxonomy of Lyngbya genus may need further
revision (Fig. 3).
consists of seven standard amino acid residues with L config-
urations and displayed no significant activity in the brine shrimp
lethality assay, cytotoxicity assays, protease inhibition assays, anti-
microbial, and anti-fungal assays.
3. Experimental
3.1. General experimental procedures
The optical rotation was measured using a PerkinElmer 241
polarimeter at 22 ꢀC in MeOH. The UV spectrum was recorded
using a Shimadzu UV spectrophotometer UV2401. The IR spectrum
was acquired on a PerkinElmer 577 IR spectrophotometer. The 1D
and 2D NMR spectra including 1H NMR, COSY, TOCSY, HSQC, HMBC,
and ROESY were acquired on a Bruker Avance DRX 600 MHz

S. Luo et al. / Phytochemistry Letters 13 (2015) 47–52
51
spectrometer, whereas a Bruker Avance AVII 900 MHz NMR
spectrometer was used to acquire the DEPTQ spectrum. 1H and
13C NMR chemical shifts were referenced to the DMSO-d5 residual
solvent signals (dH 2.50 and dC 39.51, respectively). The TOCSY
experiment was conducted using a 60 ms mixing time. The ROESY
spectrum was acquired with a 200 ms mixing time. The HSQC
reference strains Moorea producens 3L and Moorea bouillonii
PNG5-198 were chosen according to the literature by Engene
et al. (2012). The reference strains of M. wollei were selected
according to the publication by McGregor and Sendall (2015). All
the other strains were selected based upon a BLAST search of the
UIC 10445 16S rDNA partial sequence. Phylogenetic trees were
constructed using neighbor joining, maximum parsimony, and
minimum devolution methods, and all three trees showed similar
topology. The evolutionary history was inferred using the
neighbor-joining, minimum evolution, and maximum parsimony
methods. For each method, the bootstrap consensus tree inferred
from 1000 replicates was taken to represent the evolutionary
history of the taxa analyzed. Phylogenetic analysis revealed that
UIC 10445 was a member of the Microseira cluster. Combining the
morphological characters of UIC 10445, we identify UIC 10445 as
Microseira sp.
1
spectrum was recorded with the average JCH of 145 Hz and the
3
HMBC spectrum was recorded with the average JCH of 8 Hz. The
high-resolution ESI mass spectrum and LC–MS data were acquired
using a Shimadzu HPLC-IT-TOF spectrometer. The MS/MS frag-
mentation analysis was carried out on a Waters Q-TOF spectrome-
ter using ESI. The HPLC separations were performed using an
Agilent 1100 series instrumentation and a Waters instrumentation
with Waters Delta 600 pump and Waters 2487 UV detector.
3.2. Biological material and morphological identification
Microseira sp. UIC 10445 was collected from the bottom of a
shallow freshwater lake in Northern Indiana in July 2013
(N41ꢀ1802400, W85ꢀ440600). The biomass was manually cleaned to
remove debris and freeze-dried. The initial taxonomic identifica-
tion was performed by morphological observation according to the
system by Komárek et al. (2003) using a Zeiss Axiostar Plus light
microscope equipped with a Canon PowerShot A620 camera
(Komárek et al., 2003). The morphological characteristics can be
found in Supporting information.
3.5. Extraction and isolation
The lyophilized biomass (13.9 g) was extracted with a mixture
of CH2Cl2 and MeOH (1:1) and dried in vacuo to yield 1.38 g organic
extract. The extract was fractionated using Diaion HP-20SS vacuum
liquid chromatography with an IPA/water step gradient (0%, 20%,
40%, 60%, 70%, 80%, 90%, and 100%) to yield eight fractions. LC–MS
and 1H NMR dereplication indicated the presence of a potentially
new peptide with a molecular weight of 756 Da in the fraction
eluted at 40% IPA (Orjala et al., 2011). Subsequent reversed-phase
semi-preparative HPLC (Varian C18 semi-preparative column,
3.3. DNA extraction, 16S rDNA PCR amplification and sequencing
10 ꢁ 250 mm, 5
mm, 3 mL/min, 65–100% MeOH in water over
25 min) yielded microseiramide (1, 7.8 min, 1.9 mg, 0.01% of dry
A cleaned sample of Microseira sp. UIC 10445 was combined
with 1.5 mL of lysozyme buffer and 0.5 mL of 20 mg/mL lysozyme
stock solution. The cell mass was centrifuged and transferred to a
2 mL microcentrifuge tube after incubation at 35 ꢀC for 1 h. The
genomic DNA was extracted using a Wizard Genomic DNA
purification kit from Promega. A partial sequence of the 16S rDNA
gene was amplified by PCR using the cyanobacteria-specific
primers 109F and 1509R (Nubel et al., 1997). The PCR reaction
biomass).
3.6. Microseiramide (1)
White, amorphous powder; ½a D22
ꢂ ꢃ30.7 (c 0.15, MeOH); UV
(MeOH) lmax (log e) 214 (4.01), 265 (2.72) nm; IR (neat) nmax 3303
(br), 2967, 2877,1661, 1524,1439,1204,1135 cmꢃ1; 1H and 13C NMR
mixture contained DNA (1
Reaction Buffer (5 L), dNTP mix (0.5
primer (1 L each, 10 M), GoTaq DNA Polymerase (0.25
L), and sufficient nuclease free water to make the total volume
25 L. The reaction was performed in a Bio-Rad C1000 thermal
m
L, approximately 50 ng), 5 ꢁ GoTaq
L, 10 mM), 109F and 1509R
L, 2.5 u/
see Table 1; HR-ESI-TOF-MS (+) m/z 757.4258 [M + H]+ (calcd for
m
m
C37H57N8O9, 757.4249).
m
m
m
m
3.7. Determination of absolute configurations of amino acids by the
advanced Marfey’s analysis
m
cycler using the following reaction program: initial denaturation
for 2 min at 95 ꢀC, 35 amplification cycles of 50 s at 95 ꢀC, 50 s at
49 ꢀC, and 2 min at 72 ꢀC, and a final extension for 5 min at 72 ꢀC.
PCR products were purified using a MinElute PCR purification kit
from Qiagen and subjected to Sanger sequencing using the
cyanobacteria-specific primers 109F, 359F, and 1509R (Nubel
et al., 1997). The resulting 16S rDNA gene sequence was deposited
in the NCBI GenBank under the Accession No. KJ813000.
Approximately 0.2 mg of 1 was hydrolyzed in 1 mL 6N HCl for
20 h at 110 ꢀC in pressure tubes sealed with teflon tape. The cooled
hydrolysate mixtures were dried in vacuo, and traces of HCl were
removed by repeated evaporation. The resulting hydrolysate was
separated into two equal portions for derivatization with either
or -FDLA (1-fluoro-2,4-dinitrophenyl-5-leucinamide, from TCI
America). Each hydrolysate portion was dissolved in 110 L of
acetone followed by 50 L of de-ionized water, and then mixed
with 20 L of 1 N NaHCO3. Finally, 20 L of -FDLA or -FDLA
L-
D
m
m
3.4. Phylogenetic analysis of Microseira sp. UIC 10445
m
m
L
D
(10 mg/mL in acetone) was added, and the mixtures were heated to
MEGA 5.05 was used to perform the phylogenetic analysis. The
resulting Sanger sequencing chromatograms were visually
inspected, and the total sequence of 1234 nucleotides (GenBank
accession number KJ813000) was aligned with 31 cyanobacterial
species obtained from GenBank (http://www.ncbi.nlm.nih.gov).
Multiple sequence alignment was performed via the ClustalW
interface. The aligned sequences were used to construct phyloge-
netic tress in MEGA 5.05. The calculated optimum nucleotide
substitution model was Kimura 2-parameter with a discrete
Gamma distribution parameter of 0.19. The reference strains
Oscillatoria acuminate PCC6304, Oscillatoria sancta PCC7515, and
Lyngbya aestuarii PCC7419 were selected according to Bergey’s
Manual of Systematic Bacteriology (Castenholz, 2001). The
40 ꢀC for 1 h. The reaction mixtures were cooled to room
temperature, and 20
reaction. The cooled reaction mixtures were dried in vacuo, and re-
suspended in 300 L acetonitrile. LC–MS analysis was performed
using reversed-phase column (Phenomenex Kinetex C18
250 ꢁ 4.6 mm, 5 m, 1.0 mL/min) with a linear gradient from
25% to 65% aqueous acetonitrile containing 0.1% formic acid over
50 min. The extracted ion chromatograms of -FDLA and DL-FDLA
derivatives (DL-FDLA derivative was the mixture of - and -FDLA
mL of 1 N HCl was added to quench the
m
a
,
m
L
L
D
derivatives) of Marfey’s derivative of each amino acid were
compared for the assignment of amino acid configurations. The
absolute configuration of isoleucine residue was assigned by

52
S. Luo et al. / Phytochemistry Letters 13 (2015) 47–52
comparing the retention time of hydrolysate-
L
-FDLA derivative
Castenholz, R., 2001. Phylum BX. cyanobacteria. In: Boone, D., Castenholz, R. (Eds.),
Bergey’s Manual1 of Systematic Bacteriology. Springer, New York, pp. 473–599.
Engene, N., Choi, H., Esquenazi, E., Rottacker, E.C., Ellisman, M.H., Dorrestein, P.C.,
Gerwick, W.H., 2011. Underestimated biodiversity as a major explanation for the
perceived rich secondary metabolite capacity of the cyanobacterial genus
Lyngbya. Environ. Microbiol. 13, 1601–1610.
with the retention times of appropriate isoleucine stereo isomers
derivatized with -FDLA or -FDLA. The table for retention times of
L
D
each amino acid can be found in Supporting information.
Engene, N., Coates, R.C., Gerwick, W.H., 2010. 16S rRNA gene heterogeneity in the
filamentous marine cyanobacterial genus Lyngbya. J. Phycol. 46, 591–601.
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J., Gerwick, W.H., 2012. Moorea producens gen. nov., sp. nov and Moorea
bouillonii comb. nov.: tropical marine cyanobacteria rich in bioactive secondary
metabolites. Int. J. Syst. Evol. Microbiol. 62, 1171–1178.
Foss, A.J., Phlips, E.J., Yilmaz, M., Chapman, A., 2012. Characterization of paralytic
shellfish toxins from Lyngbya wollei dominated mats collected from two Florida
springs. Harmful Algae 16, 98–107.
Fujii, K., Ikai, Y., Mayumi, T., Oka, H., Suzuki, M., Harada, K., 1997a. A nonempirical
method using LC/MS for determination of the absolute configuration of
constituent amino acids in a peptide: elucidation of limitations of Marfey’s
method and of its separation mechanism. Anal. Chem. 69, 3346–3352.
Fujii, K., Ikai, Y., Oka, H., Suzuki, M., Harada, K., 1997b. A nonempirical method using
LC/MS for determination of the absolute configuration of constituent amino
acids in a peptide: combination of Marfey’s method with mass spectrometry
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3.8. Brine shrimp lethality assay
A modification of the previously described method was used to
assess lethality to the brine shrimp (Artemia salina) (Singh et al.,
1999). Approximately 10 hatched brine shrimps in 80 mL artificial
seawater (made from Sigma Sea Salts, S9883, 40 g/L) were added to
each well containing varying concentrations of microseiramide in
1
m
L DMSO and 19
mL of artificial seawater to make a total volume
of 100 L. Each concentration of sample was run in duplicate. The
m
number of dead and alive brine shrimps was counted under a
dissecting microscope after 24 h incubation at room temperature.
3.9. Cytotoxicity assays
Jones, A.C., Monroe, E.A., Podell, S., Hess, W.R., Klages, S., Esquenazi, E., Niessen, S.,
Hoover, H., Rothmann, M., Lasken, R.S., Yates, J.R., Reinhardt, R., Kube, M.,
Burkart, M.D., Allen, E.E., Dorrestein, P.C., Gerwick, W.H., Gerwick, L., 2011.
Genomic insights into the physiology and ecology of the marine filamentous
cyanobacterium Lyngbya majuscula. Proc. Natl. Acad. Sci. U. S. A.108, 8815–8820.
Kang, H.S., Krunic, A., Orjala, J., 2012. Stigonemapeptin, an Ahp-containing
depsipeptide with elastase inhibitory activity from the bloom-forming
freshwater cyanobacterium Stigonema sp. J. Nat. Prod. 75, 807–811.
Komárek, J., Komárková, J., Kling, H., 2003. 4-filamentous cyanobacteria. In: John, D.
W., Robert, G.S. (Eds.), Freshwater Algae of North America. Academic Press,
Burlington, pp. 117–196.
Leao, P.N., Pereira, A.R., Liu, W.T., Ng, J., Pevzner, P.A., Dorrestein, P.C., Konig, G.M.,
Vasconcelosa, V.M., Gerwick, W.H., 2010. Synergistic allelochemicals from a
freshwater cyanobacterium. Proc. Natl. Acad. Sci. U. S. A. 107, 11183–11188.
Leikoski, N., Fewer, D.P., Jokela, J., Wahlsten, M., Rouhiainen, L., Sivonen, K., 2010.
Highly diverse cyanobactins in strains of the genus Anabaena. Appl. Environ.
Microbiol. 76, 701–709.
Cytotoxicity assays against the MDA-MB-435 and HT-29 cancer
cell line were performed according to established protocols (Ayers
et al., 2011).
3.10. Protease inhibition assays
The inhibition assays against elastase, trypsin, and chymotryp-
sin were conducted according to established protocols (Kang et al.,
2012).
3.11. Anti-bacterial and anti-fungal assays
Luo, S., Kang, H.S., Krunic, A., Chlipala, G.E., Cai, G.P., Chen, W.L., Franzblau, S.G.,
Swanson, S.M., Orjala, J., 2014. Carbamidocyclophanes F and G with anti-
Mycobacterium tuberculosis activity from the cultured freshwater
cyanobacterium Nostoc sp. Tetrahedron Lett. 55, 686–689.
McGregor, G.B., Sendall, B.C., 2015. Phylogeny and toxicology of Lyngbya wollei
(Cyanobacteria/Oscillatoriales) from north-eastern Australia, with a description
of Microseira gen. nov. J. Phycol. 51, 109–119.
The inhibition assays against M. tuberculosis, M. smegmatis, S.
aureus, E. coli and C. albicans were performed according to
established protocols (Luo et al., 2014).
Acknowledgements
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H., Brun, R., Konig, G.M., 2008. A novel beta-amino acid in cytotoxic peptides from
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of potential anticancer agents from aquatic cyanobacteria, filamentous fungi,
and tropical plants, Bioactive Compounds from Natural Sources. Second edition
CRC Press, pp. 37–64.
This research was supported by grant PO1 CA125066 from NCI/
NIH. The 900 MHz NMR spectrometer was purchased by NIH grant
GM068944 to Dr. Peter G.W. Gettins. We thank Dr. B.E. Ramirez for
providing access to NMR spectrometers at the UIC Center for
Structural Biology (CSB). We thank Dr. M. Johnson and H. Lei for
providing access to a plate reader for protease assays. We thank Dr.
M. Federle for providing access to the thermo cycler.
Pergament, I., Carmeli, S., 1994. Schizotrin A; a novel antimicrobial cyclic peptide
from a cyanobacterium. Tetrahedron Lett. 35, 8473–8476.
Schrader, K.K., Blevins, W.T., 1993. Geosmin-producing species of streptomyces and
lyngbya from aquaculture ponds. Can. J. Microbiol. 39, 834–840.
Shih, P.M., Wu, D.Y., Latifi, A., Axen, S.D., Fewer, D.P., Talla, E., Calteau, A., Cai, F., de
Marsac, N.T., Rippka, R., Herdman, M., Sivonen, K., Coursin, T., Laurent, T.,
Goodwin, L., Nolan, M., Davenport, K.W., Han, C.S., Rubin, E.M., Eisen, J.A.,
Woyke, T., Gugger, M., Kerfeld, C.A., 2013. Improving the coverage of the
cyanobacterial phylum using diversity-driven genome sequencing. Proc. Natl.
Acad. Sci. U. S. A. 110, 1053–1058.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
phytol.2015.05.003.
Singh, I.P., Milligan, K.E., Gerwick, W.H., 1999. Tanikolide, a toxic and antifungal
lactone from the marine cyanobacterium Lyngbya majuscula. J. Nat. Prod. 62,
1333–1335.
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