Antibacterial low-molecular-weight compounds produced by the marine bacterium Rheinheimera japonica KMM 9513T

Article abstract of DOI:10.1007/s10482-017-0839-1

Strain KMM 9513^T was isolated from a sediment sample collected from the Sea of Japan seashore and selected due to its ability to inhibit indicator bacterial growth. The strain KMM 9513^T has been recently described as a novel species Rheinheimera japonica. This study was undertaken to determine which substances produced by strain KMM 9513^T could be responsible for its antimicrobial activity. Eight compounds were obtained from an ethyl acetate extract of R. japonica KMM 9513^T. The structures of five diketopiperazines (4–8) and diisobutyl-, dibutyl- and bis(2-ethylhexyl) phthalates (1–3) were established on the basis of detailed interpretation of NMR data, by Marfey method and optical rotation data. The structures of diketopiperazines were determined as cyclo-(l-valyl-l-proline), cyclo-(l-valyl-d-proline), cyclo-(l-phenylalanyl-l-proline), cyclo-(l-leucyl-l-proline), and cyclo-(l-phenylalanyl-d-proline). Compounds 1–3, 5 and 8 revealed antimicrobial activities against Bacillus subtilis and/or Enterococcus faecium and Staphylococcus aureus. In this paper, we describe the isolation and structural elucidation of the isolated compounds 1–8. This is the first report of the characterisation of low molecular weight antibacterial metabolites produced by a member of the genus Rheinheimera.

Full text of DOI:10.1007/s10482-017-0839-1

Antonie van Leeuwenhoek
DOI 10.1007/s10482-017-0839-1
ORIGINAL PAPER
Antibacterial low-molecular-weight compounds produced
by the marine bacterium Rheinheimera japonica KMM
9513T
.
Natalia I. Kalinovskaya Lyudmila A. Romanenko Anatoly I. Kalinovsky
.
Received: 19 September 2016 / Accepted: 31 January 2017
Ó Springer International Publishing Switzerland 2017
Abstract Strain KMM 9513T was isolated from a
sediment sample collected from the Sea of Japan
seashore and selected due to its ability to inhibit
indicator bacterial growth. The strain KMM 9513T has
been recently described as a novel species Rhein-
heimera japonica. This study was undertaken to
determine which substances produced by strain
KMM 9513T could be responsible for its antimicrobial
activity. Eight compounds were obtained from an ethyl
acetate extract of R. japonica KMM 9513T. The
structures of five diketopiperazines (4–8) and diisobu-
tyl-, dibutyl- and bis(2-ethylhexyl) phthalates (1–3)
were established on the basis of detailed interpretation
of NMR data, by Marfey method and optical rotation
data. The structures of diketopiperazines were deter-
mined as cyclo-(L-valyl-L-proline), cyclo-(L-valyl-D-
proline), cyclo-(L-phenylalanyl-L-proline), cyclo-(L-
proline). Compounds 1–3, 5 and 8 revealed antimicro-
bial activities against Bacillus subtilis and/or Entero-
coccus faecium and Staphylococcus aureus. In this
paper, we describe the isolation and structural eluci-
dation of the isolated compounds 1–8. This is the first
report of the characterisation of low molecular weight
antibacterial metabolites produced by a member of the
genus Rheinheimera.
Keywords Rheinheimera japonica Á Low-
molecular-weight metabolites Á Antibacterial activity Á
Diketopiperazines Á Phthalates
Introduction
Sea coastal environments are known to be of signif-
icance for marine biota nurseries and bioremediation
activities. Microbial communities that are associated
with coastal ecosystems are considered as a basic and
important component of these ecosystems function-
ing. It is becoming increasingly important to study
marine coastal microorganisms with the aim of
discovering novel taxa and the search for potential
bacteria capable of producing biologically active
substances (BAS). Marine-derived bacteria have
proven to be a rich source of BAS with antimicrobial,
cytotoxic or antineoplastic activities that could be
applied in biotechnology and pharmaceutics (Fenical
1993; Faulkner 1999; Debbab et al. 2010; Hughes and
Fenical 2010).
leucyl-L-proline),
and
cyclo-(L-phenylalanyl-D-
Electronic supplementary material The online version of
this article (doi:10.1007/s10482-017-0839-1) contains supple-
mentary material, which is available to authorized users.
N. I. Kalinovskaya Á L. A. Romanenko Á A. I. Kalinovsky
G.B. Elyakov Pacific Institute of Bioorganic Chemistry,
Far Eastern Branch, Russian Academy of Sciences,
Prospect 100 Let Vladivostoku, 159, Vladivostok, Russia
690022
L. A. Romanenko (&)
Far Eastern Federal University, Oktyabr’skaya Street, 27,
Vladivostok, Russia 690091
e-mail: lro@piboc.dvo.ru
123

Antonie van Leeuwenhoek
During a screening of microorganisms associated
with marine coastal sediments for their antimicrobial
activity, Gram-negative brown-pigmented bacteria
KMM 9512 and KMM 9513T were found to inhibit
growth of Enterococcus faecium, Staphylococcus
aureus, Staphylococcus epidermidis (weakly), Bacil-
lus subtilis, Xanthomonas sp. pv. badrii and Candida
albicans. On the basis of phylogenetic analysis,
genetic and phenotypic characterization strains
KMM 9512 and KMM 9513T were classified as a
novel species of the genus Rheinheimera, Rhein-
heimera japonica (Romanenko et al. 2015). Antimi-
crobial activity against B. subtilis, E. faecium and S.
aureus was revealed in ethyl acetate extracts of cells
and cell-free supernatants of novel isolates pointing
to the presence of low-molecular-weight metabolites,
which could be responsible for their activity. The
type strain KMM 9513T was chosen to be cultivated
for further investigation of its antimicrobial com-
pound production. It should be noted that our
knowledge regarding antimicrobial activity of Rhein-
heimera strains is very restricted. Recently Chen
et al. (2010a) have reported antimicrobial activity of
greenish-yellow pigmented Rheinheimera strain
GR5T against Gram-positive and Gram-negative
bacteria, yeast, algae, and the strain itself. Strain
GR5T was subsequently described as the novel
species Rheinheimera aquatica (Chen et al. 2010b).
This strain could synthesize a macromolecule with
antimicrobial activity due to the generation of
hydrogen peroxide and this antimicrobial effect can
be inhibited by catalase. The antimicrobial macro-
molecule was a monomeric protein with a molec-
ular mass of 71 kDa. The 19-amino acid fragment
derived from this protein revealed close similarity
to the antibacterial protein AlpP from the marine
bacterium Pseudoalteromonas tunicata D2, and to
the antibacterial protein, marinocine, from the
marine bacterium Marinomonas mediterranea
(Chen et al. 2010a).
from alkaline brackish water of the high-altitude
Pangong Lake of Ladakh, India, has been reported to
display antimicrobial activity (Gupta et al. 2011). The
genome of Rheinheimera sp. strain A13L is predicted to
contain genes for marinocine and colicin V production,
which may be responsible for the antimicrobial activity
of this strain. The present study focused on isolation,
separation and purification, and structure elucidation of
theactivecompoundsproducedbythestrainR.japonica
KMM 9513T.
Materials and methods
Isolation and cultivation of strain KMM 9513T
Strain R. japonica KMM 9513T was isolated from a
sediment sample collected from the Sea of Japan
seashore, Russia (Romanenko et al. 2015). Strain
KMM 9513T was grown in the seawater medium
(SWM), containing (g l-1): 5.0 peptone, 2.5 yeast
extract, 1.0 glucose, 0.2 K2HPO4, 0.05 MgSO4, 50%
aged filtered seawater, 50% distilled water. R. japon-
ica KMM 9513T was incubated in 1 l flasks each
containing 0.5 l of SWM for 72 h at 25 °C with
shaking at 120 rpm and a total of 10 l of culture was
obtained.
Extraction and isolation of compounds
Cell-free supernatant and cells after centrifugation
were extracted twice with an equal volume of ethyl
acetate, and the combined ethyl acetate extracts from
cells and supernatant were washed with H2O, dried
over Na2SO4 and concentrated under reduced pressure.
The resulting dark yellow oily extract (710 mg) was
dissolved in 30 ml of H2O:methanol 5:1 and extracted
twice with an equal volume of hexane and ethyl
acetate. Both phases collected separately were evap-
orated until dryness under reduced pressure. The active
ethyl acetate extract (210 mg) was subjected to
column chromatography on silica, using hexane/chlo-
roform ? chloroform/EtOAc gradient. As a result,
nine fractions were received, and three fractions of
them (hexane:chloroform 7:3, chloroform, and chlo-
roform/EtOAc 1:1) were active against Gram-positive
indicator microorganisms and used for further
investigations.
It was reported marine strains Rheinheimera spp.
HP1 and HP9 produced a blue pigment (Glaukothalin)
with a molecular formula of C34H56N4O4 and M?
584.85 m/z (Grossart et al. 2009). Strains HP1 and HP9
were able to inhibit two isolates of the Cytophaga-
Flavobacterium-Bacteroides group and one of the
Bacillus/Clostridium group. The cytotoxic tests showed
thatglaukothalinhadastrongactivityagainstcrustacean
Artemia salina. Rheinheimera sp. strain A13L isolated
123

Antonie van Leeuwenhoek
Spectroscopic measurements
standard amino acids were as follows: L-Pro (14.6), D-
Pro (17.1), L-Val (22.9), L-Leu (29.3), L-Phe (28.5).
Optical rotations were taken on a Perkin Elmer
Polarimeter Model 343. The nuclear magnetic reso-
nance (NMR) spectra of compounds obtained were
determined using NMR spectroscopy on Bruker DRX
500 spectrometer. Proton chemical shifts were deter-
mined from one-dimensional 1H NMR and from
HSQC experiments, and 13C chemical shifts were
Antimicrobial assay
The inhibitory activity against indicator bacteria,
Escherichia coli K-12 CL588, E. faecium CIP
104105, S. aureus CIP 65.8T, S. epidermidis CIP
81.55T, B. subtilis CIP 52.65T, Xanthomonas sp. pv.
badrii LMG 546, C. albicans KMM 455 obtained from
the respective Culture Collections was tested by an
agar diffusion method as described previously (Ro-
manenko et al. 2008). In addition, compounds 4–8
were tested for the antimicrobial activity against
marine bacteria isolated from the sediments of the Sea
of Japan seashore, Tamlana sedimentorum KMM
9545T, Loktanella maritima KMM 9530T, Sphin-
gorhabdus pacifica KMM 9574T, and Pseudomonas
marincola KMM 3042T from a deep sea brittle star
specimen. Four tested marine bacterial strains were
grown as a lawn each on Marine Agar 2216 (BD
Difco). All tests were carried out in three independent
experiments.
1
determined from HSQC and H–13C HMBC experi-
ments. Chemical shifts were reported relative to the
solvent peaks. HPLC was performed on Agilent 1100
Series instrument on an YMC-Pack-ODS-A column
(4.6 9 250 mm, S-5 lm, 12 nm). Silica gel L
100/160 (Lachema, Czech Republic) was used for
low pressure CC.
Marfey analysis of peptides
100 lg of 4–8 each sample was hydrolyzed at 110 °C
for 24 h with 200 ll of 6N HCl. These solutions were
evaporated to dryness, and the residues were dissolved
in 50 ll water. 20 ll of 1 M sodium bicarbonate and
then 100 ll of 1% Na-(2,4-dinitro-5-fluorophenyl)-L-
alaninamide (FDAA) in acetone were added to each of
these amino acid solutions. The solutions were
vortexed and incubated at 37 °C for 60 min. After-
ward, the reaction was quenched by the addition of
20 ll of 1N HCl, the reaction mixtures were diluted
with 810 ll of acetonitrile, and 5 ll of each solution
was analyzed by HPLC on a YMC-Pack-ODS-A
column (4.6 9 250 mm, S-5 lm, 12 nm), heated at
40 °C. For the HPLC gradient elution, mobile phase A
was prepared from 0.01 M aqueous trifluoroacetic
acid, which was adjusted to pH 3 by the addition of
ammonium hydroxide, and the mobile phase B was
acetonitrile. Linear gradients started with 25% mobile
phase B and finished with 55% mobile phase B in
35 min. The system was allowed to equilibrate for
10 min at 25% mobile phase B prior to the next
analysis. The flow rate was maintained as 1 ml/min
with the UV detection at 340 nm. In the hydrolyzates
of 4–8 the retention times of the Val FDAA, Val
FDAA, Phe FDAA, Leu FDAA and Phe FDAA were
22.7, 22.7, 28.7, 29.2 and 28.7 min, respectively,
which correspond to L-Val, L-Val, L-Phe, L-Leu and L-
Phe FDAA derivatives. The Pro in 4, 6 and 7 had the L-
configuration (14.5 min) and in 5 and 8 had the D-
configuration (16.9 min). Retention times (min) of
Results and discussion
The active fraction (hexane:chloroform 7:3) was
further subjected to reversed-phase HPLC chromatog-
raphy on YMC-Pack-ODS-A column (250 9 4.6 mm
I.D., 5 lm, 12 nm) and gave active compounds 1 (tR
15.0 min, 3.5 mg), 2 (tR 15.4 min, 5.5 mg) and 3 (tR
24.8 min, 3.2 mg). The system was operated at a flow
rate of 0.8 ml/min with solvent mixture of 76%
acetonitrile and 24% H2O, detection by UV absorption
at 226 nm. The more polar column fractions of the
EtOAc extract (chloroform, chloroform/EtOAc 1:1)
1
with typical H NMR signals for diketopiperazines
(DKPs) were further purified by a reverse phase HPLC
using a 19% acetonitrile/H2O of solvent mixture. Five
DKPs 4–8 (tR 11.3 min, 5.0 mg; tR 11.7 min, 2.5 mg;
tR 12.3 min, 11.3 mg; tR 13.4 min, 12.0 mg and tR
14.6 min, 3.0 mg) were obtained.
The structures of five diketopiperazines (4–8) were
investigated using NMR data, and by Marfey method
and optical rotation data. The DKP ring system was
evident from the characteristic 13C NMR chemical
shifts of two CONH (dC 165–170) and two a-methines
1
(dC 54–60), from the H NMR signals of the amino
123

Antonie van Leeuwenhoek
acids residues and from the HMBC. Proline was found
in all DKPs isolated from R. japonica KMM 9513T on
the basis of three broad 2H multiplets (dH 1.7–3.6) in
the 1H NMR spectra. From the NMR chemical shifts,
HMBC and 1H–1H COSY correlations, valine, valine,
phenylalanine, leucine and phenylalanine were iden-
tified as the second amino acid residue in 4–8,
respectively. The absolute configurations of the con-
stituting amino acids in isolated DKPs were deter-
mined by acid hydrolysis followed by chiral
derivatization with Marfey’s reagent (Marfey 1984).
HPLC analysis of the Marfey derivatives in compar-
ison to their analogs of authentic amino acids revealed
that all amino acids, with an exception of proline in 5
and 8, had the L-configuration. The spectroscopic and
optical rotation data for 4–8 (Supplementary Material)
were in agreement with those previously reported
(Mitova et al. 2003; Li et al. 2006; He et al. 2013) for
cyclo-(L-valyl-L-proline), cyclo-(L-valyl-D-proline),
cyclo-(L-phenylalanyl-L-proline), cyclo-(L-leucyl-L-
the diketopiperazine system is required for activity.
The antibacterial activity of cyclo-(L-Leu-L-Pro)
against B. subtilis, Escherichia coli, S. aureus and
Pseudomonas aeruginosa has been reported by Rhee
(2004). Kumar et al. (2014) found that cyclo-(D-Pro-D-
Leu) revealed a significantly higher antimicrobial
activity than that of the reported for cyclo-(L-Pro-L-
Leu). We have previously reported DKPs from the
marine mollusk-derived actinomycete Saccharothrix
espanaensis containing both DL-amino acids and LL-
amino acids, having significant inhibitory activity
against Vibrio alginolyticus and only modest activity
against Vibrio parahaemolyticus (Kalinovskaya et al.
2010). In the present study we found that compounds 5
and 8 containing D-proline showed antimicrobial
activity against S. aureus only whereas DKPs 4, 6
and 7 were inactive towards any of indicatory
microorganisms. Compounds 4–8 inhibited weakly
growth of T. sedimentorum (phylum Bacteroidetes),
and did not inhibited the growth of strains L. maritima,
S. pacifica (class Alphaproteobacteria), and P. mar-
incola (class Gammaproteobacteria) when tested
against marine bacteria.
proline),
and
cyclo-(L-phenylalanyl-D-proline),
respectively (Fig. 1). DKPs have been reported to be
isolated from microorganisms, sponges and from a
¨
variety of tissues and liquids (Fenical 1993; Strom
Compounds cyclo-(Val-Pro) have been previously
reported to be obtained from marine sponge Disco-
dermia calyx (He et al. 2013), Bacillus cereus (Kumar
et al. 2014), Streptomyces fungicidicus (Li et al. 2006)
and Aspergillus fumigatus (Niege et al. 2005). The
isolation of cyclo-(Leu-Pro) has been reported from
Pseudomonas putida (Degrassi et al. 2002), Strepto-
myces sp. (Rhee 2002), Pseudomonas fluorescens
(Santos et al. 2014) and Achromobacter xylosoxidans
(Yan et al. 2004). The cyclo-(Phe-Pro) was previously
isolated from P. aeruginosa (Jayatilake et al. 1996), S.
fungicidicus (Li et al. 2006) and B. subtilis (Wang
et al. 2010). The compound cyclo-(L-Val-L-Pro)
activated N-acyl homoserine lactone (AHL) biore-
porters (Tommonaro et al. 2012). In addition, cyclo-
(L-Val-L-Pro), cyclo-(L-Leu-L-Pro), cyclo-(L-Phe-L-
Pro) demonstrated antilarval activity towards the
barnacle Balanus amphitrite (Li et al. 2006). These
results showed that DKPs may be used as novel
antifoulants. The cyclo-(L-Val-L-Pro) and cyclo-(D-
Phe-L-Pro) have shown significant antifungal activi-
ties against five fungal strains tested and they were
more effective than the standard fungicide bavistin
(Kumar et al. 2014). Besides, Kumar et al. (2012)
found that the three antimicrobial DKPs, cyclo-(L-
Leu-L-Pro), cyclo-(L-Leu-D-Pro) and cyclo-(L-Tyr-D-
et al. 2002; Mitova et al. 2003) and to possess diverse
biological activities such as antibacterial, antifungal,
antitumor, antihyperglycemic, antioxidant, antifoul-
ing and quorum sensing signals (Holden et al. 1999;
Degrassi et al. 2002; Song et al. 2003; Li et al. 2006;
Van der Merwe et al. 2008; De Carvalho and Abraham
2012; Furukawa et al. 2012; Kumar et al. 2014). DKPs
are considered to be specific signaling molecules that
are involved in communication both within and
between species of bacteria. The concentration of a
signal molecule not only reflects cell numbers but also
regulates the expression of some biological behaviors,
for example, secondary metabolite production, conju-
gal plasmids transfer, antibiotic resistance, biofilm
maturation, virulence. However, the role of these
molecules in signal transduction still remains largely
unknown. Most DKPs isolated from natural sources
have been reported to exist in the LL form. There are a
few reports on naturally occurring DKPs derived from
non-proteinogenic D-a-amino acids. Fdhila et al.
(2003) reported that DD-DKPs from marine bacteria
associated with the mollusk Pecten maximus had
strong antibiotic activity against the pathogen Vibrio
anguillarum, whereas the LL-enantiomers were com-
pletely inactive, therefore, at least, one D-amino acid in
123

Antonie van Leeuwenhoek
Fig. 1 Structure of isolated
compounds. 1 DIBP, 2 DBP,
3 DEHP; DKPs: 4 cyclo-(L-
Val-L-Pro), 5 cyclo-(L-Val-
D-Pro), 6 Cyclo-(L-Phe-L-
Pro), 7 cyclo-(L-Leu-L-Pro),
8 cyclo-(L-Phe-D-Pro)
Pro), exhibited strong synergy effects in inhibiting the
growth of pathogenic microorganisms, when they
were used in combination. At the same time Jayatilake
et al. (1996) reported that cyclo-(L-Val-L-Pro), cyclo-
(L-Leu-L-Pro) and cyclo-(L-Phe-L-Pro) were inactive
as antibiotics or cytotoxins. It has been reported that
cyclo-(L-Leu-L-Pro) could inhibit the aflatoxin pro-
duction by Aspergillus parasiticus (Yan et al. 2004),
and exhibited antifungal activity especially against
rice blast fungus Pyricularia oryzae (Rhee 2003) and
bactericidal activity against S. aureus and P. aerug-
inosa, and showed cytotoxic activity towards HEp-2
tumour cells (Santos et al. 2014). The cyclo-(L-Phe-L-
Pro) has been reported to be involved in quorum
sensing mechanisms, which synchronize bacterial
physiological responses in a cell density-dependent
manner (Holden et al. 1999). Thus, DKPs are consid-
ered to be an important class of biologically active
substances which have a promising future. Due to their
chiral, rigid and functionalized structure, DKPs can
bind to a large variety of receptors with a high affinity
providing a broad range of biological activities.
Analysis of the 1H, 13C, DEPT, HSQC and COSY
NMR data established the structure of the active
compounds 1–3 as diisobutyl-, dibutyl- and bis(2-
ethylhexyl) phthalates (Fig. 1). The spectroscopic data
for 1–3 (Supplementary Material) were in agreement
with those previously reported (Hoang et al. 2008) for
diisobutyl-, dibutyl- and bis(2-ethylhexyl) phthalates.
A lot of phthalate derivatives have been isolated from
terrestrial and marine organisms including plants,
bacteria and fungi. The dibutylphthalate (DBP) has
123

Antonie van Leeuwenhoek
Table 1 Antimicrobial activity of cell-free supernatant, ethyl acetate extract and isolated compounds produced by the strain
Rheinheimera japonica KMM 9513T
Compounda
Inhibitory zone (mm)
Bacillus
subtilis
Staphylococcus Staphylococcus
aureus
Enterococcus Escherichia Xanthomonas sp. Candida
faecium
epidermidis
coli
pv. badrii
albicans
1
2
3
4
5
6
7
8
15
16
–
–
–
15
12
–
–
–
–
–
–
–
–
–
–
–
–
Wb
21
–
W
–
–
–
–
–
–
–
–
–
–
–
17
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
15
20
–
–
–
–
Cell-free
supernatant
17
W
28
14
18
Ethyl acetate 18
extractc
20
W
15
–
12
–
a
Antimicrobial activity was assayed at a concentration of 0.5 mg/ml of each compound
W weak activity, inhibition zones were observed as zones with reduced growth
Antimicrobial activity was tested at a concentration of 1 mg/ml
b
c
been isolated from Streptomyces strains (El-Naggar
aureus and S. epidermidis, and DEHP (compound 3)
was active against S. aureus (Table 1).
1997; Roy et al. 2006), and a marine Pseudomonas
strain (Hoang et al. 2008). The di(2-ethylhexyl)
phthalate (DEHP) was previously isolated from
Streptomyces sp. (Uyeda et al. 1990), Pseudomonas
rhizosphaerae (Qi et al. 2009a) and marine fungus
Cladosporium sp. F14 (Qi et al. 2009b). Two
cathepsin B inhibitors, DBP and DEHP, were
obtained from the culture supernatant of Pseu-
domonas sp. PB01 isolated from marine source and
showed dose-dependent cathepsin B inhibitions
(Hoang et al. 2008). Antimicrobial activity of DBP
and glucosidase inhibition by DBP has been reported
from Streptomyces strains (El-Naggar 1997; Lee
2000). DBP is also could be used as an endocrine
disruptor with estrogenic activity (Marchetti et al.
2002). DEHP exhibited antimicrobial activities
against mainly Gram-positive bacteria and yeasts
whereas the inhibition of Gram negative bacteria was
lower, and could display cytotoxic activities against
some human carcinoma cells (El-Sayed 2012) and a
high antilarval settlement activity towards B. amphi-
trite (Qi et al. 2009a, b). In the present study DIBP
and DBP (compounds 1 and 2, respectively) revealed
antimicrobial activity against B. subtilis and E.
faecium, and latter inhibited weakly growth of S.
In conclusion, five diketopiperazines and three
phthalate compounds DBP, DIBP and DEHP were
isolated from the novel marine bacterium R. japonica
KMM 9513T and their structures were established.
Compounds 1–3, 5 and 8 revealed antimicrobial
activities against B. subtilis and/or E. faecium and
Staphylococcus aureus. To our knowledge this is the
first report on isolation of low-molecular-weight
metabolites with antibacterial activity from a member
of the genus Rheinheimera. Our results demonstrate
that marine sediments of the Sea of Japan represent an
untapped source of bacteria capable of producing
antibacterial substances. It is conceivable that these
antimicrobial metabolites are of significance for
competition for space and nutrient within microbial
communities colonizing seashore sediments. Addi-
tionally, this antimicrobials-producing bacterium has
a potential for biotechnology and may have potential
as a biological control agent against Gram-positive
microorganisms in mariculture. The study extends our
knowledge of microbial processes and interactions via
active metabolites production in the marine coastal
environment and in the finding of novel strains
producing BAS.
123

Antonie van Leeuwenhoek
Acknowledgements This study was supported by a grant
from the RSF ‘‘Biodiversity and biotechnological potential of
marine bacteria and fungi’’, No. 14-14-00030.
Holden MTG, Chhabra SR, Denys R, Stead P, Bainton NJ, Hill
JP, Manefield M, Kumar N et al (1999) Quorum sensing
cross talk: isolation and chemical characterization of cyclic
dipeptides from Pseudomonas aeruginosa and other Gram-
negative bacteria. Mol Microbiol 33:1254–1266
Hughes CC, Fenical W (2010) Antibacterials from the sea.
Chem Eur J 16:12512–12525
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