Received: 28 May 2018

|
DOI: 10.1002/jobm.201800252
Revised: 24 September 2018
| Accepted: 30 November 2018

RESEARCH PAPER

Antimicrobial potential of haloalkaliphilic Nocardiopsis sp. AJ1

isolated from solar salterns in India

John Selesteen Charles Adlin Jenifer | Mariavincent Michaelbabu |

Chinnadurai Lelin Eswaramoorthy Thirumalaikumar | Selva Raj Jeraldin Nisha |
Ganapathi Uma | Thavasimuthu Citarasu

Centre for Marine Science and Technology,

Manonmaniam Sundaranar University, Antagonistic haloalkaliphilic Nocardiopsis sp. AJ1 (GenBank JX575136.1), isolated
Rajakkamangalam, Kanyakumari District, and identified from the saline soil of Kovalam solar salterns was able to produce
Tamilnadu, India
antimicrobial secondary metabolites and effectively suppressed several bacterial and
Correspondence fungal pathogens. The metabolite extracted from ethyl acetate precipitation
Thavasimuthu Citarasu, Centre for Marine suppressed the bacterial and fungal pathogens to the range between 2.14 and
Science and Technology, Manonmaniam
Sundaranar University, Rajakkamangalam
20.14 mm and also controlled the shrimp killer virus WSSV by 83% than the control
629502, Kanyakumari District, Tamilnadu, and significantly (p < 0.05) differed. GC-MS analysis revealed that, the ethyl acetate
India. precipitation contains pyrrolo (1,2-A(pyrazine-1,4-dione, hexahydro-3-(2-methyl-
Email: [email protected]
propyl)-) and actinomycin C2. Non ribosomal peptide synthetase (NRPS) was
Funding information amplified by PCR with the amplicon size of 750-800 bp length and further predicted
Department of Science and Technology
the secondary structure by Iterative Threading Assembly Refinement (I-TASSER)
(DST), India, Grant number: DO NO. SR/
SO/HS-0161/2012 dated 28.07.2014 bioinformatics approach. I-TASSER prediction helped to find out the secondary, 3-D
structure, and ligand binding sites. The top ten modelling concluded that, the NRPS
gene is closely similar to surfactin synthesizing gene, surfactin A synthetase C
(SRFA-C). The findings revealed that, the active compounds from the secondary
metabolites effectively suppressed the pathogenic bacteria, fungi, and virus and
useful to develop antimicrobials.

KEYWORDS
actinobacteria, antimicrobials, haloalkaliphils, Nocardiopsis sp. AJ1, non ribosomal peptide synthetase
(NRPS)

1 | INTRODUCTION walls contain meso-2,6-diaminopimelic acid but do not

contain diagnostically important carbohydrates. Whole cell
Members of the genus Nocardiopsis were first described by hydrolysates of this genus do not show the presence of
Meyer [1] and they belong to the class Actinobacteria; madurose or nocardomycolic acids and their genomes have
subclass Actinobacteridae; order Actinomycetales and family high contents of guanine and cytosine [4].
Nocardiopsaceae [2]. On the basis of the distinct phylogenetic Mostly, the Actinobacteria are predominantly found in
position, morphological features, and chemotaxonomic different types of soils including desert, saline, hypersaline,
properties displayed by Nocardiopsis sp., Rainey et al. [3] and alkaline origins [5,6]. Hypersaline environments such as
created a new family for the genus and referred as soda lakes and salterns, are the habitats that harbor
“Nocardiopsaceae.” The genus Nocardiopsis includes aero- Nocardiopsis species along with other Actinobacteria
bic, Gram-positive, non acid-fast catalase-positive. Their cell including Streptomyces sp [7]. Under these circumstances,
J Basic Microbiol. 2019;1–14. www.jbm-journal.com © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim | 1
2
| ADLIN JENIFER ET AL.

Nocardiopsis genus may be playing a degradative role in same agar slants at 4 °C as well as at 20% (v/v) glycerol stock
mediating the breakdown of naturally occurring complex at −80 °C for future use.
polymers [6]. To aid their survival under these conditions,
they mainly produce extremozymes, compatible solutes, and
2.2 | In vitro antagonistic screening
surfactants [6]. Important antimicrobial agents obtained from
this genus include polyketides, phenzines, quinoline alka- Antagonistic screening was performed against the pathogenic
loids, terphenyls, proteins, thiopeptides, and amines [8,9]. bacterial and fungal species (Escherichia coli, Staphylococ-
Several isolates obtained from soil, marine forms, marine cus aureus, Pseudomonas aeruginosa, Vibrio parahaemoly-
sediments, and mine tailings also produce different types of ticus, Aeromonas hydrophila, Aspergillus niger, Fusarium
antibiotics. The Nocardiopsis genus has the gene clusters for sp., and Pythium sp.) based on the method described by
polyketide synthases (PKSs) and non-ribosomal peptide Shomurat et al. [16] using the isolated Actinobacterial sp. The
synthetases (NRPSs) and many of the natural isolates may be Actinobacterial isolates were spot inoculated in starch casein
producing antibiotics [5,10]. A broad range of biologically agar (pH 9, 0.0614 M) for 4 days. After 4 days, they were
active polyketide and peptide compounds with applications overlaid with 5 ml of sloppy agar (0.6%) layer previously
in medicine, agriculture, and biochemical research are seeded with the test microbes, bacteria, and fungi. Further this
synthesized by type-I polyketide synthases (PKS-I) and was incubated for 24 h at 37 °C and the diameter of the
non ribosomal peptide synthetases (NRPS). Structurally, both incubation zone was measured using ruler and recorded in
PKS-I and NRPS are multifunctional polypeptides encoded millimeters. For bacterial pathogen, the major inhibition zone
by a variable number of modules with multiple enzymatic was calculated as above 10 mm and the minor calculated to
activities [11]. Marine Nocardiopsis species produced cyclic below 10 mm. For fungal pathogens, major inhibition zone
hexapeptides have antimicrobial activity [12]; soil-derived was calculated as above 5 mm and minor calculated to below
N. trehalosei produced a novel antibiotic 3-trehalosamine; 5 mm of zone of inhibition.
N. mutabilis produced dopsisamine and Nocardiopsis sp.
TFS65-07 isolated from marine sediments secreted thiopeptide
2.3 | Characterization and identification
antibiotics [13]. To assess the capacity of Nocardiopsis sp. AJ1
in secondary metabolism, the present study is performed to The potent Actinobacteria isolates selected from primary
analyze the structural and molecular characterization of the antagonistic screening were characterized by morphological,
antimicrobial secondary metabolites, their antimicrobial biochemical, and physiological methods. The morphological
influence, and the molecular characterization of biosynthetic method consists of macroscopic and microscopic characteri-
cluster gene Nonribosomal Peptide Synthetase (NRPS). The zation. Macroscopically the Actinobacterial isolates were
genome sequence data of Nocardiopsis sp. AJ1 suggested its differentiated by their colony characters, for example, size,
high potentiality for the secondary metabolism and gave shape, colour, consistency, etc. For the microscopy, the
important information for the discovery of new bioactive isolates were grown by a cover slip culture method [17]. The
compounds. morphology of spore chain, pigment production, color of
aerial mycelium, color of substrate mycelium, consistency,
Gram's staining, and growth of Actinobacteria were done by
2 | MATERIALS AND METHODS
following the method of Lim et al. [18].
The highly antagonistic strain, Nocardiopsis sp. AJ1 was
2.1 | Sampling, isolation, and maintenance of
further identified by 16S rRNA sequencing. The genomic
actinobacterial cultures
DNA was extracted from the Nocardiopsis sp. AJ1 by high
Soil samples were collected from the man made solar salterns salt method. The single colony of the strain was inoculated in
in Kovalam (8°05′04.35″N 77°31′17.07″E), Kanyakumari to the 5 ml of starch casein broth (pH 9, 0.08 M salinity). The
District, Tamilnadu, India in sterile plastic bags at a depth of cells were harvested by centrifugation at 10,000 rpm for
4–5 cm. The salinity (7.14 M) and pH (11) were also 25 min. To the collected pellet, added 100 µl of TKM I
measured by using hydrometer and pH meter, respectively. solution containing 5 µl Triton X100 and incubated for 25 min
Upon arrival of the sample to the laboratory, serial dilution, at room temperature. The suspended cells were once again
and plating technique was performed using starch casein agar collected by centrifugation at 10,000 rpm for 10 min. The
medium (pH 9, 0.0614 M) [14], then the plates were incubated collected pellet was again added with TKM II solution and
at 28 °C for 7 days. The suspected colonies were picked up 20 µl of 10% SDS, vortexed and incubated at 55 °C for
and streaked on the same agar media and incubated at room 10 min. Then added 125 µl of 5 M NaCl and centrifuged at
temperature for 7 days based on the protocol followed by 10,000 rpm for 10 min. The supernatant was transferred to a
International Streptomyces Project (ISP-2) [15]. The pure microfuge tube and added double the volume of 100% ethanol
culture of the isolates were sub-cultured and maintained in the with 1/10 volume of 3 M sodium acetate and kept at −20 °C
ADLIN JENIFER ET AL..
| 3

overnight. The suspended pellet was washed with 100 µl of 2.6 | Minimum inhibitory concentration (MIC)
70% ethanol by centrifugation at 12,000 rpm for 5 min and the and minimum bactericidal concentration
collected pellets were allowed to dry at 37 °C for 30 min. (MBC)
Then, the pellet was suspended in TE buffer and run on 1%
The minimum inhibitory concentration (MIC) was deter-
agarose gel. The genomic DNA was extracted from the gel by
mined as the lowest concentration, and the highest dilution,
genomic DNA extraction buffer.
which completely inhibited the growth of bacteria. To
The extracted DNA was amplified by PCR using 16S
determine the minimum bactericidal concentration (MBC),
rRNA universal primers, further cloned into the “T” vector
the two lowest concentrations which inhibited bacterial
pTZ57R and used to transform E. coli DH5α as described in
growth were plated out on a Müller Hinton Agar and
Sambrook et al. [19]. The transformants were sequenced
incubated at 37 °C for 24 h. The MIC and MBC were carried
using an ABI 3700 automated DNA sequencer and the
out against V. parahaemolyticus using the ethyl acetate
sequences were compared with other 16S rRNAs obtained
extract based on the highest activity.
from GenBank using the BLAST program. The phylogenetic
tree was constructed by MEGA X software and evolutionary
history was inferred using Neighbor-Joining method [20]. 2.7 | Analytical characterization of secondary
metabolites
2.4 | Extraction of antimicrobial secondary Based on the better antimicrobial activity, the metabolite was
metabolites extracted from the ethyl acetate solvent and characterized by
Nocardiopsis sp. AJ1 was inoculated into starch casein broth Gas Chromatography-Mass Spectrometry (GC-MS) analysis
(pH 7, 0.0614 M) and incubated at 28 °C on a shaker to identify the active compounds. Agilent GC–MS 5975 Inert
(200–250 rpm) for 7 days. After 7 days of incubation, the XL MSD (United States) gas chromatography equipped with
culture was spun at 5000×g for 30 min and the supernatant J and W 122–5532G DB–5 ms 30 × 0.25 mm × 0.25 μm and
was filtered through a 0.22 μm membrane filter. The filtrate mass detector (EM with replaceable horn) that operated in
was extracted with the equal volume of ethyl acetate, ethanol, EMV mode was used to characterize the compounds.
benzene, methanol, and chloroform. The extraction process
was repeated for four times and the extract was collected.
2.8 | Molecular characterization of
Then the collected extract was concentrated in a rotary
biosynthetic cluster gene NRPS
evaporator, lyophilized and stored at 4 °C.
Degenerate PCR primers A3F (5′-GCSTACSYSATSTA-
CACSTCSGG-3′), A7R (5′-SASGTCVCCSGTSCGGTAS-
2.5 | Antimicrobial influence of secondary
3′), was used [11], to amplify the NRPS gene from the
metabolites
genomic DNA of Nocardiopsis sp. AJ1. PCR reactions were
The different extracts of the secondary metabolite (benzene, performed to a final volume of 50 µl containing 10% of
methanol, ethyl acetate, chloroform, and ethanol) were extracted DNA, 0.4 µM of each primer, 0.2 mM of each of the
screened against pathogenic bacteria, fungi, and White four dNTPs, 5 µl extracted DNA, 1 U Taq polymerase with its
Spot Syndrome Virus (WSSV). In vitro antibacterial activity recommended reaction buffer. The PCR conditions were
was performed by using different solvent extract against few 95 °C for 5 min of initial denaturation, 95 °C for 30 s of
pathogenic bacteria by agar diffusion method. For antifungal denaturation, 59 °C for 2 min of annealing, 72 °C for 4 min
activity, known quantity of fractions was poured into a well and 72 °C for 10 min for final elongation. Amplification was
made in the centre of Potato Dextrose Agar (PDA) plates and resolved in 1% agarose gels stained with ethidium bromide.
fungal spores (approximately 10 spores) were inoculated onto The PCR product was purified by using gel extraction kit
the plates and incubated at 28 °C for 48 h. The zone of (Medox Biotech India Pvt. Ltd.) and sequenced using an ABI
inhibition was recorded [21]. 3700 automated DNA sequencer by M13+ and M13− primers.
Antiviral activity was performed against WSSV following Sequences were compared with other NRPS gene obtained
the method of Balasubramanian et al. [22]. The secondary from GenBank using the BLAST program. Nucleotide
metabolite incubated WSSV suspensions (29 °C for 3 h) were sequence alignment and identity were analyzed by using
injected intramuscularly to the Indian white shrimp, Fenner- Clustal X (1.8.1) software (European Bioinformatics Insti-
openaeus indicus. Hemolymph was bled from the shrimps after tute). The phylogenetic tree was constructed by MEGA X
the 3rd day of injection, and extracted the genomic DNA [23]. software and tree was built by using the Neighbor Joining
Double step diagnostic PCR were performed from the genomic method. The genetic distance was also calculated [20].
DNA template using the WSSV VP28 primer [24] and standard The secondary structure and the function of the NRPS
PCR protocols were followed [25]. protein were predicted by using Iterative Threading Assembly
4
| ADLIN JENIFER ET AL.

Refinement (I-TASSER) online bioinformatics software the other sequences in the NCBI database confirmed the AJ1
(http://zhanglab.ccmb.med.umich.edu/I-TASSER/). This al- isolates as Nocardiopsis sp. AJ1. The sequence was deposited
gorithm modeled and worked based on LOMETS multiple- in NCBI database (GenBank Accession No: JX575136.1).
threading alignment and TASSER iterative simulation. I- Nocardiopsis sp. AJ1 was closely related (>90%) to the other
TASSER server results predicted the accurate structure and Nocardiopsis sp. including Nocardiopsis aegypptia-DY-R11,
function base on state-of-the-art algorithm [26,27]. After Nocardiopsis sp. NH2, N. dassonvillei ADVJ1 (97%), N.
running the sequence, the protein 3D structure was down- aegypptia-TGT-R2, N. aegypptia-SCAU-A-11 (94%),
loaded and visualized using molecular visualization tool Nocardiopsis sp. 6–1, and N. flavescens SA-6 (93%) (Fig. 2).
RasMol. The quality of the predicted protein models was
estimated using C Score and the calculation is based on Z-
3.3 | Antimicrobial influence of secondary
Score of threading alignment in LOMETS multiple-threading
metabolites
alignment and cluster density of I-TASSER simulation. TM
and C Score determined the structure similarity and confidence The secondary metabolites precipitated by different solvents
between the predicted protein model and the native protein (benzene, methanol, ethyl acetate, chloroform, and ethanol)
structure [28]. For binding the drug molecules to bind the revealed that, the ethyl acetate extract proved the maximum
cluster, the ligand binding site was also determined. inhibitory zones against tested bacterial and fungal
pathogens followed by methanol, ethanol, chloroform,
and benzene extracts. The secondary metabolites precipi-
3 | RESULTS
tated by the ethyl acetate solvents showed significant
antibacterial activity of 20.14, 17.15, 13.57, 13.25, and
3.1 | In vitro antagonistic screening
10.88 mm of zone of inhibition against V. parahaemolytics,
Among the ten isolates (AJ1–AJ10) isolated; only four strains P. aeruginosa, S. aureus, A. hydrophila, and E. coli,
(AJ1, AJ2, AJ3, and AJ4) were selected for antagonistic respectively. The same metabolites also highly influenced
screening. The actinobacteria AJ1 exhibited most significant to suppress the fungal growth by 13.14, 8.33, and 6.43 mm
activity against bacterial and fungal pathogens. The isolate AJ2 of zone of inhibition against A. niger, Fusarium sp and
showed minimum activity and the other isolates AJ3 and AJ4 Pythium sp., respectively. The other extractions including
had low activity against the tested pathogens. The significant ethanol, chloroform, methanol, and benzene had only minor
zone of inhibition was observed against the bacterial pathogens antimicrobial activities (Table 3). Two way ANOVA
V. parahaemolyticus (19.5 mm), P. aeruginosa (16.5 mm), and revealed that, the values were significantly differed from
A. hydrophila (12.2 mm). In addition, the antifungal activity of each other's (F = 2.72; p < = 0.05–column and (F = 24.38;
12.4, 8.2, and 6.8 mm of zone of inhibition was observed p < = 0.001–row).
against A. niger, Fusarium sp., and Pythium sp., respectively, The ethyl acetate precipitation also suppressed the shrimp
by the actinobacteria AJ1 (Table 1). killer virus WSSV by 83% when compared with the control
group. 100% PCR positive signals were observed in the genomic
DNA template used for PCR from F. indicus treated with WSSV
3.2 | Characterization and identification
alone without Nocardiopsis sp. AJ1s’ secondary metabolites
The actinobacterial isolate AJ1was able to grow well on incubation. The different solvent precipitations with WSSV
nutrient agar and starch casein agar media supplemented with incubations gradually decreased the PCR positive signals at a
9 and 10% NaCl and had well-developed branched aerial significant (p < 0.05) level of 70, 41, 37, 28, and 17% by double
hyphae. The aerial hypha were white, long, and at the step PCR detections in methanol, benzene, ethanol, chloroform,
beginning of sporulation. Spores were elongated and have and ethyl acetate precipitations, respectively (Fig. 3).
smooth surfaces. A good growth was observed on starch The MIC and MBC of the five extraction methods against
casein agar which is supplemented with 10% NaCl, glucose as V. parahaemolyticus were given in the Table 4. The ethyl
sole carbon sources and alanine as nitrogen source and grown acetate extraction helped to control V. parahaemolyticus at
at pH 9 (Table 2). The isolate was the characteristics of the minimal level of 20 μg due to the presence of
aerobic, Gram-positive, non-acid-fast, and non-motile which antimicrobial active principles whereas the other extractions
forms an extremely branched substrate mycelium that had the range of 50–70 μg.
fragments into rod and coccal elements. The substrate
mycelium bears the aerial hyphae, which frequently form
3.4 | Analytical characterization of secondary
chains of smooth spores was observed under scanning
metabolites
electron microscope (Fig. 1).
Multiple sequence alignment and graphical phylogenetic The GC peak of secondary metabolites from Nocardiopsis sp.
tree analysis of the 16S rRNA gene sequence compared with AJ1 confirmed two different compounds with the help of
ADLIN JENIFER ET AL..
| 5

TABLE 1 In vitro antagonistic screening of dominant haloalkaliphilic actinobacterial colonies isolated from Kovalam solar salt works against
bacterial and fungal pathogens
Dominant Actinomycetes strains screened

Bacterial/fungal pathogens AJ1 AJ2 AJ3 AJ4

Escherichia coli 10.3 ± 0.50 6.4 ± 0.4 4.1 ± 0.05 1.98 ± 0.05
Staphylococcus aureus 13.7 ± 0.33 5.2 ± 0.1 3.5 ± 0.01 1.02 ± 0.07
Pseudomonas aeruginosa 16.5 ± 0.12 3.1 ± 0.6 2.3 ± 0.4 0.8 ± 0.02
Vibrio parahaemolyticus 19.5 ± 0.65 6.8 ± 0.1 3.9 ± 0.7 1.12 ± 0.04
Aeromonas hydrophila 12.2 ± 0.06 4.6 ± 0.7 1.8 ± 0.1 0.0 ± 0.0
Aspergillus niger 12.4 ± 0.15 3.6 ± 0.5 1.2 ± 0.03 0.0 ± 0.0
Fusarium sp. 8.2 ± 0.42 1.5 ± 0.1 0.0 ± 0.0 0.0 ± 0.0
Pythium sp. 6.8 ± 0.07 1.9 ± 0.3 0.0 ± 0.0 0.0 ± 0.0

NIST library database. The peak at the retention times of 3.5 | Molecular characterization of
17.26, 17.91, and 21.86 was confirmed as pyrrolo [1,2-A] biosynthetic cluster gene NRPS
pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl). The
other peak with the retention times of 18.07 and 18.21 was A set of forward and reverse degenerated primers (A3F/
confirmed as actinomycin C2 (Fig. 4). A7R) helped to amplify the NRPS gene product at the

TABLE 2 Phenotypic identification of the highly antagonistic Actinobacteria strain AJ1 isolated from Kovalam solar salt works
Sl. no Characteristics Variables Confirmation
1 Biochemical Gram's staining Gram positive
Colony colour Puffy white
2 Cultural characteristics Nutrient agar ++
Starch casein agar ++
Actinomycetes isolation agar +
Malt extract agar +
Glucose aspargine agar +
3 Utilization of carbon source Glucose ++
Starch ++
Fructose +
Dextrose +
Lactose +
4 Utilization of nitrogen source D-alanine ++
L-histidine +
Potassium nitrate +
5 pH 7 ++
8 ++
9 ++
6 NaCl tolerance (%) 7 -
8 +
9 ++
10 ++
7 Enzyme activity Protease ++
Lipase ++
Amylase -
Xylanase -

++, Major; +, Minor; −, low.

6
| ADLIN JENIFER ET AL.

3.6 | Protein modeling using I-TASSER

The NRPS protein had the amino acid length of 229 (Fig. 6a)
and the predicted secondary structure by I-TASSER is given
in the Fig. 6b. Using the target sequence Nocardiopsis sp. AJ1
NRPS protein the highly similar threading template protein
3D structure was obtained. Confidence score (C score) is used
to estimate the quality of the predicted model. The top five
models predicted territory structure for NRPS protein by I-
TASSER and the C-score were −0.25, −1.54, −2.44, −3.50,
and −3.74, respectively. Among the five models generated
based on C score model 5 with C score −3.74 is considered as
the best 3D structure (Fig. 6c). The multiple binding site
ligands from different PDB hits (4d4jA, 4g37A, 5burA,
2wd9C, and 3IVRA) were confirmed as the ligand name of
ANP, SLU, ATP, IBP, and 3IVRA02 etc. (Table 5). The
FIGURE 1 Scanning electron micrograph (SEM) view of
predicted EC numbers of the PDB hits were given in the
Nocardiopsis lucentensis AJ1
Table 6. Based on the results, it was revealed that the five top
most PDB enzyme hits were dipeptidyl carboxypeptidase
Dcp, mouse acetylcholinesterase, Cys-418 thiylradical,
range of 750-800 bp fragment from the genomic DNA human acetylcholinesterase and recombinant human butyr-
template of Nocardiopsis sp. AJ1. Phylogenetic and ylcholinesterase. Top 10 proteins structurally close to the the
evolutionary analysis of the NRPS gene sequence target NRPS protein were identified in the PDB by TM-align.
revealed that, Nocardiopsis sp. AJ1 shared more than They were 2vsqA- surfactin A synthetase C (SRFA-C);
80% similarity to other Streptomyces sp. and Actino- 3e7Wa- D-alanine: D-alanyl carrier protein liagase; 2cb4A-
planes sp. (Fig. 5a). Multiple sequence alignment by catalytic domain of mosquitocidal toxin; 4oxiA- Adenylation
clustalX analysis revealed that, the NRPS gene of domain AlmE in complex with glycyl adenosine 5′
Nocardiopsis sp. AJ1 was more identical to the other phosphate; 4gr5A-Crystal structure of SlgN1 delta Asub in
NRPS family suc as Streptomyces hiroshimensis complex with AMPcPP and 4gr4A- crystal structure of
NBRC14693, S. stramineus NBRC16131, S. fimbriatus SlgN1deltasub (Table 7). The PDB hit 2vsqA was highly
NBRC15-5, Actinoplanes sp. MN07-A0325, and Actino- similar with our target NRPS protein at the rate of four times
planes sp. MN07-A0349 (Fig. 5b). among the top five numbers. So it is confirmed that, our target

FIGURE 2 Graphical phylogenetic tree analysis of Nocardiopsis lucentensis AJ1 based on 16S rRNA gene sequence data compare with
other Nocardiopsis sp
ADLIN JENIFER ET AL..
| 7

TABLE 3 Antimicrobial screening of N. lucentensis AJ1s’ secondary metabolites extracted from different solvent system against bacterial and
fungal pathogens
Solvent extraction

Microbial pathogens Benzene Methanol Ethyl acetate Chloroform Ethanol

E.coli* 5.16 ± 0.34 3.18 ± 0.17 10.88 ± 0.12 7.16 ± 0.34 6.24 ± 0.18
S. aureus* 4.32 ± 0.21 8.15 ± 0.65 13.57 ± 0.67 5.12 ± 0.65 7.16 ± 0.5
P. aeruginosa* 3.17 ± 0.12 7.16 ± 0.18 17.15 ± 1.56 3.18 ± 0.68 4.15 ± 0.84
V. parahaemolyticus* 5.68 ± 0.84 5.15 ± 0.05 20.14 ± 1.98 4.14 ± 0.13 4.18 ± 0.67
A. hydrophila* 6.14 ± 0.76 3.16 ± 0.23 13.25 ± 1.55 4.55 ± 0.12 4.15 ± 0.14
A. niger** 6.12 ± 0.17 3.12 ± 0.15 13.14 ± 1.87 2.14 ± 0.11 2.12 ± 0.56
Fusarim sp.** 3.50 ± 0.34 2.40 ± 0.19 8.33 ± 0.54 3.10 ± 0.17 1.80 ± 0.14
Pythim sp.** 2.15 ± 0.15 3.87 ± 0.05 6.43 ± 0.54 3.32 ± 0.11 2.15 ± 0.01

*, Bacterial pathogens; **, Fungal pathogens. The values significantly differed from each other's (F = 2.72; p <= 0.05 – column and (F = 24.38; p <= 0.001 – row) – two
way ANOVA.

NRPS may be belonging the family of surfactin A synthetase species N. lucentensis sp. nov. DSM 44048 from a salt marsh
C (SRFA-C) (Fig. 7). soil sample near Alicante, Spain and they produced yellowish
to yellowish brown substrate mycelium and a white aerial
mycelium. N. salina sp. nov YIM 90010T, a novel halophilic
4 | DISCUSSION
actinomycete isolated from saline soil in China produced
abundant aerial mycelia grown well in 10% NaCl and
Members of the genus Nocardiopsis are generally encoun-
7 ± 2 pH [32]. The Nocardiopsis strains isolated from saline
tered in locations that are inherently extreme. They are
environments were able to tolerate or abundantly in the NaCl
present in frozen soils, desert sand, compost, saline, or
concentrations of maximum 10% and withstand high
hypersaline habitats (marine systems, salterns, and soils) and
alkaline pH. The Nocardiopsis sp. AJ1 grown well at 7 to
alkaline places [9]. They are frequently isolated from habitats
9 pH and 9 to 10% NaCl concentrations due to the alkalophilic
with moderate to high salt concentrations such as saline soil or
nature. Nocardiopsis alkaliphila YIM 80379T sp. nov., a
marine sediments [29] and salterns [30]. Recently, our team
novel alkaliphilic actinobacteria isolated from desert soil in
has isolated a few Nocardiopsis strains including N. salina,
Egypt produced substrate and aerial mycelia on different
N. alba, N. lucentensis, and Nocardiopsis sp. from the solar
media, with an optimum pH for growth of 9 ± 0.5 to 10 [33].
salterns at Thamaraikulam, Kovalam, Puthalum, and Thar-
N. halotolerans sp. nov., isolated from salt marsh soil in
uvaikulam of southern India and which are having potential
Kuwait produced substrate and aerial mycelium and grew
bioactivities. Yassin et al. [31] isolated and identified a new
well in the NaCl concentrations of 0–15% [29].
Hypersaline environments are extreme habitats; their
actinomycete inhabitants are largely unexplored for discovery
of novel bioactive secondary metabolites [34]. Members of
the genus Nocardiopsis produce a wide array of antimicrobial
and cytotoxic agents. Due to the presence of NRPS and PKS
gene clusters and respective antibiotics, the Nocardiopsis
species was highly influenced to control different bacterial,
fungal pathogens, and had potent anticancer activities [32,35].
The potential strain Nocardiopsis sp. AJ1 showed a
significant antibacterial and antifungal activity at the range
of 7 to 19 mm of zone of inhibition against human and aquatic
important bacterial and fungal pathogens due to the
antimicrobial secondary metabolite production. The MIC
and MBC results also confirmed that the ethyl acetate
FIGURE 3 Double step PCR detection in the genomic DNA extraction controlled the V. parahaemolyticus pathogen at a
template of F. indicus injected with WSSV incubated the secondary minimal level due to the presence of active compounds.
metabolites of N. lucentensis AJ1s’. Means with the same superscripts Pyrrolo[1,2-a]pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl)
(a–e) do not differ from each other (p < 0.05)–One Way ANOVA characterized from Streptomyces albus CN-4 had
8
| ADLIN JENIFER ET AL.

TABLE 4 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the N. lucentensis AJ1s’ secondary
metabolites extracted with different organic solvents against V. parahaemolyticus
Extraction

Benzene Methanol Ethyl acetate Chloroform Ethanol

Concentration (µg) MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC
10 ++++ ++++ ++ ++++ ++++
20 +++ ++++ 20 + ++++ ++++
30 +++ +++ - ++++ +++
40 ++ ++ - +++ +++
50 50 + 50 + - +++ ++
60 - - - ++ ++
70 - - - 70 + 70 +
80 - - - -

-, No growth; +, Minimum growth; ++, Intermediate growth; >+++, Maximum growth.

effectively controlled several bacterial and fungal pathogens line in the Nocardiopsis sp. The Nocardiopsis sp. AJ1closely
including Staphylococcus aureus, Pseudomonas aerugi- related to most of the Nocardiopsis sp. and N. flavescens SA6.
nosa, and Fusarium solani [36]. This compound was also Nocardiopsis lucentensis (lu. cen. ten'sis. M. L. adj,
identified from the secondary metabolites of Stretomyces lucentensis, referring to Lucentum, the ancient Latin name
werraensis KBR01 [37] and Streptomyces sp. UPMRS4 of Alicante, a city in Spain, where the type strain was
which had antimicrobial activities [38]. Likewise, actino- isolated). The discovery of many natural products from
mycin C complex extracted from S. pratensis effectively biosynthetic gene clusters in microbial genomes is very
controlled S. aureus [39]. Actinomycin C2 and actinomycin interesting and recently genomic sequencing has been used to
C3 isolated from red soil Actinomyces effectively inhibit the unveil the hidden production of natural products from
growth of Micrococcus luteus and S. aureus [40]. Nocar- microbes [43]. The NRPS gene sequenced from the
diopsis sp. isolated from Puducherry coast had 18, 20, and Nocardiopsis sp. AJ1was more identity to the other NRPS
15 mm zone of inhibition against E. coli, P. aeruginosa, and family of Streptomyces and Actinoplanes actinomyces genus.
K. pneumonia, respectively [41]. Saccharopolyspora salina Most of the nucleotides showed 100% similarity among the
VITSDK4, isolated from a saltpan soil sample collected at compared sequences of the actinomyces genus only. The
the Marakkanam coast of the Bay of Bengal, India, was sequencing and further bioinformatics approach of the NRPS
profoundly antagonistic with fungal and Gram positive gene of Nocardiopsis sp. AJ1helped to characterize the
bacterial pathogens [42]. bioactive molecules. Multiple gene clusters for secondary
The comparison of 16S rRNA sequence of Nocardiopsis metabolism appear also to be a hallmark of several non-
sp. AJ1with corresponding homologous DNA sequences Streptomyces filamentous actinobacteria [44] and of the
clearly revealed that the test organism form a distinct phyletic myxobacteria [45]. Chakrabortti et al. [46] identified eight

FIGURE 4 GC peak of partially purified secondary metabolites from N. lucentensis AJ1. *: pyrrolo [1,2-A]pyrazine-1,4-dione, hexahydro-3-
(2-methylpropyl)- (retention times 17.26, 17.91, and 21.86); **: actinomycin C2 (retention times 18.07 and 18.21)
ADLIN JENIFER ET AL..
| 9

FIGURE 5 (a) Phylogenetic analysis of the nucleotide sequence of NRPS gene of N. lucentensis AJ1 with that reported in other
Actinobacteria sp. by Neighbor Joining method. (b) Nucleotide alignment identities of NRPS gene of N. lucentensis AJ1 with its homologues.
Asterisks indicate 100% similarity among the nucleotide sequences

NRPS gene clusters and two PKS gene clusters in the genome to the diverse of antimicrobial molecules in the secondary
of N. jinanensis. metabolites of Nocardiopsis sp. AJ1, they were effectively
The secondary metabolites were extracted from the inhibiting the bacterial and fungal growth. Halophilic
Nocardiopsis sp. AJ1 fermentation with different solvent Nocardiopsis gilva YIM 90087, produced three p-terphenyl
systems, and the solvent ethyl acetate helped to precipitate the derivatives including the antimicrobial 60-hydroxy-
potent antimicrobial compounds. The extracts effectively 4,20,30,4″-tetramethoxyp-terphenyl, novobiocin, nine cyclo-
suppressed the multiplication of bacterial, fungal growth, and dipeptides, and two aromatic acids [47]. Novobiocin is an
arrest the replication of the killer shrimp virus WSSV. aminocoumarin antibiotics that inhibits bacterial gyrases [48]
Generally, the gene clusters in the Nocardiopsis sp. had the and act as a good antifungal against Fusarium avenaceum,
ability of synthesizing potent antimicrobial compounds. Due F. graminearum, F. culmorum, and C. albicans [49].
10
| ADLIN JENIFER ET AL.

FIGURE 6 (a) FASTA format of NRPS protein query sequence of N. lucentensis AJ1. (b) Secondary structure prediction of the NRPS
protein of N. lucentensis AJ1. (Color indications: H Helix; S Strands and C Coil). (c) The predicted 3D model and the estimated global and local
accuracy of the NRPS protein of of N. lucentensis AJ1

Novobiocin [47] isolated from Nocardiopsis sp. showed bacteria, fungi and helped arrest the transcription, translation
antibacterial activity against B. subtilis and Staphylococcus leading to arrest the multiplication of WSSV. It reflected in
aureus with a MIC of 16 and 64 µg ml−1, respectively. the lowest (<20%) PCR positive detected by double step PCR
Griseusins obtained from an alkalophilic Nocardiopsis sp. detection against the ethyl acetate treated group. K-252a, the
(YIM DT266) displayed antibacterial activity toward Micro- protein kinase C inhibitors are representative bioactive
coccus luteus, S. aureus ATCC 29213, and B. subtilis [50]. alkaloids derived from Nocardiopsis sp. K-252a also
The NRPS based molecules produced by Nocardiopsis sp. displayed antiviral effects [51]. The replication of vesicular
AJ1 helped to disturb the cell wall synthesis in the pathogenic somatitis virus in baby hamster kidney cells line (BHK-21)
ADLIN JENIFER ET AL..
| 11

TABLE 5 Showing the ligand binding sites of the NRPS protein of N. lucentensis AJ1
Rank C-score Cluster size PDB hit Ligand name Downloaded complex Ligand binding site residues
1 0.43 58 4d4jA ANP Rep. Mult 7,8,41,103,104,105,130,132,133,
135,162,227
2 0.41 55 4g37A SLU Rep. Mult 41,42,43,47,100,101,102,103,104,105,
130,131,132,133,134,135,138,
139,140,227
3 0.04 6 5burA ATP Rep. Mult 8,9,10,12,17,103,104,131,132,133,
134,135,227
4 0.02 4 2wd9C IBP Rep. Mult 41,42,43,102,133,134,135
5 0.02 3 3IVRA 3IVRA02 Rep. Mult 101,105,106,130,131,160,162

was specifically inhibited by K-252a and its derivative structural studies the two effective compounds having high
KT5926. antimicrobial properties have been identified. Further second-
Structural analysis by GC-MS analysis revealed that two ary structure prediction of the NRPS protein may help to
peptide based compounds including actinomycin C and identify some more molecules.
pyrrolo[1,2-A]pyrazine-1,4-dione, hexahydro-3-(2-methyl- The I-TASSER analysis for secondary structure, ligand
propyl)- were present in the secondary metabolites of binding sites and top most similarity results clearly revealed
Nocardiopsis sp. AJ1. Actinomycin was first isolated from a that, the NRPS based metabolites is most resemble to
culture of Streptomyces antibioticus by Waksman and surfactin A synthetase C (SRFA-C). The PDB hit no 2vsqA is
Woodruff [52] and later its derivatives were characterized responsible for surfactin A synthetase C (SRFA-C) and it
and designated as A, B, C, D, I, J, and X [53]. Actinomycin C, displayed four times among the top 10 hits. The SRFA-C
actinomycin C2, and C3, are differed from other actinomycin peptides are involving in the synthesis of lipopeptide
groups in the presence of D-alloisoleucine in the peptide part of biosurfactant and surfactin molecules. Surfactin is mostly
the molecule [54]. Actinomycin is a cyclic polypeptide- identified in the Bacillus sp. and actinobacteria. The surfactin
containing antibiotic that binds to DNA and inhibits RNA biosynthetic assembly line consists of three large NRPSs:
synthesis [55]. Due to the DNA binding and RNA synthesis SrfA-A, SrfA-B, and SrfA-C, comprising of a total of seven
inhibition properties of actinomycin C in the secondary modules. These modules are arranged into core and
metabolites of Nocardiopsis sp. AJ1, they are effectively elongation domains which are responsible for lipopeptide
inhibiting the transcription and translation of the bacterial, chain formation [59]. SrfA-C bears a thioesterase domain,
fungal, and viral pathogens. Like actinomycin C, pyrrolo 1,2-a SrfA-C-TE, located at its C-terminal end. The srfA-C-TE
pyrazine group is a group of potent naturally occurring gene, one of the genes required for surfactin biosynthesis, is
antibiotics from various Streptomyces species, which are directly involved in surfactin biosynthesis [60]. Surfactin is
among the most promising types of lead compounds [56]. one of the most powerful lipopeptide biosurfactants produced
Pyrone derivatives are a class of important secondary by various strains of Bacillus subtilis, having exceptional
metabolites synthesized via such PKS gene clusters [57]. surface activity as well as antiviral, antibacterial and
The pyrones derivatives including nocardiopyrone A and antitumor properties [61]. Surfactin is considered to be
nocardiopyrone B were effectively suppressing different antibiotics due to their broad antimicrobial spectrum [62].
pathogenic bacteria and fungi species [6]. Pyrrolo derivatives The crude surfactin possesses antimicrobial activity against
including pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2- antibiotic resistant Staphylococcus aureus, E. coli strains and
methylpropyl); pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- the pathogenic yeast Candida albicans [63]. Surfactin and its
3-(phenylmethyl) characterized from Streptomyces sp. by GC- derivatives also act as antiviral agents, antibiotics, antitumor
MS analysis had potent antifungal activity [58]. Based on the agents, immunomodulators, or specific toxin-inhibitors [64].

TABLE 6 The predicted enzyme commission numbers and active sites of the NRPS protein
Rank C-scoreEC PDB hit TM-score RMSDa IDENa Cov EC number Active site residues
1 0.357 3fccA 0 .820 2.32 0.127 0.926 6.1.1.13 NA
2 0.324 3e7wA 0.848 2.07 0.153 0.939 6.1.1.13 NA
3 0.323 3e7xA 0.845 2.15 0.157 0.943 6.1.1.13 103,136
4 0.321 1amuA 0.826 2.11 0.153 0.913 5.1.1.11 NA
5 0.319 318cB 0.824 2.41 0.112 0.939 6.1.1.13 6,136
12
| ADLIN JENIFER ET AL.

TABLE 7 Top 10 proteins structurally close to the the target NRPS In conclusion, Haloalkaliphilic Nocardiopsis sp. AJ1
protein identified in the PDB by TM-align isolated from the solar salterns had the ability of producing
PDB NRPS based secondary metabolites with potent antagonistic
Rank Hit Top 10 identical protein by structure activity against pathogenic bacteria and fungi. The
1 2vsqA Surfactin A synthetase C (SRFA-C) metabolites were found to control several pathogenic
2 2vsqA Surfactin A synthetase C (SRFA-C) bacteria, fungi, and the killer virus shrimp white spot
syndrome virus (WSSV) very effectively. Structural
3 3e7Wa D-alanine: D-alanyl carrier protein liagase
elucidation and prediction revealed that, different peptide
4 2cb4A Catalytic domain of mosquitocidal toxin
compounds such as pyrrolo [1,2-A]pyrazine-1,4-dione,
5 2vsqA Surfactin A synthetase C (SRFA-C)
hexahydro-3-(2-methylpropyl), actinomycin C2 and surfac-
6 4oxiA Adenylation domain AlmE in complex with tin A synthetase C (SRFA-C) were present in the
glycyl adenosine 5’ phosphate
metabolites.
7 2vsqA Surfactin A synthetase C (SRFA-C)
8 4oxiA Adenylation domain AlmE in complex with
glycyl adenosine 5’ phosphate ACKNOWLEDGMENTS
9 4gr5A Crystal structure of SlgN1 delta Asub in
complex with AMPcPP
The authors acknowledge the Department of Science and
Technology (DST), New Delhi, Government of India for
10 4gr4A Crystal structure of SlgN1deltasub
funding (DO NO. SR/SO/HS-0161/2012 dated 28.07.2014)
and also thank Dr. Yang Zhang's Research Group, Depart-
In our study, the I-TASSER analysis clearly confirmed that ment of Computational Medicine and Bioinformatics,
the sequenced NRPS PCR products was more resemble to University of Michigan, MI 48109–2218, USA for perform-
SRFA-C gene which indicates the presence of SRFA-C gene ing the I-TASSER analysis for the study.
in the Nocardiopsis sp AJ1 genome, which responsible for the
production of surfactin. Surfactin is formed by non-ribosomal
peptide synthetases (NRPSs), which govern all necessary CONFLICT OF INTEREST
steps in this process [65]. All authors declare no financial conflicts of interest.

ORCID
Thavasimuthu Citarasu http://orcid.org/0000-0001-6166-
620X

REFERENCES
[1] Meyer J. Nocardiopsis, a new genus of the order Actinomyetales.
Int J Syst Bacteriol 1976;26:487–93.
[2] Sun H, Lapidus A, Nolan M, Lucas S. Complete genome sequence
of Nocardiopsis dassonvillei type strain (IMRU 509T). Stand
Genomic Sci 1976;3:325–36.
[3] Rainey FA, Rainey NW, Kroppenstedt RM, Stackebrandt E. The
genus Nocardiopsis represents a phylogenetically coherent taxon
and a distinct Actinomycete lineage: proposal of Nocardiopsaceae
fam. nov. Int J Syst Bacteriol 1996;46:1088–92.
[4] Kroppenstedt RM, Evtushenko LI. The family nocardiopsaceae.
In: Dworkin M, Falkow S, Rosenberg E, Schleifer KH, editors. The
prokaryotes: a handbook on the biology of bacteria. New York:
Springer; 2006. 1:pp 754–95.
[5] Zitouni A, Boudjella H, Lamari L, Badji B. Nocardiopsis and
Saccharothrix genera in Saharan soils in Algeria: isolation,
biological activities and partial characterization of antibiotics.
Res Microbiol 2005;156:984–93.
FIGURE 7 NRPS enzyme, surfactin A synthetase C (SRFA-C) [6] Bennur T, Ravi Kumar A, Zinjarde SS, Javdekar V. Nocardiopsis
responsible for biosurfactant synthesis is closely related to our target species: a potential source of bioactive compounds. J Appl
NRPS by PDB analysis Microbiol 2015;120:1–6.
ADLIN JENIFER ET AL..
| 13

[7] Mwirichia R, Muigai AW, Tindall B, Boga HI. Isolation and Penaeus japonicus Bate and systemic ectodermal and mesodermal
characterisation of bacteria from the haloalkaline Lake Elmenteita, baculovirus (SEMBV) DNA in Penaeus monodon Fabricus. J Fish
Kenya. Extremophiles 2010;14:339–48. Dis 1996;19:399–403.
[8] Li HW, Zhi XY, Yao JC, Zhou Y. Comparative genomic analysis [26] Roy A, Kucukural A, Zhang Y. I-TASSER: a unified platform for
of the genus Nocardiopsis provides new insights into its genetic automated protein structure and function prediction. Nat Protoc
mechanisms of environmental adaptability. PLoS ONE. 2013;8: 2010;5:725–38.
e61528. [27] Zhang Y. I-TASSER server for protein 3D structure prediction.
[9] Bennur T, Kumar AR, Zinjarde S, Javdekar V. Nocardiopsis BMC Bioinform 2008;9:40.
species as potential sources of diverse and novel extracellular [28] Barrett A. Nomenclature committee of the international union of
enzymes. Appl Microbiol Biotechnol 2014;98:9173–85. biochemistry and molecular biology (NC-IUBMB). enzyme
[10] Meklat A, Sabaou N, Bouras N, Zitouni A. A novel strain of nomenclature. recommendations 1992, supplement 4: corrections
Actinopolyspora mortivallis with antibacterial activity isolated and additions. Eur J Biochem 1997;250:1–6.
from a Saharan soil. Ann Microbiol 2012;62:1049–57. [29] Al-Zarban SS, Abbas I, Al-Musallam AA, Steiner U. Nocardiopsis
[11] Ayuso-Sacido A, Genilloud O. New PCR primers for the screening halotolerans sp. nov., isolated from salt marsh soil in Kuwait. Int J
of NRPS and PKS-I systems in Actinomycetes: detection and Syst Evol Microbiol 2002;52:525–9.
distribution of these biosynthetic gene sequences in major [30] Chun J, Bae KS, Moon EY, Jung SO. Nocardiopsis kunsanensis sp.
taxonomic groups. Microb Ecol 2005;49:10–24. nov., a moderately halophilic actinomycete isolated from a saltern.
[12] Wu ZC, Li S, Nam SJ, Liu Z. Nocardiamides A and B, two Int J Syst Evol Microbiol 2000;50:1909–13.
cyclohexapeptides from the marine-derived actinomycete Nocar- [31] Yassin AF, Galinski EA, Wohlfarth A, Jahnke KD. A new
diopsis sp. CNX037. J Nat Prod 2013;76:694–701. actinomycete species, Nocardiopsis lucentensis sp. nov. Int J Syst
[13] Engelhardt K, Degnes KF, Kemmler M, Bredholt H. Production of Bacteriol 1993;43:266–71.
a new thiopeptide antibiotic, TP-1161, by a marine Nocardiopsis [32] Li WJ, Park DJ, Tang SK, Wang D. Nocardiopsis salina sp. nov., a
species. Appl Environ Microbiol 2010;76:4969–76. novel halophilic actinomycete isolated from saline soil in China.
[14] Johnson LF, Curl EA, Bond JH, Fribourg HA. Methods for Int J Syst Evol Microbiol 2004;54:1805–9.
Studying Soil Microflora-Plant Disease Relationships. Burgess, [33] Hozzein WN, Li WJ, Ibrahim M, Ali A. Nocardiopsis alkaliphila
Minneapolis, 15 Minn. USA 1960; 178 p. sp. nov., a novel alkaliphilic actinomycete isolated from desert soil
[15] Waksman SA. The Actinomycetes, Classification, Identification in Egypt. Int J Syst Evol Microbiol 2004;54:247–52.
and Description of Genera and Species. Baltimore: Baltimore: The [34] Hamedi J, Mohammadipanah F, Ventosa A. Systematic and
Williams and Wilkins Company; 1961. 2: pp 61–92. biotechnological aspects of halophilic and halotolerant actino-
[16] Shomurat T, Yoshida J, Amano S, Kojima M. Studies on mycetes. Extremophiles 2013;17:1–3.
actinomycetales producing antibiotics only on agar culture I. [35] Komaki H, Ichikawa N, Hosoyama A, Fujita N. Draft genome sequence
Screening taxonomy and morphology-productivity relationship of of marine derived Actinomycete Nocardiopsis sp. strain TP-A0876, a
Streptomyces halstedii, strain SF-. J Antibiot 1993;32:427–35. producer of polyketide pyrones. Genome Announc 2014;2:e00665–14.
[17] Kawato M, Shinobu R. A simple technique for the microscopical [36] Narasaiah BC. Structural elucidation and antimicrobial activity of
observation. Memoirs of the Osaka University Liberal Arts and secondary metabolites from Streptomyces albus CN-4. IJREAS
Education 1959;8:114. 2016;6:77–89.
[18] Lim SW, Kim JD, Kim BS, Hwang B. Isolation and numerical [37] Singh T, Wahla V. GC-MS analysis of antifungal compounds
identification of Streptomyces humidus strain S5-55 antagonistic derived from soil actinobacteria. Int Res J Pharm 2018;9:81–4.
to plant pathogenic Fungi. Plant Pathol J 2000;16:189–99. [38] Awla HK, Kadir J, Othman R, Rashid TS. Bioactive compounds
[19] Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: a produced by Streptomyces sp. isolate UPMRS4 and antifungal
Laboratory Manual. Cold Spring Harbor, New York: Cold Spring activity against Pyricularia oryzae. Am J Plant Sci 2016;7:
Harbor Laboratory Press; 1989. 1077–85.
[20] Saitou N, Nei M. The neighbor-joining method: a new method for [39] Shah AM, Mushtaq S, Hassan QP. Isolation and characterization of
reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406–25. actinomycin C complex from Streptomyces pratensis: A potent
[21] Bauer AW, Kirby WM, Sherries JC, Turck M. Antibiotic antimicrobial agent against Staphylococcus aureus. Ist interna-
susceptibility testing by a standardized single disk method. Am J tional Conference on Recent Dvelopments in Science, Humanities
Clin Pathol 1966;45:493–6. and Management-2018 (ICRDSHM-18), Srinagar, 17-18th
[22] Balasubramanian G, Sarathi M, Rajesh Kumar S, Sahul Hameed April 2018, India, 2018. 387 p.
AS. Screening the antiviral activity of Indian medicinal plants [40] Falkinham JO, Wall TE, Tanner JR, Tawaha K. Proliferation of
against white spot syndrome virus in shrimp. Aquaculture antibiotic-producing bacteria and concomitant antibiotic produc-
2006;263:15–9. tion as the basis for the antibiotic activity of Jordan's red soils. Appl
[23] Chang CF, Su MS, Chen HY, Lo CF. Effect of dietary β 1,3-glucan Environ Microbiol 2009;75:2735–41.
on resistance to white spot syndrome virus (WSSV) in postlarval [41] Vimal V, Benita Mercy R, Kannabiran K. Antimicrobial activity of
and juvenile Penaeus monodon. Dis Aquat Organ 1999;36:163–8. marine actinomycete, Nocardiopsis sp. VITSVK 5 (FJ973467).
[24] Namita R, Kumar S, Jaganmohan S, Murugan V. DNA vaccines Asian J Med Sci 2009;1:57–63.
encoding viral envelope proteins confer protective immunity [42] Suthindhiran K, Kannabiran K. Cytotoxic and antimicrobial
against WSSV in black tiger shrimp. Vaccine 2007;25:2778–86. potential of actinomycete species Saccharopolyspora salina
[25] Takahashi Y, Itami T, Maeda M, Suzuki N. Polymerase chain VITSDK4 isolated from the Bay of Bengal coast of India. Am J
reaction (PCR) amplification of bacilliform virus (RVPJ) DNA in Infect Dis 2009;5:90–8.
14
| ADLIN JENIFER ET AL.

[43] Hoshino Y, Chiba K, Ishino K, Fukai T. Identification of pyrrole and imidazole polyamide conjugates. Org Biomol Chem
nocobactin NA biosynthetic gene clusters in Nocardia farcinica. 2003;1:3327–42.
J Bacteriol 2011;193:441–8. [57] Busch B, Hertweck C. Evolution of metabolic diversity in
[44] Sosio M, Bossi E, Bianchi A, Donadio S. Multiple peptide polyketide-derived pyrones: using the non-colinear aureothin
synthetase gene clusters in actinomycetes. Mol Gen Genet 2000; assembly line as a model system. Phytochemistry 2009;70:
264:213–21. 1833–40.
[45] Gerth K, Pradella S, Perlova O, Beyer S. Myxobacteria: proficient [58] Awla HK, Kadir J, Othman R, Rashid TS. Bioactive compounds
producers of novel natural products with various biological produced by Streptomyces sp. isolate UPMRS4 and antifungal
activities − past and future biotechnological aspects with the activity against Pyricularia oryzae. Am J Plant Sci 2016;7: 1077–85.
focus on the genus Sorangium. J Biotechnol 2003;106: 233–53. [59] Pratap SS, Seema G, Neetu P, Nimisha S. Surfactin. 2013. A review
[46] Chakrabortti A, Li J, Liang ZX. Draft genome sequence of on novel microbial surfactant. Int J Bioassays 2013;02: 740–5.
Nocardia jinanensis, an opportunistic bacterial pathogen that [60] Schneider A, Stachelhaus T, Marahiel MA. Targeted alteration of
causes cellulitis. Genome Announc 2016;4:e00593–16. the substrate specificity of peptide synthetases by rational module
[47] Tian SZ, Pu X, Luo G, Zhao LX. Isolation and characterization of swapping. Mol Gen Genet 1998;257:308–18.
new p-terphenyls with antifungal, antibacterial, and antioxidant [61] Isa MHM, Shannaq MAF, Mohamed N, Hassan AR. Antibacterial
activities from halophilic actinomycete Nocardiopsis gilva YIM activity of surfactin produced by Bacillus subtilis MSH1. Trans Sci
90087. J Agric Food Chem 2013;61:3006–12. Technol 2017;4:402–7.
[48] Heide L. Aminocoumarins Mutamynthesis, chemoenzymatic [62] Mory M, Spętana M, Dziubek K, Paraszkiewicz K. Antimicrobial,
synthesis, and metabolic engineering (chapter 18). Methods antiadhesive and antibiofilm potential of lipopeptides synthesised
Enzymol 2009;459:437–55. by Bacillus subtilis, on uropathogenic bacteria. Acta Biochim
[49] Liu JK. Natural terphenyls: developments since 1877. Chem Rev Polon 2015;62:725–32.
2006;106:2209–23. [63] Ndlovu T, Rautenbach M, Vosloo JA, Khan S. Characterisation
[50] Ding ZG, Griseusins FG. Spiro-naphthoquinones from a tin mine and antimicrobial activity of biosurfactant extracts produced by
tailings-derived alkalophilic Nocardiopsis species. J Nat Prod Bacillus amyloliquefaciens and Pseudomonas aeruginosa isolated
2012;75:1994–8. from a wastewater treatment plant. AMB Expr 2017; 7:1–9.
[51] Kim YS, Sagara J, Kawai A. Studies on the antiviral activity of [64] Meena KR, Sharma A, Kanwar SS. Microbial lipopeptides and
protein kinase inhibitors against the replication of vesicular their medical applications. Ann Pharmacol Pharmaceutics
stomatitis virus. Biol Pharm Bull 1995;18:895–9. 2017;2:1–5.
[52] Waksman SA, Woodruff HB. Bacteriostatic and bactericidal [65] Siwak E, Jewgiński M, Wójcicka IK. Biological activity of
substances produced by a soil actinomyces. Exp Biol Med 1940; surfactins − a case of a biosurfactant produced by Bacillus subtilis
45:609–14. PCM 1949. Acta Biochim Polon 2015;62:875–8.
[53] Waksman SA. Actinomycin. I. Historical nature and cytostatic
action. Antibiot Chemother (Northfield) 1954;4:502–10.
[54] Dalgliesh CE, Johnson AW, Todd AR, Vining LC. Actinomycin. 1. How to cite this article: Adlin Jenifer JSC,
amino acid content. J Chem Soc 1950;2946–52. https://pubs.rsc. Michaelbabu M, Lelin C Thirumalaikumar C,
org/en/Content/ArticleLanding/1950/JR/jr9500002946#! Jeraldin Nisha SR, Uma G, Citarasu T.
divAbstract
Antimicrobial potential of haloalkaliphilic
[55] Sobell HM. Actinomycin and DNA transcription (actinomycin
Nocardiopsis sp. AJ1 isolated from solar salterns in
mechanism of action/transcriptional complex/melton/premelton).
Proc Natl Acad Sci 1985;82:5328–31. India. J Basic Microbiol. 2019;1–14.
[56] Kumar R, Lown JW. Design, synthesis and in vitro cytotoxicity https://doi.org/10.1002/jobm.201800252
studies of novel pyrrolo [2,1][1,4] benzodiazepine-glycosylated