Figure 8 Profile of active compounds from fraction BA-13, BA-17 and BA-18 on
silica gel plate merck 60 F
254
Arrow head.
Figure 9 Antimicrobial activity of fifteen compos ite fractions purified using PTLC technique; C- = negative control ethyl acetate.
Morphological and Molecular Identification Bas ed on 16S rDNA Analysis
Morphological characterization of isolates SAB E-31, SAB E-41 and SAB E-57 was carried out using Gram staining bacteria. Gram staining results showed
that three marine bacterial isolates were rod-shaped, motile, formed spores and Gram-pos itive bacteria. These isolates have the same characteristics with the genus
of Bacillus Figure 10.
Figure 10 Gram staining of three bacterial isolates coded as: A. SAB E-31; B. SAB E-41 and C. SAB E-57.
BA-13 BA-17
BA-18
Active Co mpounds Rf 0.87
Active Co mpounds Rf 0.50
Active Co mpounds Rf 0.41
Active Co mpounds Rf 0.12
Active Co mpounds
Rf 0.93
Active Co mpounds
Rf 0.12 Active
Co mpounds Rf 0.93
Active Co mpounds
Rf 0.25
λ 366 nm λ 366 nm
λ 254 nm
A. B.
C.
2 µm 2 µm
2 µm
Molecular analysis of 16S rDNA was carried out for the further identification of those three isolates. PCR product of 16S rDNA sequences was
about 1300 bp. Sequences analysis of 16S rDNA showed a high similarity level 97-98 to various strains of Bacillus compared to those available in GenBank
database Table 8 .
Table 8 Similarity of 16S rDNA sequences from isolates SAB E-31, SAB E-41 and SAB E-57 compared
with GenBank Database
Bacte rial Isolates
Similarity Ide ntity
E- Value
Accession
SAB E-31 Bacillus pumilus strain KD3 98
0.0 EU500930.1
SAB E-41 Bacillus amyloliquefaciens strain zy2 98
0.0 JN160740.1
SAB E-57 Bacillus subtilis strain YRL02 97
0.0 EU373407.1
Phylogenetic analysis of 16S rDNA sequences showed that isolates SAB E- 31, SAB E-41 and SAB E-57 formed a different clade with the reference strains of
Bacillus in GenBank database. Clade-1 was formed by the reference strains of Bacillus while clade-2 was formed by those three isolates with Bacillus sp. DF49
Figure 11.
Figure 11 Phylogenetic tree of isolates SAB E-31, SAB E-41 and SAB E-57 with
the reference strains based on 16S rDNA sequences. Numbers at the node s indicate the levels of bootstrap support based on 1000 re-sampled
data sets. The scale bar indicates 0.1 substitutions per nucleotide pos ition.
Amplification of DNA Frag me nts Encoding KS and A Domai n
DNA fragments of three bacterial isolates encoding KS and A domain were successfully amplified using PCR. Three bacterial isolates coded as SAB E-31,
SAB E-41 and SAB E-57 had a DNA fragment encoding A do main whereas only two bacterial isolates coded as SAB E-41 and SAB E-57 had a DNA fragment
encoding KS do main. Visualization of the PCR amplicon for DNA fragments was
analyzed using agarose gel electrophoresis 1 bv. DNA fragment encoding KS
domain was in a size of 700 bp while for A domain was in a size of 1000 bp Figure 12
.
Figure 12 Agarose gel electrop horesis of DNA fragments encod ing KS Domain
and A Domain.
Cloning a nd Bioinformatics Analys is
DNA fragments of those three isolates encoding KS and A domain were successfully cloned into T-Vector pMD20 TaKaRa Bio Inc. and named pMD20-
KS domain and pMD20-A domain. White colonies of E. coli DH5- α carrying the
recombinant plasmid had been isolated and for the plasmid excision was do ne using a combination of restriction enzymes, Bam HI and XbaI. The cropped
recombinant plasmid DNA resulted in two bands which were approximately of 2736 bp that show ed the size of T- vector pMD20 and 700 bp was the size of DNA
fragment encoding KS domain and 1000 bp for DNA fragment encoding A do main Figure 13
.
1 k
b L
a d
d e
r
S AB
E -3
1 S
AB E
-3 1
S AB
E -4
1 S
AB E
-5 7
S AB
E -4
1 S
AB E
-5 7
1 kb
250 500
750 1000
A Do main ~1000 bp KS Do ma in ~700 bp
bp
A B
Figure 13 Restriction of recombinant plasmids digested with BamHI + XbaI; A. pMD20-KS domain and B. pMD20-A do main.
Bioinformatics sequences analysis of DNA fragment encoding KS domain from two bacterial isolates coded as SAB E-41 and SAB E-57 using BlastX
program showed that isolate SAB E-41 had a similarity level of 97 with type I PKS from Bacillus amyloliquefaciens LL3 while isolate SAB E-57 had a similarity
level of 98 with putative polyketide synthase pksL from B. amyloliquefaciens subsp. plantarum CAU-B946 Table 9.
Table 9 Bioinformatics sequences analysis of DNA fragment encoding KS domain using BlastX program
Bacte rial Isolates
Similarity Ide ntity
E- Value
Accession
SAB E-41 Type I PKS; B.
amyloliquefaciens LL3 97
2e-130 YP_005545643.1
SAB E-57 Putative polyketide synthase
pksL; B. amyloliquefaciens subsp. plantarum CAU-B946
98 2e-143
YP_005130937.1
Sequences analysis of DNA fragment encoding A domain showed that isolate SAB E-31 had a similarity level of 81 with bacitracin synthetase 1 from B.
pumilus ATCC 7061. Isolate SAB E-41 had a similarity level of 80 with surfactin
19329 7743
6223 4254
3472 2690
1882 1489
925 421
19329 7743
6223 4254
3472 2690
1882 1489
925 421
A Do main ~1000 bp
T-Vector pMD20
2736 bp T-Vector
pMD20 2736 bp
KS Do ma in ~700 bp
λ M
ar ke
r C
o n
tr o
l S
AB E
-4 1
S AB
E -5
7 λ
M ar
ke r
C o
n tr
o l
S AB
E -4
1 S
AB E
-3 1
S AB
E -5
7 bp
bp
synthetase B from B. amyloliquefaciens subsp. plantarum CAU-B946 while isolate SAB E-57 had a similarity level of 81 with surfactin synthetase A from B.
amyloliquefaciens subsp. plantarum CAU-B946 Table 10.
Table 10 Bioinformatics sequences analysis of DNA fragment encoding A domain using BlastX program
Bacte rial Isolates
Similarity Ide ntity
E- Value
Accession
SAB E-31 Bacitracin synthetase 1; B.
pumilus ATCC 7061 81
1e-154 ZP_03054623.1 SAB E-41
Surfactin synthetase B; B. amyloliquefaciens subsp.
plantarum CAU-B946 80
1e-139 YP_005129035.1 SAB E-57
Surfactin synthetase A; B. amyloliquefaciens subsp.
plantarum CAU-B946 81
1e-129 YP_005129034.1
Amino acid sequences of KS domain from isolates SAB E-41 and SAB E- 57 were aligned and compared with the reference strains in GenBank database.
Two bacterial isolates have a homology of conserved region with the reference strains Figure 14. Phylogenetic analysis of amino acid sequences of KS domain
showed that isolates SAB E-41 and SAB E-57 formed a different clade with the other reference strains Figure 15.
Figure 14 Alignment of amino acid sequences of KS domain from isolates SAB E-
41 and SAB E-57 with the reference strains in GenBank Database using ClustalW program. Shaded area showed the similarity of amino acid
sequences. Black bo x showed a homology of conserved region.
Figure 15 Phylogenetic tree of isolates SAB E-41 and S AB E-57 with the reference strains based on amino acid sequences of KS domain. Numbers at the
nodes indicate the levels of bootstrap support based on 1000 re-sampled data sets. The scale bar indicates 0.1 substitutions per nucleotide
pos ition.
Clade-1
Clade-2
Amino acid sequences of A domain from isolates SAB E-31, SAB E-41 and SAB E-57 were also aligned and compared with the reference strains in GenBank
database. These bacteria have a homology of conserved region with the other reference strains Figure 16. Phylogenetic analysis of amino acid sequences of A
domain showed that isolate SAB E-41 was formed a similar clade with B. amyloliquefaciens FZB42 while isolates SAB E-31 and SAB E-57 formed a same
clade Figure 17.
Figure 16 Alignment of amino acid sequences of A domain from isolates SAB E- 31, SAB E-41 and SAB E-57 with the reference strains in GenBank
Database using ClustalW Program. Black box showed a homology of conserved region for amino acid sequences.
Figure 17 Phylogenetic tree of isolates SAB E-31, SAB E-41 and SAB E-57 with
the reference strains based on amino acid sequences of A domain. Numbers at the nodes indicate the levels of bootstrap support based on
1000 re-sampled data sets. The scale bar indicates 0.2 substitutions per nucleotide pos ition.
DISCUSSION
Antimicrobial compounds of three bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 can be directly extracted using ethyl acetate solvent. The
selection of this solvent system for the extraction process is based on the polarity difference of the liquid culture of bacteria. This solvent is non-polar so that the
separation of liquid culture of bacteria from ethyl acetate layer was more easily done. Jeffery et al. 1989 stated that antimicrobial compounds contained in
bacterial crude extract have different solubility prope rties in each of solvent system. The selection of an appropriate solvent in the extraction process is largely
determined by the solubility properties of the antimicrobial compounds, type of
substrate, the partition coefficient and the distribution ratio of solvent system.
Crude extracts of those three isolates showed different antimicrobial activity against non-pathogenic and pathogenic microbes. The highest activity
shown by the bacterial crude extracts of isolate SAB E-41 which indicated from the large diameter of the inhibition zone. Lay 1994 stated that physical and chemical
properties of antimicrobial compounds will affect the resulting of clear zone. The greater of the molecular weight of bioactive compounds, the more enlarge of the
inhibition zone. The other factors that also affecting the inhibition zone are the density of cells, the sensitivity of microbial test strains to antimicrobial compounds,
the component of antimicrobial compounds, the diffusion rate of antimicrobial compounds into media and the expos ure time of microbial test strains to
antimicrobial compounds.
On the ot her hand, crude extracts of those three isolates showed the highest activity against S. aureus whereas the lowest activity shown by the test strains of C.
albicans and C. tropicalis. The antimicrobial compounds from three bacterial crude extracts couldn’t optimally inhibit the growth of pa thogenic fungal because of the
lower concentration. For the comparison in tested their antimicrobial activity, ampicillin was used as a positive control whereas ethyl acetate solvent was used as
a negative control. Ampicillin which included in - lactam group of antibiotics had a broad spectrum activity so it was chos e as a positive control for this study. This
ant ibiotic unable to inhibit the growth of enteropathogenic E. coli K1-1 because of
the -lactamase enzyme that produced by this microbe while interestingly, our crude extracts from three bacterial isolates which associated with sponge Jaspis sp.
are able to inhibit the growth of this microbe as seen from the diameter average of inhibition zone.
Anand et al. 2006 reported that antimicrobial activity of marine bacteria associated with sponges from the waters off the coast of South East India which
guided fractionation of the broth showed that the ethyl acetate extract of strain SC3 demonstrated activity against the bacterial and fungal test strains. The strain SC3
showed the highest activity against test strains of B. subtilis and E. coli with an inhibition zone of 26 mm and for C. albicans of 15 mm.
TLC analysis and bioautography test were performed for three bacterial crude extracts. The TLC analysis aims to separate the constituent components of
bacterial crude extract based on the difference of absorption, partition and solubility of the chemical components that will be moved with the polarity of
eluent whereas bioautography test aims to quickly detect which spots were the active compounds or impurities compounds. Detection of the spots was performed
by administering the chromatogram plates under UV light with 254 nm and 366 nm wave- length. All of the detection methods that used in this study are expected to
quickly detect the presence of active compo unds without needed to detect the spo ts one by one on silica gel plate. In this study, detection of active compounds was
only performed with these methods so that not all patches of the active compounds contained in bacterial crude extract can be detected.
Sudirman 2005 reported that the rapid detection of active compounds contained in bacterial crude extract can be done using bioautography method
besides the UV irradiation and the spraying of color-forming reaction. Bioautography technique is a combination of chemical methods chromatography
with the microbiological method, the chromatogram plates covered with media that contained microbial test strains and incubated according to the growth temperature
of test strains. Betina 1964 stated that bioautography method can be directly detected the activity and the minimum number of active compounds that contained
in the bacterial crude extract. In addition, this method can be directly detected the
specify location of active compounds based on the position or the Rf values on the chromatogram plate.
Four active spotsfractions with the Rf values of 0.62; 0.55; 0.44; 0.23 isolate SAB E-31, Rf 0.66; 0.60; 0.46; 0.27 isolate SAB E-41 and Rf 0.65; 0.56;
0.45; 0.31 isolate SAB E-57 were successfully detected using these methods and respectively showed antimicrobial activity against P. aeruginosa, while two active
spotsfractions with the Rf values of 0.77; 0.66 isolate SAB E-41 and Rf 0.75; 0.56 isolate SAB E-57 respectively displayed antimicrobial activity against S.
aureus. No active spotsfractions from isolates SAB E-31 can inhibit the growth of S. aureus.
Banoet 2011 reported that at least one active spotfraction was successfully detected using TLC analysis and bioautography detection. Two
spotsfractions with the Rf value of 0.31; 0.81 from ethyl acetate extract of isolate HAL-13 and one spotfraction with the Rf value of 0.85 from n-butanol extract of
isolate HAA-01 and Rf 0.28 from the same extract of isolate HAL-74 displayed antimicrobial activity against S. aureus and enterop athogenic E. coli K1-1.
Bacterial crude extract of isolate SAB E-41 was further fractionated using column chromatography techniques. The selection of this crude extract for further
testing due to the be st activity against non-pathogenic and pathogenic microbes compared to the other bacterial crude extracts. Purification of antimicrobial
compounds via column chromatography techniques is based on the polarity of eluent. Crude extract was slowly injected into the silica gel-column
chromatography and this extract will be separated based on the difference of the polarity of eluent system that used d uring the elucidation process.
Hurtubise 2010 mentioned that chromatography column was a classic chromatography method which used to separate the constituent components of
antimicrobial compounds for the large quantities by adsorption and partition mechanism. The principles of this process were based on the different polarity of
active compounds that contained in the bacterial crude extract. The right selection of stationary phase absorbance and mobile phase eluent in fractionation process
will be determined the successful of the separation of crude extract. The solvent was allowed to flow through the column due to the gravity or the pressure. The
compounds will move through the column at the different rates, separated and collected as the fractions out of the column base.
Two hundred and five fractions were collected from the fraction collector. TLC analys is was performed to know the fractions which have the same
chromatogram. Fractions with the same chromatogram can be combined into one fraction. A total of 30 composite fractions were successfully obtained after TLC
analysis. Antimicrobial activity of thirty composite fractions was tested to P. aeruginosa, S. aureus, enteropathogenic E coli K1-1, C. albicans and C. tropicalis.
Fifteen composite fractions coded as BA-1, BA-2, BA-3, BA-4, BA-5, BA-6, BA- 7, BA-8, BA-11, BA-12, BA-13, BA-14, BA-15, BA-17 and BA-18 showed
different antimicrobial activity against non-pathogenic and pa thogenic microbes. Fraction BA-2 demonstrated a broad spectrum of inhibition compared to the other
fractions. Fraction BA-13 displayed significant activity against S. aureus followed by fraction BA-17.
Anand et al. 2006 mentioned that further fractionation of the ethyl acetate extract from strain CS3 was undertaken by reverse phase HPLC. Fifteen fractions
had been successfully detected based on the HPLC trace. Fraction 11 was found to be active against test strains of E. coli, B. subtilis and C. albicans. Fractions 5 and 7
were found to have trace activity against E. coli. The remaining fractions were found that not possess antimicrobial properties.
Crude extract of isolate SAB E-41 showed antifungal activity against C. albicans and C. tropicalis. After the fractionation process, the fractions are only
active against the test strain of C. albicans. The active compounds in these fractions are allegedly lost due to the purification process. Antifungal activity of
crude extracts from isolate SAB E-41 was a combination factor of several active compounds which can be lost during the fractionation process. Sudirman 2005
stated that during the purification process, most of the active compounds separated from one to another or the active compounds are separated from the pollutant
compound so that the antimicrobial activity from bacterial crude extract can be different between before or after the purification process.
Fifteen composite fractions that showed antimicrobial activity were further purified using PTLC techniques. The basic selection of this technique as the
purification method was due to the less quantity of active compo unds. The principle of this technique was the active compounds that had already known their
position of Rf values were scraped off from silica gel plate and dissolved in chloroform- methanol-ethyl acetate solvent mixtures 1:1:1.
Antimicrobial activity test for active compounds, obtained from PTLC technique were tested to a representative group of Gram-positive, Gram- negative
bacteria and yeast. Three kinds of pathogenic microbes that selected to test their antimicrobial activity were S. aureus, enteropathogenic E coli K1-1 and C.
albicans. These microbes have an impor tant aspect in public health because of their pathogenicity to humans. S. aureus is a group of Gram pos itive bacteria, aerobic
facultative and cause skin infections or infection on the upper respiratory system while enteropathogenic E. coli K1-1 is a group of Gram- negative bacteria that are
resistant to -lactam class of antibiotics because it possess a -lactamase activity enzyme. C. albicans is the pa thogenic yeast that causes candidiasis disease.
Liu 2009 stated that S. aureus is one of the bacteria that cause minor skin infections, pneumonia and meningitis. This strain can be found at skin mucosal
surface, nasal passage and gastrointestinal system. Other bacteria that also cause a seriously infection disease was EPEC K1-1. This strain can cause infection at
gastrointestinal system. Budiarti 1997 found that 55 of diarrhea disease in Indo nesia, mostly caused by EPEC K1-1. C. albicans was a pathogenic strain that
caused a serious problem of infection disease. This strain can be found normally at mucosal surface and urogenital system as human microflora. Many of genitally
diseases or related to human immune systems diseases such HIV-AIDS are mostly caused b y this strain Kortig et al. 1999.
Fraction BA-13 that was eluted by chloroform- methanol 50 -50 solvent system demonstrated as the most antimicrobial compound followed by
fraction BA-17 and BA-18. Four active compounds with the Rf values of 0.87; 0.50; 0.41 and 0.12 respectively obtained from fraction BA-13 and each of those
displayed significant activity against S. aureus and enteropathogenic E. coli K1-1. Two active compounds with the Rf values of 0.93; 0.12 were successfully obtained
from fraction BA-17 while two active compounds with the Rf values of 0.93; 0.25
were successfully obtained from fraction BA-18. Both of the active compounds from fraction BA-17 and BA-18 have antimicrobial activity against S. aureus.
Banoet 2011 had purified active compounds for bacterial crude extract of isolate HAL-13 which associated with sponge Haliclona sp. Fraction BS13-5
displayed as the most active compounds. Four active compounds with the Rf values of 0.35; 0.41; 0.72 and 0.87 were collected using PTLC technique. Two of these
active compounds with the Rf values of 0.35 and 0.41 showed the best activity against enteropathogenic E. coli K1-1. The diameter of inhibition zone that formed
by these two active compounds was 12 mm. Morphology of isolates SAB E-31, SAB E-41 and SAB E-57 was
characterized using Gram staining procedure. The results showed that these bacteria were rod-shaped, Gram positive, motile and formed spores. The
characteristic of these isolates are same with the genus of Bacillus. Anand et al. 2006 reported that the morphological and physiological characterization of strain
SC3 isolated from India waters showed that it to be a Gram-positive, motile, catalase and oxidase-positive rod. Molecular identification of this strain also
indicated it to be a member of the Bacillus genera. Molecular genetic analysis of 16S rDNA sequences were done for the
further identification of those three isolates. Three bacterial isolates which associated with sponge Jaspis sp. were included in the genus of Bacillus based on
16 rDNA analysis. These sequences were in a size of approximately 1300 bp and have conserved and variable regions Appendix 1. This gene is quite large with the
polymorphism between species that can be used as a tool to distinguish between species Woese 2006; Clarridge 2004. Analysis of 16S rDNA is an important
standard for bacterial identification. This identification method was based on the most suitable sequences of bacteria with all of the 16S rDNA sequences that are
known in the GenBank database. Partial analysis of 16S rDNA sequences showed that isolate SAB E-31 had
98 of homology level with B. pumilus strain KD3 Accession No. EU500930.1 Appe ndix 4, isolate SAB E-41 had 98 of homology level with B.
amyloliquefaciens strain zy2 Accession No. JN160740.1 Appe ndix 5 and isolate
SAB E-57 had 97 of homology level with B. subtilis strain YRL02 Accession No. EU373407.1 Appendix 6.
Phylogenetic analys is of 16S rDNA sequences showed that three bacterial isolates formed a different clade with the other reference strains. First clade was
dominated formed by the reference strains of Bacillus while second clade was formed by those three isolates with Bacillus sp. DF49. This strain was in the same
clade with isolate SAB E-41. Isolate SAB E-57 formed a closely relationship clade with isolates SAB E-31, SAB E-41 and Bacillus sp. DF49. This phylogenetic result
was different from the BlastN result. Although the result was different but three bacterial isolates were still include in the ge nus of Bacillus. The formation of
different clade between three bacterial isolates and other reference strains meant that these isolates were assumed in a new species of Bacillus. Santos et al. 2010
reported that molecular identification by partial 16S rRNA gene sequencing and phylogenetic analysis showed that the majority of bacterial isolates isolated from
Brazilian spo nges could be subdivided into three phylogenetically different clusters. Five strains were affiliated with Firmicutes genera Bacillus and
Virgibacillus, three with α-Proteobacteria Pseudovibrio sp. and four with -
Proteobacteria Pseudomonas and Stenotrophomonas. Marine Bacillus species are often isolated from sediments, invertebrates and
marine sponges Pabel et al. 2003. The species of this genus is known to generate spores under adverse conditions, such as those encountered in marine ecosystems
Hentschel et al. 2001. In the marine environment, members of the genus Bacillus are known for their production of metabolites with antimicrobial, antifungal or
generally cytotoxic property. They were regularly isolated from invertebrates and thus display a high potential in the search for new antimicrobial substances
Muscholl-Silberhorn et al. 2008. Many antibiot ics including cyclic peptides, cyclic lipopeptides and novel thiopeptides have been reported from this strain
Nagai et al. 2003. Most of the bioactive compounds that produced by marine bacteria didn’t
getting loose from the invo lvement of two multifunc tional enzymes named polyketide synthases PKS and non-ribo somal
peptide synthetases NRPS. These two multifunctional enzymes mostly involved in the biosynthesis of bioactive
compounds. The simplest functional PKS mod ule consists of a ketosynthase KS, an acyltransferase AT, an acyl carrier protein ACP and a thioesterase TE
domain Schirmer et al. 2005. Besides that, the simplest NRPS module consists of
an adenylation A, a thiolation T, a peptidyl carrier protein PCP and a conde nsation C do main Schwarzer et al.
2003. In this study, the presence of KS and A domain in the cluster of PKS and
NRPS genes from three marine bacterial isolates became one of the most important domains to be investigated. These two domains were analyzed using PCR
amplification. PCR products that indicated the presence of KS domain will show the DNA fragment with the length of 700 bp Appendix 2 whereas for A domain
will show the length of 1000 bp Appe ndix 3. Kim Fuerst 2006 mentioned that
the common feature of complex PKS gene is ketosynthase KS domain that usually present in each module and exhibits the highest degree of conservation
among all domains. Likewise, Schirmer et al. 2005 stated that adenylation A domain become the most conserved domain of NRPS gene compared to the others.
Therefore, the KS and A do main are especially well suited for phyl ogenetic analyses of PKS and NRPS gene diversity.
Isolates SAB E-41 and SAB E-57 possessed DNA fragments encod ing KS and A do main in the cluster of PKS and NRPS genes whereas only isolate SAB E-
31 possessed DNA fragment encoding A do main in the cluster of NRPS gene. These meant that, by detecting one of the domains, whether KS domain of PKS
gene or A domain of NRPS gene could be ensured that they can synthesize the bioactive compounds. Zhao et al. 2008 stated that the modular PKS and NRPS
have been involved in natural prod uct synt hesis in many microor ganisms. On the other hand, the presence of bo th KS and A domain in the cluster of PKS and NRPS
genes at isolates SAB E-41 and SAB E-57 meant that these isolates ha ve the much broader potential in generating many kinds of bioactive compounds. Besides that,
we also assumed that they formed the complex hybrid of PKS-NRPS genes. Interestingly, the existence of these hybrid PKS-NRPS systems will enlarge the
variation of each mod ule in forming an immense variety of bioactive compounds. Many kinds of natural prod ucts are for med through the combination of
PKS-NRPS hybrid systems such as yersiniabactin, one of the iron transport
systems of Yersinia pestis that acts as a virulence factor for pathogenic strains and generated from a hybrid assembly line containing 3 NRPS modules and 1 PKS
module Cane Walsh 1999 and bleomycin BLM, a family of anticancer antibiotics produced by Streptomyces verticillus and generated from BLM
megasynthetase that consist of 10 NRPS modules and 1 PKS module Shen et al. 2001. Donadio et al. 2007 stated that PKS, NRPS or both are molecular
assembly lines that direct product formation on a protein template. Both systems accomplish their task by maintaining reaction intermediates covalently bound as
thioesters on the same phosphopantetheine prosthetic group. In PKS assembly lines, the monomers are acetyl-CoA, malonyl-CoA or methylmalonyl-CoA
whereas the monomers for NRPS assembly are prot einogenic and nonproteinogenic amino acids and other carboxylic acids such as aryl acids.
DNA fragment of KS and A domain were sub-cloned into T-Vector pMD20 Appendix 7 and transformed into competent E. coli
DH5α. Recombinant plasmid that carrying the DNA fragment were named pMD20-KS domain and pMD20-A
domain. This recombinant plasmid was isolated and digested with a combination of restriction enzymes, BamHI and XbaI. Several steps like PCR sequenc ing and
purification of PCR product were done for DNA sequencing. M13 primer RV and M13 primer M4 were used for PCR sequencing and Big Dye
®
X Terminator
T M
Sequences analysis of DNA fragment encod ing KS domain showed that isolates SAB E-41 and SAB E-57 have a feature of subject sequences that similar
to polyketide synthase. Isolate SAB E-41 had 97 of homology level with type I PKS from B. amyloliquefaciens LL3 Appe ndix 8. Weitao et al. 2011 found that
the complete genome sequence of B. amyloliquefaciens LL3 that isolated from Korean fermented food presented the glutamic acid- independent production of
poly- -glutamic acid. This compound is a capsular component or extracellular secretion of Bacillus and a few other organisms that widely used in medicine,
cosmetics, food and wastewater treatment. -PGA is a natural polyamide consisting of D- and L-glutamic acid units connected by -amide linka ges Ashiuchi
Misono 2002; Candela et al. 2009. purification kit was used for the purification of PCR product.
Isolate SAB E-57 had 98 of homology level with putative polyketide synthase from B. amyloliquefaciens subsp. plantarum CAU-B946 Appendix 9.
Borriss et al. 2011 reported that strain CAU B946 that isolated from the rice rhizosphere, was identified by 16S rRNA gene and gyrA gene sequencing and by
physiological and biochemical analysis as being B. amyloliquefaciens subsp. plantarum. Due to its capability to produce antibiotics, some products developed
from strain CAU B946 had already been applied as biofungicides to control several plant diseases such as tobacco black shank, rice sheath blight, cotton fusarium wilt,
cotton verticillium wilt, and wheat scab. Likewise, bioinformatics sequences analysis of DNA fragment encoding A
dom ain showed that isolates SAB E-31, SAB E-41 and SAB E-57 have a feature of subject sequences that similar to nonribosomal peptide synthetase. Isolate SAB E-
31 had 81 of homology level with bacitracin synthetase 1 from B. pumilus ATCC 7061 Appendix 10. Awais et al. 2008 isolated a Bacillus species from soil that
collected from different areas and identified as B. pumilus according to Bergey’s Manual of Determinative Bacteriology. The antibiot ic that prod uced by the
identified B. pumilus strain was designated as bacitracin. This compound was active against Micrococcus luteus and S. aureus. Bottone and Peluso 2003
produced an antifungal compound from B. pumilus that is active against Mucoraceae and Aspergillus species. The active compound inhibited Mucor and
Aspergillus spore germina tion, aborted elongating hyphae and presumable inducing a cell-wall lesion.
Isolate SAB E-41 had 80 of homology level with surfactin synthetase B from B. amyloliquefaciens subsp. plantarum CAU-B946 Appendix 11 while
isolate SAB E-57 had 81 of homology level with surfactin synthetase A from the same strain of bacteria, CAU-B946 Appendix 12. Prokofyeva et al. 1996
mentioned that some of bacteria from Bacillus group produce biologically active lipopeptides that are modified by a fatty acid. One of them was surfactin that has a
large spectrum of biological activity. Surfactin is a powerful lipopeptide that commonly used as an antibiotic. This antibiotic contains a - hydroxy fatty acid a nd
synthesized by a linear nonribosomal peptide synthetase. Besides that, surfactin has
surface active properties directed against microbial adhesion and disrupt ive the permeability of membrane cell of Gram pos itive and Gram negative bacteria.
Besides the surfactin peptides, recently, Blom et al. 2012 reported that the genome of the rhizoba cterium B. amyloliquefaciens subsp. plantarum CAU B946
was 4.02 Mb in size and harbored 3,823 genes coding sequencesCDS. Nine giant gene clusters were dedicated to nonribosomal synthesis of antimicrobial
compounds. This strain also possessed a gene cluster that involved in synthesis of iturin A. Mizumoto et al. 2007 mentioned that iturin A is a cyclolipopeptide
containing seven residues of α-amino acids L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-
Asn-L-Ser- and one residue of a β-amino acid is likely to be the active agents in
biological control. The alignment of amino acid sequences encod ing KS domain showed that
isolates SAB E-41 and SAB E-57 have a conserved region of amino acid sequences with the other reference strains but the similarity number of amino acid sequences
from isolate SAB E-41 with B. amyloliquefaciens LL3 and isolate SAB E-57 with strain CAU B946 was very low. These meant that isolates SAB E-41 and SAB E-
57 have the new feature of amino acid sequences encoding polyketide synthase enzyme. Besides that, phylogenetic analysis also proved that these two bacterial
isolates formed an own clade and differed to the other reference strains. Polyketide synthase enzyme from isolate SAB E-41 was closely related to isolate SAB E-57
and differ to the other reference strains based on phylogenetic tree result. Meanwhile, the alignment of amino acid sequences encod ing A domain also
showed that isolates SAB E-31, SAB E-41 and SAB E-57 have a conserved region of amino acid sequences as shown in figure 16. The similarity number of amino
acid sequences encoding A domain from three bacterial isolates was also low. Isolate SAB E-31 has a low similarity number of amino acid sequences with B.
pumilus ATCC 7061 and so do isolates SAB E-41 and SAB E-57 with strain CAU B946. These meant that three bacterial isolates have a new feature of amino acid
sequences encoding nonribosomal peptide synthetase enzyme. Phylogenetic analysis showed that isolates SAB E-31 and SAB E-57
formed a different clade with the other reference strains. Nonribosomal peptide synthetase enzyme from isolate SAB E-31 was closely related to isolate SAB E-57
while for isolate SAB E-41 was closely related to B. amyloliquefaciens FZB42 based on phylogenetic tree result. Fortman and Sherman 2005 stated that few
marine NRPS genes have been revealed compared with PKS genes. Zhang et al. 2009 reported that
the NRPS genes from the bacteria associated with South China Sea Sponges cluster together
forming two groups , which means that these NRPS genes
are different from the other marine NRPS genes. Twelve NRPS genes
grouped together showed a high similarity to three NRPS relatives of sponge-
associated bacteria. In summary, combining the antimicrobial activity test and detection the
occurrence of KS and A domain of PKS and NRPS genes based on molecular approach could be applied to efficient screening of the potent marine bacterial
isolates and also predicting their related compounds. These related compounds could be developed and applied in pharmaceutical industry in order to treat the
resistant microbes.
CONCLUSION AND SUGGESTION
Conclusion
Crude extracts of isolates SAB E-31, SAB E-41 and SAB E-57 showed different antimicrobial activity against non-pathogenic and pathogenic microbes.
Bacterial crude extract of isolate SAB E-41 demonstrated the best antimicrobial activity compared to the other bacterial crude extracts. Three marine bacterial
isolates were included in the ge nus of Bacillus based on molecular genetic analysis of 16S rDNA. Both isolates SAB E-41 and SAB E-57 possessed KS and A domain
in the cluster of PKS and NRPS genes and only isolate SAB E-31 possessed A domain in the cluster of NRPS gene.
Suggestion
Further purification of active compounds for four active fractions Rf 0.87, 0.50, 0.41 and 0.12 from fraction BA-13 was needed to be conducted in order to
identify the group of these active compo unds and the molecule structure elucidation. By knowing the molecule structure elucidation can help to synthesize
the new antimicrobial substances and also can be applied in pharmaceutical industry in the future.
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APPENDIXES
Appendix 1 The 16S rDNA sequences of three marine bacterial isolates A.
SAB E-31
XbaI ↓
TCTAGAGGATCTACTAGTCATATGGATTGGGCGGTGTGTACAAGGCCCGGGAACGTATTC ACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGA
CTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTAAACCTTGCGGTCTCGCAGCCC TTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGAC
GTCATCCCCACCTCCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATG CTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACAC
GAGCTGACGACAACCATGCACCACCTGTCACTCTGTCCCCGAAGGGAAAGCCCCTATCTC TAGGGTTGTCAGAGGATGGTCAAGGACCCTGGTAAGGTTCTTCGCGTTGCTTCAGAAATT
AAACCCCCACATGCTCCCACCCGCTTGTGCGGGCCCCCCGTCAATTCCTTTGAGTTTCAG TCTTGCGACCGTACTCCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGG
CGGAAACCCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAA TCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTT
CGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCT CCTCTTCTGCACTCAAGTTTCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGGCT
TTCACATCAGACTTAAGAAAACCGCCTGCGAGCCCCTTTACGCCCAATAAATTCCGGACA ACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGGTAGTTAGCCGTGGCTTTCTGG
TTAGGTACCGTCAAGGTGCAAGCAGTTACTCTTGCACTTGTTCTTCCCTAACAACAGAGC CTTTACGATTCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTACGTCCA
TTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGT GTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCAC
CAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGACAGCCGAAACCGTCTTTCATCCT TGAACCATGCGGTTCAAGGAACTATCCGGTATTAGCTCCGGTTTCCCGGAGTTATCCCAG
TCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCCGGGAGCAAG CTCCCTTCTGTCCGCTACGACTTGCATGTGTTAGGCCCTGAATCGGATCC
↑ BamHI
B. SAB E-41
XbaI ↓
TCTAGAGGACTACTAGTCATATGGATTGGGCGGTGTGTACAAGGCCCGGGAACGTATTCA CCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGAC
CGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCT TTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACG
TCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGC TGGCAACTAAGATCAAGGGTTGCGCATCGTTGCGGGACTTAACCCAACATCTCACGACAC
GAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCT AGGATTGTCAGAGGATGTACAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAAAC
CACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACC GTACTCCCCAGGGCGGAGTGCTTTAATGCGTTTAGCTGACAGCACTAAAGGGGCGGAAAC
CCCCTAACAACACTATAGCAACTCTATCGTTTACGGGCGTGGACTACCAGGGTATCTAAT CCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTC
GCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATATCCACTCT CCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGGTTGAGCCGGGGGCT
TTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACG CTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAG
GTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTTCCCTAACAACAGAGCTTT ACGATCCGAAAACAACTTCATCACTCACGCGGCGTTGCGTCCGTCAGACTTTCGTCCATT
GCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGT GGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCA
ACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTCTG AACCATGCGGTTCAGACAACCATCCGGTATTAGCCCCGGTTCCCGGAGTTATCCCAGTCT
TACAGGCAGTTACCCACGTTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCC ATCTGTCCGCTCGACTTGCATGTGTTAGGCCCTGAATCGGATCC
↑ BamHI
C. SAB E-57
XbaI ↓
TCTAGAGGGATCTACTAGTCATATGGGATTGGGCGGGGTGGTACAGGCCCGGGGAACGTA TTCACCGCGGCATGCTGATCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCA
GACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGC CCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTG
ACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAA TGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGAC
ACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATTC TCTAGGATTGTCAGAGGATGTCAAGACCCTGGTAAGGTTCTTCGCGTTGCTTACGAAATT
AAACCCACATGCTCCCACCGCTTGTGCGGGCCCCCCGTCAATTCCTTTGAGTTTCAGTCT TGCGACCGTACTCCCCCAGGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGGCG
GGAAACCCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAAT CCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTC
GCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGGAATTCCACTCT CCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCCTCCCCCGGTTGAGCCGGGGGC
TTTCACATCAGACTTAAGAAACCAGCCTGCGAGCCCTCTTACGCCCAATAATATCCGGAC AACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGG
TTAGGTACCGTCAAGGTGCCGCCCTATTTGAATCGGCACTTGTTCTTCCCTAACAACAGA GCTATTACGATCCGAAAACCTTCATCACTCATCGCGGCGTTGCTCCGTCAGACTTTCGTC
CATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCA GTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTC
ACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACTTTTATTC TGAACCTTGCGGTCAGACAACCTCCGGTTTAGCCCCGGTTCCCGGAGTTTCCCAGTCTAC
CAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGACCAAGCTCCC ATCTGTCCGCTCGACTTCCATGTGTTAGCCCTGAATCGGATCC
↑ BamHI
Appendix 2 DNA sequences of KS domain of PKS gene from two bacterial isolates A.
SAB E-41
XbaI ↓
TCTAGAGGATCTACTAGTCATATGGATTGTGCCGGTGCCGTGGGCCTCGACATAACTGAC GGTCCGCGGGCTGATACCTGCTTTTTTCAGACAGGCCTTAATGACTTCTGCCTGTGCAGC
AGGACTCGGGACGGTAATTCCGCTTACTTTCCCGACGTGGTTAACGGCGCTTCCTTTAAT GACCGCGTAAATGCGGTCGCCGTCTTGTTCGGCTTTTTCCAGCGGCTTGAGCAAGACCGC
ACCGACACCTTCTCCGGAAACGTAGCCGTCCCCGCCCTCGCCGAATGTGCGGCAGCGGCC GTCACTTGAGTGCATCCCTACGCTTCCGTAGCTGAGATATTTCGCCGGGTGCAGCGACAA
GTTCACCCCTCCCGCAAGCGCGGCTTCACATTCGCCGCGGCGGATGCTTTCAATGGCCAG ATGAACGGCGGTCAATGATGAGGAACAAACGGTATCCACCGCGATGCTCGGCCCGTGGAA
GTCACAATAATAGGACACTCTGTTGGCGATCTGCGCATAATTCAGTGAAACCGGAAAAGG ATCAGCTTCAGATAATTGTTCTGCGCCGATTAAGGAATAATCTTTATGCATCACCCCTGC
AAATACGCCGATCGGATGCTGTTTCTCCCCTTTATTCCCCAGCGTTTCAGGCGTATACCC CGCATCTTCAATCGTTTCCCAGCATGTTTCTAAAAACAGCCGCTGCTGCGGATCCATCGC
ATCGGATCC
↑ BamHI
B. SAB E-57
XbaI ↓
TCTAGAGGATCTACTAGTCATATGGATTGTGCCCGTGCCATGCCCTTCCACCATTTGAAT GGTTTCCGGATTAATGTGAAAAGTATCATAGACATGCCGTTCCAATCGTTCTTGAGAAAG
CGCGCTTGGAGCGGTTATTCCGTTTGTAGCGCCGTCCTGATTCATTGCAGAGCCTTTTAT GACGCCGTACACATGATCTCCGTCACTGACGGCGTCGCTAAGACGTTTCAGCACCACCGC
TCCCACACCTTCACCCGGCACGAAGCCGTCCGCGCTTTGATCAAACGTATGGCAGCGCCC GGTCGGTGACAGCATATTCGCCTTATTTGACGACTGATAAAAAGCGGGAGTGGATTGAAT
GAAAACCCCGCCCGCCACCGCCATTTCGGTTTCTTTCGTCCAGAGCCCCTGACATGCCAA ATGAATGGCTGTCAGCGAACTGGAACATGCTGTATCAACAGTGATCGCCGGCCCTTGTAG
ATTAAGGTGATAGGCGATCCTTGCCGGAGTGACGGAATTGTGATTGCCCCAAAAAGCCTG CGCGGGCCCCTGCTGTTTAAAAATGGTCTGGTAATCTCCGCCGCAGGAGCCGGCATACAC
GCCGCATTCCCGGCCTCTTACCGAGTCTCCCGCATATCCCGCATCTCCAAGCGCTTTCCA CGATTCTTCCAGAAACACCCGCTGCTGCGGGTCCCATCATCGGATCC
↑ BamHI
Appe ndix 3 DNA sequences of A domain of NRPS gene from three bacterial isolates
A. SAB E-31
XbaI ↓
TCTAGAGGATCTACTAGTCATATGGATTCCGCGGATTTTGACTTGATGATCGATTCGCCC AATGAATTCAATATCTCCATTTGACAGCCATCGTGCGAGATCGCCCGTTTTGTACATGAC
TTCACCAGGTCGGAAAGGATTTTCAACAAACTCTTCGCTCGTTAACTCTGGCTGGCGATA ATACCCCTTGACCAGTCCGTCACCTGCGACACAAAGCTCTCCAGCTACTCCCGGCGGCTG
CACATGTCCAAACTCATCAACAATATAAACAGCCGTTTGACTCACCGGCTTTCCGATCGG AATCGATAGTGCTTGTTCTTCAATGTGATTCACCGGATAATACGTTGTGAAAATCGTGCT
TTCAGAAGGTCCATACATATGAATAAGTTTGTCTTCTCCAACCGTTTCAAGGGCTGCCAC AACATGCGGCACAGAAGCACGTTCCCCGCCAAACAGCACTTTTCTGACGTTTTTCAGGCT
GCCTTTTTTCATATCAATCAGTAAGTGAAATAGAGCGGTCGTGATCATTAAAATACTGAC TTTTTCCTTCTCAATCGCGCCAGAAAGCTCATTCATATTTAAAATGTGATCCTTTGGCAA
AACAATGAGTTTCGCTCCGTTCAATAAAGCGCCAAACACATCAAACATAAATGCATCAAA TACATAGTTTGAAAGGCTCATCACCGTGTCTTCATGATGAATGGTGAGATAATTCGACTG
CTTCACTGTTCTCAAAATGTTCCGATGCGTCACCATGTTCCCCTTTAGGTTTTGCCTGTC GTACCAGCATGTGGTAGGTCAAAATTCGCCCAAGTCCCAAAGGCGAAACACATACAAACT
GGGATTGCTCTCTGACTGCTGATCAACGGCTTGGATCTTCTGTTTCTATGATTTTCCCCC TTCAAACGCAGTAAGCACAGAGCGATGACGCAGCACCTGGATGGGGTCAGGGACAAACTG
TGCACCACTATCTTGTCAAAAAGTGCCTTGAATGCGCTCATCCAGGGAAGTCCAGGATCG ATTGGGACACATACGCCACCCACCCGCCAATCGGATCC
↑ BamHI
B. SAB E-41
XbaI ↓
TCTAGAGGATCTACCTAGTCCATATGGATTTGCGGGCGGTGCTTATGTGCCCGATTGATC CCGGTCTTTTGCCGGGAGGACCGTCTCCGCTTTATGGGCGGCAGACAGCTCGATTCGGCT
CGTGCTGACAGTTCAGGACTATCAAAGAACAAGCGGGCACATTGCAAGTCCCGATTGTCA TGCTGGATGAAAAGCGCGGATGAAACGGTAAGCGGAACAGACTTGAATCTTCCGGCCGGC
GGGCAACGACTTGGCGTATATCATGTATACATCCGGATCGACCGGCAAACCGAAAGGCGT CATGATTGAACCACAGAAATATCATCAGGCTCGTCAAACATTCGAATTACGTGCCGGTTC
ATGAAGAAGACCGGATGGCGCAAACGGGAGCCGTCAGCTTTGATGCCGGAACCTTCGAAG TCTTCGGTGCATTGCTGAACGGAGCCGCGCTGCACCCGGTGAAAAAAGAGACACTGCTTG
ACGCCGGACGATTCGCCCAATTTCTGAAAGAGCAGCGGATCACGACCATGTGGCTGACGT CTCCGCTGTTTAATCAGCTTGCCCAAAAGGATGCGGGCATGTTTAACACGCTCCGGCACC
TCATCATCGGCGGTGATGCGCTTGTGCCGCATATCGTCAGCAAAGTGAGGAAGGCATCAC CGGAGCTGTCGCTTTGGAACGGCTACGGGCCGACGGAGAATACGACGTTTTCGACGAGTT
TTCTCATTGATCAGGACTGCGACGGCTCGATCCCGATCGGCAAGCCGATCGGAAATTCCA CTGCGTACATTATGGACGAAAACCGCAACCTCCAGCCGATCGGCGCTCCCGGTGAGCTGT
GCGTCGGCGGAAGCGGAGTGGCAAGAGGCTATGTGAATCTGCCTGAATTAACGGAGAAGC AGTTTGTCCGCGATCCGTTCAGACCGGAGAAAAGATATACCCGGACGGGGGACTTGGCGA
AAGATGGCTTCCCGGCGGCCCGAACGAGTTTTTTGGCCGAAATGGCCACCCAAGAAAAAA TCGGATCC
↑ BamHI
C. SAB E-57
XbaI ↓
TCTAGAGGATCTACTAGTCATATGGGATTGCCGCGGATTTTGACCTGGATCATCTTCACG GCCGAATATATTCGATCGTCCCGTCCGGCAGCCAGCGCGCCATATCACCCGGTGCGGTAC
ATAGCGGCCGCTTCCGTTAAACGGATCTTGCAAAAAACTTCTCTGCGGGTCAAATCCGGA AAGATTTAAATAGCCGCGGCCTACACCGTCACCCGCGATATACAGCTCACCGGCCGTCCC
GTCGGGCTGAAGCCTCTGGTGCTTATCCAATATATACAGACGGGCGTTGCCGAGCGGTTT TCCGATCGGAACGTACGCCGCCTGTTGATTCATTCCGTTATCGGCTGACACCTGATGCAC
GGACGCATCTACGCACGTTTCTGTCGGCCCGTAGACATTCGTCAGACGCGGCGCCCTGCC CGATTGATGAAAGAGGTTCATCAGCTGTTCAGCAACAGCGGCGGACAGGCCCTCTCCCCC
AATGAGCATGTGGCGCAATTCAATTCCGCTGACATCTCCCGCCGCAACCATCATCTGCAG ATGCGCAGGGGTTCCGTCAGTGGCTTCAATCCGGTTTTGACGATAATAGTCCAGCAGTGC
CGAGCCATTCGTTACAGTCGTTTTCGGCACGATATAAAGCGTCTGTCCCAAAAGAAGCGA GGCAAAAATCTGTTTGACGGACGCATCAAAATGTAACGGAGCCAAAAGCGCCATTCTTAA
TGTCTGCTCACCGCATTGATAAATCTCCTGCTGCAGTGATTGCACCAGATGATGAACATT TGGGCCGGTGCTCAATCATCACGCCCTTCGGCCGCCCCCGTTGTTACCTGACGTGTAGAT
GATGTAAAGCCACGCCCGGTCTGATTTGTTGTTACTTGACAACCGTCCCGCCAGCCCGTT TTCAAAACTTGAAACAAGCCCTCCGTCAAAAATTCAAATACCGTTTCGGGCAATCCCGCC
TGCCACGGCGCCCTGTTATTTCTCGGGCCCTCCTGTTCAGGAAACAAGACCTTACCGCCT TCCCGCCTTGTTCCCTTCCTCCAAAAATGGGTAACCTTGGAAATTCCCGGAATTCCCCGC
CCCGGGAAAAAGGTCCAAAGGGAAAGTCTTAATTCGGGGAAACCAATTATATTGCCAACC CAACCCCCGCGCCAATCGGATCC
↑ BamHI
Appendix 4 Alignment of 16S rDNA sequences from isolate SAB E-31 using BlastN program
gb|EU500930.1| Bacillus pumilus strain KD3 16S ribosomal RNA gene, partial sequence
Length=1502
Score = 2234 bits 1162, Expect = 0.0 Identities = 12941320 98, Gaps = 211320 2
Strand=PlusMinus
Query 1 GGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTA 60 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1385 GGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTA 1326 Query 61 GCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGT 120
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1325 GCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGT 1266
Query 121 GGGATTGGCTAAACCTTGCGGTCTCGCAGCCCTTTGTTCTGTCCATTGTAGCACGTGTGT 180 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1265 GGGATTGGCTAAACCTTGCGGTCTCGCAGCCCTTTGTTCTGTCCATTGTAGCACGTGTGT 1206 Query 181 AGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTCCCTCCGGTTTGTCA 240
||||||||||||||||||||||||||||||||||||||||||||| |||||||||||||| Sbjct 1205 AGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCA 1146
Query 241 CCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTC 300 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1145 CCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTC 1086 Query 301 GTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGT 360
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1085 GTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGT 1026
Query 361 CACTCTGTCCCCGAAGGGAAAGCCCCTATCTCTAGGGTTGTCAGAGGATGGTCAAGGACC 420 |||||||||||||||||||||| |||||||||||||||||||||||||| |||||| |
Sbjct 1025 CACTCTGTCCCCGAAGGGAAAG-CCCTATCTCTAGGGTTGTCAGAGGAT-GTCAAG--AC 970 Query 421 CTGGTAAGGTTCTTCGCGTTGCTTCAGAAATTAAACCCCCACATGCTCCCACCCGCTTGT 480
||||||||||||||||||||||||| ||||||| ||||||||||| |||||||| Sbjct 969 CTGGTAAGGTTCTTCGCGTTGCTTC--GAATTAAA---CCACATGCTCC--ACCGCTTGT 917
Query 481 GCGGGCCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCCAGGCGGAG 540 ||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 916 GCGGG-CCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCCAGGCGGAG 858 Query 541 TGCTTAATGCGTTAGCTGCAGCACTAAGGGGGCGGAAACCCCCCTAACACTTAGCACTCA 600
||||||||||||||||||||||||||| |||||||||| ||||||||||||||||||||| Sbjct 857 TGCTTAATGCGTTAGCTGCAGCACTAA-GGGGCGGAAA-CCCCCTAACACTTAGCACTCA 800
Query 601 TCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCC 660 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 799 TCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCC 740 Query 661 TCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTAC 720
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 739 TCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTAC 680
Query 721 GCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTTCCCAGTTT 780 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 679 GCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTTCCCAGTTT 620 Query 781 CCAATGACCCTCCCCGGTTGAGCCGGGGGGCTTTCACATCAGACTTAAGAAAACCGCCTG 840
|||||||||||||||||||||||| |||||||||||||||||||||||| |||||||||| Sbjct 619 CCAATGACCCTCCCCGGTTGAGCC-GGGGGCTTTCACATCAGACTTAAG-AAACCGCCTG 562
Query 841 CGAGCCCCTTTACGCCCAATAAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGC 900 |||| ||||||||||||||| |||||||||||||||||||||||||||||||||||||||
Sbjct 561 CGAG-CCCTTTACGCCCAAT-AATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGC 504
Query 901 TGCTGGCACGGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCAAGCAGTTA 960 ||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 503 TGCTGGCAC-GTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCAAGCAGTTA 445 Query 961 CTCTTGCACTTGTTCTTCCCTAACAACAGAGCCTTTACGATTCCGAAAACCTTCATCACT 1020
||||||||||||||||||||||||||||||| |||||||| ||||||||||||||||||| Sbjct 444 CTCTTGCACTTGTTCTTCCCTAACAACAGAG-CTTTACGA-TCCGAAAACCTTCATCACT 387
Query 1021 CACGCGGCGTTGCTCCGTCAGACTTACGTCCATTGCGGAAGATTCCCTACTGCTGCCTCC 1080 ||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||
Sbjct 386 CACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCC 327 Query 1081 CGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTA 1140
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 326 CGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTA 267
Query 1141 CGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATC 1200 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 266 CGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATC 207 Query 1201 TGTAAGTGACAGCCGAAACCGTCTTTCATCCTTGAACCATGCGGTTCAAGGAACTATCCG 1260
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 206 TGTAAGTGACAGCCGAAACCGTCTTTCATCCTTGAACCATGCGGTTCAAGGAACTATCCG 147
Query 1261 GTATTAGCTCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTAC 1320 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 146 GTATTAGCTCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTAC 87
Appendix 5 Alignment of 16S rDNA sequences from isolate SAB E-41 using BlastN program
gb|JN160740.1| Bacillus amyloliquefaciens strain zy2 16S ribosomal RNA gene, partial sequence
Length=1449 Score = 2267 bits 1179, Expect = 0.0
Identities = 13201343 98, Gaps = 221343 2 Strand=PlusMinus
Query 1 GGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGA 60 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1381 GGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGA 1322 Query 61 TTCCAGCTTCACGCAGTCGAGTTGCAGACCGCGATCCGAACTGAGAACAGATTTGTGGGA 120
||||||||||||||||||||||||||||| |||||||||||||||||||||||||||||| Sbjct 1321 TTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGA 1262
Query 121 TTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCC 180 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1261 TTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCC 1202 Query 181 CAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGG 240
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1201 CAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGG 1142
Query 241 CAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCATCGTT 300 |||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||
Sbjct 1141 CAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGC-TCGTT 1083 Query 301 GCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCAC 360
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1082 GCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCAC 1023
Query 361 TCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTACAAGACCTGGTA 420 ||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||
Sbjct 1022 TCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGT-CAAGACCTGGTA 964 Query 421 AGGTTCTTCGCGTTGCTTCGAATTAAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGT 480
||||||||||||||||||||||||||| |||||||||||||||||||||||||||||||| Sbjct 963 AGGTTCTTCGCGTTGCTTCGAATTAAA-CCACATGCTCCACCGCTTGTGCGGGCCCCCGT 905
Query 481 CAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGGCGGAGTGCTTTAATGCGT 540 ||||||||||||||||||||||||||||||||||||||||| |||||||||| |||||||
Sbjct 904 CAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGG-CGGAGTGCTT-AATGCGT 847 Query 541 TTAGCTGACAGCACTAAAGGGGCGGAAACCCCCTAACAACACTATAGCAACTCTATCGTT 600
| ||||| ||||||||| |||||||||||||||||||| || ||||| ||| |||||| Sbjct 846 T-AGCTG-CAGCACTAA-GGGGCGGAAACCCCCTAACA---CT-TAGCA-CTC-ATCGTT 796
Query 601 TACGGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAG 660 ||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 795 TACGG-CGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAG 737 Query 661 CGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCAT 720
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 736 CGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCAT 677
Query 721 TTCACCGCTACACGTGGAATATCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCA 780 |||||||||||||||||||| |||||||||||||||||||||||||||||||||||||||
Sbjct 676 TTCACCGCTACACGTGGAAT-TCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCA 618 Query 781 ATGACCCTCCCCGGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAG 840
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 617 ATGACCCTCCCCGGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAG 558
Query 841 CCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGG 900 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 557 CCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGG 498
Query 901 CACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGG 960 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 497 CACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGG 438 Query 961 CACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACAACTTCATCACTCACGCG 1020
|||||||||||||||||||||||||||||||||||||||||| |||||||||||||||| Sbjct 437 CACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAAC--CTTCATCACTCACGCG 380
Query 1021 GCGTTGCGTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAG 1080 ||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 379 GCGTTGC-TCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAG 321 Query 1081 GAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCAT 1140
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 320 GAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCAT 261
Query 1141 CGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAA 1200 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 260 CGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAA 201 Query 1201 GTGGTAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAGACAACCATCCGGTATT 1260
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 200 GTGGTAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAGACAACCATCCGGTATT 141
Query 1261 AGCCCCGG-TTCCCGGAGTTATCCCAGTCTTACAGGCA-GTTACCCACGT-TTACTCACC 1317 |||||||| ||||||||||||||||||||||||||||| ||||||||||| |||||||||
Sbjct 140 AGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACC 81 Query 1318 CGTCCGCCGCTAACATCAGGGAG 1340
||||||||||||||||||||||| Sbjct 80 CGTCCGCCGCTAACATCAGGGAG 58
Appendix 6 Alignment of 16S rDNA sequences from isolate SAB E-57 using BlastN program
gb|EU373407.1| Bacillus subtilis strain YRL02 16S ribosomal RNA gene, partial sequence
Length=1518 Score = 2127 bits 1106, Expect = 0.0
Identities = 12981334 97, Gaps = 251334 2 Strand=PlusMinus
Query 14 GTACAGGCCCGGGGAACGTATTCACCGCGGCATGCTGATC-GCGATTACTAGCGATTCCA 72 ||||| | || ||||||||||||||||||||||||||||| |||||||||||||||||||
Sbjct 1400 GTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCA 1341 Query 73 GCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGC 132
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1340 GCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGC 1281
Query 133 TTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGT 192 ||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||
Sbjct 1280 TTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATCGTAGCACGTGTGTAGCCCAGGT 1221 Query 193 CATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTC 252
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1220 CATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTC 1161
Query 253 ACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGA 312 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1160 ACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGA 1101 Query 313 CTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCC 372
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1100 CTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCC 1041
Query 373 CCCGAAGGGGACGTCCTATTCTCTAGGATTGTCAGAGGATGTCAAGACCCTGGTAAGGTT 432 |||||||||||||||||| |||||||||||||||||||||||||||| ||||||||||||
Sbjct 1040 CCCGAAGGGGACGTCCTA-TCTCTAGGATTGTCAGAGGATGTCAAGA-CCTGGTAAGGTT 983 Query 433 CTTCGCGTTGCTTACGAAATTAAACCCACATGCTCCCACCGCTTGTGCGGGCCCCCCGTC 492
||||||||||||| || ||||||| ||||||||| |||||||||||||||| |||||||| Sbjct 982 CTTCGCGTTGCTT-CG-AATTAAA-CCACATGCT-CCACCGCTTGTGCGGG-CCCCCGTC 928
Query 493 AATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCCAGGGCGGAGTGCTTAATGCGTT 552 ||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||
Sbjct 927 AATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCCA-GGCGGAGTGCTTAATGCGTT 869 Query 553 AGCTGCAGCACTAAGGGGGCGGGAAACCCCCCTAACACTTAGCACTCATCGTTTACGGCG 612
|||||||||||||||||||||| || ||||||||||||||||||||||||||||||||| Sbjct 868 AGCTGCAGCACTAAGGGGGCGG--AAACCCCCTAACACTTAGCACTCATCGTTTACGGCG 811
Query 613 TGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTT 672 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 810 TGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTT 751 Query 673 ACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGC 732
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 750 ACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGC 691
Query 733 TACACGTGGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCC 792 ||||||| |||||||||||||||||||||||||||||||||||||||||||||||| |||
Sbjct 690 TACACGT-GGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGAACCC 632 Query 793 TCCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCAGCCTGCGAGCCCTCT 852
| |||||||||||||||||||||||||||||||||||||||||| ||||||||||||| | Sbjct 631 T-CCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACC-GCCTGCGAGCCCT-T 575
Query 853 TACGCCCAATAATATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACG 912 ||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 574 TACGCCCAATAAT-TCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACG 516 Query 913 TAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAATCGGCAC 972
||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||| Sbjct 515 TAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAA-CGGCAC 457
Query 973 TTGTTCTTCCCTAACAACAGAGCTATTACGATCCGAAAACCTTCATCACTCATCGCGGCG 1032 |||||||||||||||||||||||| ||||||||||||||||||||||||||| |||||||
Sbjct 456 TTGTTCTTCCCTAACAACAGAGCT-TTACGATCCGAAAACCTTCATCACTCA-CGCGGCG 399 Query 1033 TTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGT 1092
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 398 TTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGT 339
Query 1093 CTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTC 1152 ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 338 CTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTC 279 Query 1153 GCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGG 1212
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 278 GCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGG 219
Query 1213 TAGCCGAAGCCA-CTTTTAT-TCTGAACCTTGCGG-TCAGACAACC-TCCGGTTTAGCCC 1268 |||||||||||| ||||||| |||||||| ||||| ||| |||||| |||||| ||||||
Sbjct 218 TAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAAACAACCATCCGGTATAGCCC 159 Query 1269 CGG-TTCCCGGAGTT-TCCCAGTCTACCAGGCAGGTTACCCACGTGTTACTCACCCGTCC 1326
||| ||||||||||| ||||||||| ||||||||||||||||||||||||||||||||| Sbjct 158 CGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCC 99
Query 1327 GCCGCTAACATCAG 1340 ||||||||||||||
Sbjct 98 GCCGCTAACATCAG 85
Appendix 7 Plasmid map of T-Vector pMD20 TaKaRa Bio Inc.
Appendix 8 Alignment of DNA sequences encoding KS domain of PKS gene from isolate SAB E-41 using BlastX program
ref|YP_005545643.1| baeL gene product [Bacillus amyloliquefaciens LL3]
gb|AEB63415.1| bacillaene synthesis; polyketide synthase of type I [Bacillus amyloliquefaciens LL3]
Length=3513
GENE ID: 12204614 baeL | bacillaene synthesis; polyketide synthase of type I [Bacillus amyloliquefaciens LL3]
Score = 417 bits 1072, Expect = 2e-130 Identities = 221229 97, Positives = 225229 98
Gaps = 0229 0, Frame = -3
Query 689 MDPQQRLFLETCWETIEDAGYTPETLGNKGEKQHPIGVFAGVMHKDYSLIGAEQLSEADP 510 MDPQ+RLFL+TCWETIEDAGYTPETLGNK KQ P+GVFAGVMHKDYSLIGAEQLSE DP
Sbjct 472 MDPQERLFLQTCWETIEDAGYTPETLGNKKNKQRPVGVFAGVMHKDYSLIGAEQLSETDP 531 Query 509 FPVSLNYAQIANRVSYYCDFHGPSIAVDTVCSSSLTAVHLAIESIRRGECEAALAGGVNL 330
FPVSLNYAQIANRVSYYCDFHGPS+AVDTVCSSSLTAVHLAIESIRRGECEAALAGGVNL Sbjct 532 FPVSLNYAQIANRVSYYCDFHGPSLAVDTVCSSSLTAVHLAIESIRRGECEAALAGGVNL 591
Query 329 SLHPAKYLSYGSVGMHSSDGRCRTF
geggdgyvsgegv GAVLLKPLEKAEQDGDRIYAVI 150
SLHPAKYLSYGSVGMHSSDGRCRTFGEGGDGYVSGEGVGAVLLKPLEKAEQDGDRIYAVI Sbjct 592 SLHPAKYLSYGSVGMHSSDGRCRTFGEGGDGYVSGEGVGAVLLKPLEKAEQDGDRIYAVI 651
Query 149 KGSAVNHVGKVSGITVPSPAAQAEVIKACLKKAGISPRTVSYVEAHGTG 3 KGSAVNHVGKVSGITVPSPAAQAEVIKACLKKAGISPRTVSYVEAHGTG
Sbjct 652 KGSAVNHVGKVSGITVPSPAAQAEVIKACLKKAGISPRTVSYVEAHGTG 700
Appendix 9 Alignm ent of DNA sequences encoding KS domain of PKS gene from isolate SAB E-57 using BlastX program
ref|YP_005130937.1| putative polyketide synthase pksL PKS [Bacillus amyloliquefaciens subsp. plantarum CAU B946]
emb|CCF05742.1| putative polyketide synthase pksL PKS [Bacillus amyloliquefaciens subsp. plantarum CAU B946]
Length=2071 GENE ID: 11698078 dfnJ | putative polyketide synthase pksL PKS
[Bacillus amyloliquefaciens CAU-B946] Score = 451 bits 1160, Expect = 2e-143
Identities = 218222 98, Positives = 219222 99 Gaps = 0222 0, Frame = -2
Query 666 DPQQRVFLEESWKALGDAGYAGDSVRGRECGVYAGSCGGDYQTIFKQQGPAQAFWGNHNS 487 DPQQR+FLEESWKAL DAGYAGDSVRGRECGVYAGSCGGDYQ IFKQQGPAQAFWGNHNS
Sbjct 954 DPQQRLFLEESWKALEDAGYAGDSVRGRECGVYAGSCGGDYQAIFKQQGPAQAFWGNHNS 1013 Query 486 VTPARIAYHLNLQGPAITVDTACSSSLTAIHLACQGLWTKETEMAVAGGVFIQSTPAFYQ 307
VTPARIAYHLNLQGPAITVDTACSSSLTAIHLACQGLWTKETEMAVAGGVFIQSTPAFYQ Sbjct 1014 VTPARIAYHLNLQGPAITVDTACSSSLTAIHLACQGLWTKETEMAVAGGVFIQSTPAFYQ 1073
Query 306 SSNKANMLSPTGRCHTFDQSADGFVPGEGVGAVVLKRLSDAVSDGDHVYGVIKGSAMNQD 127 SSNKANMLSPTGRCHTFDQSADGFVPGEGVGAVVLKRLSDAVSDGDHVYGVIKGSAMNQD
Sbjct 1074 SSNKANMLSPTGRCHTFDQSADGFVPGEGVGAVVLKRLSDAVSDGDHVYGVIKGSAMNQD 1133 Query 126 GATNGITAPSALSQERLERHVYDTFHINPETIQMVEGHGTGT 1
GATNGITAPSALSQERLERHVYDTFHINPETIQMVE HGTGT Sbjct 1134 GATNGITAPSALSQERLERHVYDTFHINPETIQMVEAHGTGT 1175
Appendix 10 Alignment of DNA sequences encoding A domain of NRPS gene from isolate SAB E-31 using BlastX program
ref|ZP_03054623.1| bacitracin synthetase 1 BA1 [Bacillus pumilus
ATCC 7061] gb|EDW21930.1| bacitracin synthetase 1 BA1 [Bacillus pumilus ATCC
7061] Length=3570
Score = 492 bits 1266, Expect = 1e-154 Identities = 263323 81, Positives = 281323 87
Gaps = 6323 2, Frame = -1
Query 969 DERIQGTFQDSGAQFVPDPIQVLRHRSVLTAFEGGKSKQKIQAVDQQSESNPSLYVFR 790 DER++ F DSGAQF+ QVLRHRSVL +FEG + + + + QQS+SN + V
Sbjct 1588 DERVKH-FLTDSGAQFLLTH-QVLRHRSVLASFEGTII-ETEDRGIVQQSDSNIDIRVLP 1644 Query 789 LWDLGEFPTTCWYDRQNLKGNMVTHRNILRTVKQSNYLTIHHEDTVMSLSNYVFDAFMF 610
DL T+ + KGNMVTHRNILRTVKQSNYL IHHEDTVMSLSNYVFDAFMF Sbjct 1645 E-DLANLTYTSGTTGKP--KGNMVTHRNILRTVKQSNYLAIHHEDTVMSLSNYVFDAFMF 1701
Query 609 DVFGALLNGAKLIVLPKDHILNMNELSGAIEKEKVSILMITTALFHLLIDMKKGSLKNVR 430 DVFGALLNGAKLIVLPKDHILNMNELSGAIEKEKVSILMITTALFHLLIDMKK SLKNVR
Sbjct 1702 DVFGALLNGAKLIVLPKDHILNMNELSGAIEKEKVSILMITTALFHLLIDMKKDSLKNVR 1761 Query 429 KVLFGGERASVPHVVAALETVGEDKLIHMYGPSESTIFTTYYPVNHIEEQALSIPIGKPV 250
KVLFGGERASVPHV+ ALETVGE KL+HMYGPSESTIFTTYYPVNHIEEQALSIPIGKPV Sbjct 1762 KVLFGGERASVPHVMTALETVGEGKLVHMYGPSESTIFTTYYPVNHIEEQALSIPIGKPV 1821
Query 249 SQTAVYIVDEFGHVQPPGVAGELCVAGDGLVKGYYRQPELTSEEFVENPFRPGEVMYKTG 70 SQTAVYIVDEFG +QPPGVAGELCVAGDGLVKGYY QP+LTSE+FVENPFRPGEVMYKTG
Sbjct 1822 SQTAVYIVDEFGQLQPPGVAGELCVAGDGLVKGYYGQPKLTSEKFVENPFRPGEVMYKTG 1881 Query 69 DLARWLSNGDIEFIGRIDHQVKI 1
DLARWLSNG+IEFIGRIDHQVKI Sbjct 1882 DLARWLSNGEIEFIGRIDHQVKI 1904
Appendix 11 Alignment of DNA sequences encoding A do main of NRPS gene from isolate SAB E-41 using BlastX program
ref|YP_005129035.1| surfactin synthetase B [Bacillus amyloliquefaciens subsp. plantarum CAU B946]
emb|CCF03840.1| surfactin synthetase B [Bacillus amyloliquefaciens subsp. plantarum CAU B946]
Length=3586 GENE ID: 11700595 srfAB | surfactin synthetase B
[Bacillus amyloliquefaciens CAU-B946] Score = 449 bits 1154, Expect = 1e-139
Identities = 236295 80, Positives = 246295 83 Gaps = 6295 2, Frame = +1
Query 112 KEQAGTLQVPIVMLDEKRG--NGKRNRLESSGRRATTWRISCIHPDRPANRKASLNHR 285 +EQAGTLQVPIVMLDE +G L + G + +P K + HR
Sbjct 573 QEQAGTLQVPIVMLDESADETVSGTDLNLPAGGNDLAYIMYTSGSTGKP---KGVMIEHR 629 Query 286 NIIRLVKHSNYVPVHEEDRMAQTGAVSFDAGTFEVFGALLNGAALHPVKKETLLDAGRFA 465
NIIRLVKHSNYVPVHEEDRMAQTGAVSFDAGTFEVFGALLNGAALHPVKKETLLDAGRFA Sbjct 630 NIIRLVKHSNYVPVHEEDRMAQTGAVSFDAGTFEVFGALLNGAALHPVKKETLLDAGRFA 689
Query 466 QFLKEQRITTMWLTSPLFNQLAQKDAGMFNTLRHLIIGGDALVPHIVSKVRKASPELSLW 645 QFLKEQRITTMWLTSPLFNQLAQKDAGMFNTLRHLIIGGDALVPHIVSKVRKASPELSLW
Sbjct 690 QFLKEQRITTMWLTSPLFNQLAQKDAGMFNTLRHLIIGGDALVPHIVSKVRKASPELSLW 749 Query 646 NGYGPTENTTFSTSFLIDQDCDGSIPIGKPIGNSTAYIMDENRNLQPIGAPGELCVGGSG 825
NGYGPTENTTFSTSFLIDQD DGSIPIGKPIGNSTAYIMDENRNLQPIGAPGELCVGGSG Sbjct 750 NGYGPTENTTFSTSFLIDQDYDGSIPIGKPIGNSTAYIMDENRNLQPIGAPGELCVGGSG 809
Query 826 VARGYVNLPELTEKQFVRDPFRPEKRYTRTGDLAKDGFPAARTSFLAEMATQEKI 990 VARGYVNLPELTEKQFVRDPFRP++ RTGDLAK P FL + Q K+
Sbjct 810 VARGYVNLPELTEKQFVRDPFRPDETIYRTGDLAK-WLPDGTIEFLGRIDNQVKV 863
Appendix 12 Alignment of DNA sequences encoding A do main of NRPS gene from isolate SAB E-57 using BlastX program
ref|YP_005129034.1| surfactin synthetase A SrfA [Bacillus amyloliquefaciens subsp. plantarum CAU B946]
emb|CCF03839.1| surfactin synthetase A SrfA [Bacillus amyloliquefaciens subsp. plantarum CAU B946]
Length=3584 GENE ID: 11700594 srfAA | surfactin synthetase A SrfA
[Bacillus amyloliquefaciens CAU-B946] Score = 420 bits 1080, Expect = 1e-129
Identities = 227280 81, Positives = 236280 84 Gaps = 15280 5, Frame = -1
Query 845 NNKSDRAWLYIIYTS gnngggr
RALSTGPNVHHLVQSLQQEIYQCGEQTLRMALLAPL 666 + +SDR YIIYTSG G + + VHHLVQSLQQEIYQCGEQTLRMALLAP
Sbjct 593 STQSDRL-AYIIYTSGTTGRPKGV--MIEHRQVHHLVQSLQQEIYQCGEQTLRMALLAPF 649 Query 665 HFDASVKQIFASLLLGQTLYIVPKTTVTNGSALLDYYRQNRIEATDGTPAHLQMMVAAGD 486
HFDASVKQIFASLLLGQTLYIVPKTTVTNGSALLDYYRQNRIEATDGTPAHLQMMVAAGD Sbjct 650 HFDASVKQIFASLLLGQTLYIVPKTTVTNGSALLDYYRQNRIEATDGTPAHLQMMVAAGD 709
Query 485 VSGIELRHMLIGGEGLSAAVAEQLMNLFHQSGRAPRLTNVYGPTETCVDASVHQVSADNG 306 VSGIELRHMLIGGEGLSAAVAEQL+NLFHQSGRAPRLTNVYGPTETCVDASVHQVSADNG
Sbjct 710 VSGIELRHMLIGGEGLSAAVAEQLLNLFHQSGRAPRLTNVYGPTETCVDASVHQVSADNG 769 Query 305 MNQQAAYVPIGKPLGNARLYILDKHQRLQPDGTAGELYIAGDGVGRGYLNLSGFDPQRSF 126
MNQQAAYVPIGKPLGNARLYILDKHQRLQPDGTAGELYIAGDGVGRGYLNL ++ F Sbjct 770 MNQQAAYVPIGKPLGNARLYILDKHQRLQPDGTAGELYIAGDGVGRGYLNLPDLTAEK-F 828
Query 125 LQDPFNGSGRYVPHRVIWRAG-----CRTGRSNIFGREDD 21 LQDPFNGSGR ++R G G GREDD
Sbjct 829 LQDPFNGSGR------MYRTGDMARWLPDGTIEYIGREDD 862
INTRODUCTION
Background
The improper and uncontrolled uses of antibiotics against pathogenic bacteria induced and accelerated the occurrence of multi drugs resistant MDR
strain. The number of infection cases by MDR strains in Indonesia remains high. Some of infection cases were tuberculosis, HIV, malaria, diarrhea and infection on
upper respiratory system Ditjen PP PL Depkes RI 2011. In 2009, Indo nesia was ranked eighth of 27 countries with the highest of multi drugs resistant cases in
the world WHO 2010
Nowadays, the exploration of bioactive compounds has been carried out in many kinds of resources such as medicinal plants, animals, aquatic organisms as
well as microorganisms in unique ecosystem in order to find the new bioactive compounds which can treat the MDR strains. Indonesia was one of the hotspot
countries possessing many natural resources and almost 70 of the area was covered by coastal area. Considering that, the exploration of new bioactive
compounds in an aquatic area is very promising for the new inve ntion of .
The increase of MDR cases has encouraged many scientists to find the new bioactive compounds in order to solve the MDR problem.
chemot herape utic agents
Marine sponges are one of the evolutionary multicellular organisms that have been reported very potential in producing many kinds of bioactive
compounds. Some of the bioactive compounds showed antibacterial, antifungal, antiviral, anticancer, antifouling and cytotoxic properties Taylor et al. 2007. The
limitation of sponge biomass is the main factor for isolating the large scale of bioactive compounds. Therefore, alternative and ecologically sound sources of
bioactive compounds are needed. Marine microorganisms have contributed to the majority of bioactive compounds. They can produce the same metabolite
compo unds as their host Proksch et al. 2002. which can be developed and applied in pharmaceutical
industry in the future.
The surfaces and internal spaces of marine sponges are unique microhabitat and more nutrient rich than seawater or most sediments, thus they would likely be a
unique niche for the isolation of diverse microor ganisms Friedrich et al. 1999.
Isolation and screening the potent bacterial isolates from marine sponge, identifying the antibiotic-encoding genes in active microorganisms, cloning in
amenable host and characterize the bioactive compounds were the main strategy for producing large amounts of new metabolites Webster Hill 2001.
Three bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 have been isolated from sponge Jaspis sp. at Waigeo Island, Raja Ampat District, West
Papua Province. Each of these isolates indicated different activity against Staphylococcus aureus, Escherichia coli, enteropathogenic E. coli K1-1,
Pseudomonas aeruginosa, Candida albicans and C. tropicalis by using multilayer technique Abubakar 2009. Extraction, fractionation and purification of
antimicrobial compounds for these isolates were important for this study in order to characterize their antimicrobial compounds. Analysis of 16S rDNA and detection
of KS domain of PKS and A domain of NRPS genes were important for identifying these bacteria as well as ensuring their capability in synthesizing the bioactive
compounds.
Aims and Scope of the Study
The aims of this study were to determine the antimicrobial activity of bioactive compounds, analyze the 16S rDNA and detect the occurrence of KS and
A domain of PKS and NRPS genes of those three bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 that symbiosis with sponge Jaspis sp.
LITERATURES
Marine Natural Products
The ocean covers more than 70 of earth surface and is considered as a great reservoir of natural resources. However, the extent of marine biodiversity,
especially of microor ganisms, is barely known. Marine microbial communities are composed of ubiquitous members that can be found not only in the sur face waters
of the sea, but also in the lower and abyssal depths from coastal to the offshore regions Larsen et al. 2005. Several studies have repor ted the discovery of new
bioactive compounds from marine organisms, focusing mainly on chemistry of secondary metabolites, which include now more than 15,000 structurally diverse
bioactive compounds isolated during the last 30 years Salomon et al. 2004. Given the diversity of marine or ganisms and habitats, marine natural
products encompass a wide variety of chemical classes such as terpenoid, polyketides, acetogenins, peptides and alkaloids of varying structures representing
biosynthetic schemes of stunning variety Wright 1998. Marine sponges are one of the benthic organisms that play a potential role as natural compounds producer.
They also became a host for a wide range of microbes. The
role of these diverse microbes in sponge biology varies from
the digestion of microbes as a food source to mutualistic
symbiosis with the spo nge. On the ot her hand, sponge is believed to
provide shelter from predators, a substrate for colonization, access to sunlight for
photosynthetic microbes and a supply of nutrients Taylor et al. 2007.
The availability of sponge biomass is the main factor for isolating marine natural prod ucts. Therefore, marine microor ganisms which associated with marine
sponges became one of the alternative ways to solve that problem. They have contributed to the majority of marine natural prod ucts and produced the same
metabolite compounds as their hos t. Friedrich et al. 2001 reported that many sponges contain enormous amounts of bacteria within their tissues, sometimes
occupying 40 to 60 of the total biomass equivalent to 10
8
to 10
10
bacteria per gram. Considering to the rich diversity of microor ganisms in their tissues and the
growth of microor ganisms were more rapidly, therefore isolation and cultivation of
associated microorganism producer of bioactive compounds could help to solve the recognized problem of development of potential sponge-derived drugs.
Convincing evidence for the involvement of microor ganisms in natural product synthesis has been complied for the tropical sponges Dysidea herbacea and
Theonella swinhoei, in which the producing microbe is a cynobacterium in the former and a bacterium in the latter Proksch et al. 2002. Thus an alternative
strategy targeting the microor ganisms associated with sponges for the screening of bioactive natural products may prove to be an effective approach to circumvent the
associated difficulties of dealing with the organism itself.
Marine Sponge -Associated Bacteria
Sponges are filter feeders animal which live in areas with strong c urrents or wave action. Most carnivorous animals avoid sponges because of the splinter-like
spicules and toxic chemicals producedsequestered by the sponge. Sponges are organized around a system of pores, ostia, canals and chambers that are used to
canalize the large flow of water that is pumped through spo nges. The water enters the sponge through the inhalant canals and exits by the oscules. A sponge is
constituted of three layers. The first layer comprises pinacocytes and is called the pinacoderm. Under the pinacoderm is the mesohyl region that contains canals and
choanocyte chambers. This is where the sponge metabolisms, reproduction and nutrient transfer occur. The third layer is the choa nod erm and contains
choanocytes. They are flagellated cells possessing a collar of cytoplasmic tentacles. It is through the movement of these tentacles that the flow of water is created
bringing in nutrients Wilkinson 1992. There are mainly three classes of sponges, namely the Calcarea 5 orders
and 24 families, Demospongiae 15 orders and 92 families and Hexactinellida 6 orders and 20 families. So far about 15.000 species of sponges have been
described, but their true diversity may be higher Fieseler et al. 2004. Most of the species are placed under the class Demospongiae. Since sponges are simple and
sessile organisms; during e volut ion they have de velope d pot ent chemical defensive mechanism to protect themselves from competitors and predators as well as
infectious microorganisms Belarbi et al. 2003.
Interaction between marine sponges and living aquatic microorganisms are so variously and had many important roles. Many microorganisms were found that
growth commensally in the surface and also found inside of the other microorganisms such as in the food digestive system. Porus from sponges contain
diverse bacteria. Reinheimer 1991 found that there were many kinds of bacteria such as Pseudomonas, Bacillus, Micrococcus, Aeromonas, Vibrio, Achromobacter,
Flavobacterium and Corynebacterium in Microcionia prolifera sponge. There is a symbiotic connection between sponges and a number of bacteria
and algae. Sponges give protection for the symbionts and the symbionts give nutrition for sponges. Algae that symbiotic with sponges give nutrient from their
photosynthesis product Taylor et al. 2007. Suryati et al. 2000 reported that the formation of bioactive compounds from sponges was depend on the precursor of
enzyme, nutrient and product of symbiotic with another biota that contain bioactive compounds such as bacteria, mold and another kinds of dinoflagellata that can spur
on producing bioactive. Suryati et al. 2000 found a number of sponge types living in Spermonde seawater, South Sulawesi, the diversity of mold and symbiotic
bacterial with sponges are so variously and usually dominated by Aeromonas, Flavobacterium, Vibrio, Pseudomonas, Acinetobacter and Bacillus Table 1.
Table 1 Bacterial isolates identification from marine sponges Suryati et al. 2000
No. Marine Sponges
Bacte rial species
1. Acanthela clethera
Flavobacterium sp., Aeromonas sp. 2.
Aplisina sp. Aeromonas sp.
3. Callyspongia sp.
Pseudomonas sp. 4.
Clathria bacilana Aeromonas sp.
5. Clathria reinwardhi
Aeromonas sp. 6.
Jaspis sp. Flavobacterum sp.
7. Phakelia aruensis
Bacillus sp., Aeromonas sp. 8.
Phyllospongia sp. Vibrio sp., Pseudomonas sp., Aeromonas sp.
9. Reniochalina sp.
Acinetobacter sp. 10.
Thionella cilindrica Aeromonas sp.
11. Stylotella aurantiorum
Aeromonas sp., Vibrio sp. 12.
Xestospongia sp. Enterobacteriaceae sp., Aeromonas sp.
Experimental evidence suggests that there are qualitative and quantitative variations in secondary metabolites produced by some organisms. There is a
tende ncy to explain these variations ecologically with environmental factors
influencing the biochemical profile of the organisms. There are many examples of marine invertebrates with chemical defenses. The synthesis and storage of these
substances favors the survival of such organisms in a complex environment and gives such species a selective advantage due to the genetic transmission of the
capacity for chemical defense synthesis Chr istop hersen 1991. Menezes et al. 2010 reported that microbial diversity associated with
algae, ascidians and sponges from the north coast of Sao Paulo State, Brazil had been dominated by phylum Firmicutes, Bacillus spp., together with Ruegeria spp.
Bacillus was the most abundant genus recovered, with 33 isolates, followed by Ruegeria with 31 isolates and Micrococcus with 23 isolates. All of them revealed
broad distribution among the marine macroor ganisms sampled. 16S rDNA sequencing-based analysis showed that marine-derived bacteria were related to 41
genera distributed among the phyla Proteobacteria 35.4, Actinobacteria 30.4, Firmicutes 28.7 and Bacteroidetes 1.1.
Bioactive Compounds
Marine sponges are pre-eminent producers of bioactive secondary metabolites and their repertoire includes peptides, terpenes and sterols. Many of
these compounds showed a functional diversity of actions including antimicrobial, antiviral and cytotoxic activities Table 2. Bioactive compounds of sponge origin
have been used as basic for the synthesis of analogs, for example is glycolipids produced by bacteria that live associated with the marine spo nge Agelas sp. and the
antibacterial agelasines isolated from the marine sponge Agelas nakamurai Bakkestuen et al. 2005.
Kimura et al. 1998 had isolated 1-Methyherbipoline from Halisulfate-1 and suvanin as a serine protease inhibitor from Coscinoderma mathewsi sponge.
Bioactive compounds such as macrocyclic peptide had isolated from Theonella swinhoei comes from water area at Japan. These bioactive compounds known as
Cyclotheonamida A and B that have inhibitory activity to serine protease like thrombin and contains vinyl ogous tyrosine V-Tyr and
α-ketoarginine residu which is still unknown amino acid in nature.
Table 2 Bioactive compounds produced by marine sponges Soediro 1999;
Simmons et al. 2005
Pharmace utical Activity
Bioactive Compounds Types of Sponge
Cytotoxic 3,6 epoksieikosa acid
Swinholida A Vaskulin
Halisilindramida A Jasplakinolide
Jaspicamides Hymeniacidon hauraki
Theonella swinhoei Cribrocalina vasculum
Halichondria caveolata Jaspis johnstoni
Jaspis sp.
Anticancer Agelasfin AGL
Agelas muritianus Anti blood cancer
Kurasin A Amfidinolid B1, B2, B3, N, Q
Triangulinat acid Lingbya majuscule
Amphidinium sp. Pellina triagulata
Antiviral HIV 1 Trikendiol
Trikentrion loeve Antimicrobial
Hormotamnim Diskodermin E-H
Wondosterols Hormothamnion
Discodermia kiiensis Jaspis wondoensis
Antibacterial Lokisterolamin A and B
Corticium sp. Antifungal
kortikatat acid A,B,C Leukasandrolida
Halisilindramida Petrosia corticata
Leucasandra caveolata Halichondria cylindrical
Imunomodulator Agelasflin 10 and 12
Agelas muritianus
Anti-inflammatory Manualida
Luffariella variabilis Unknown substances
still research Halisiklamina A
Bastadin A and B Klatirimin
Halisiklamina B Haliclona sp.
Lanthella basta Clathria basilana
Xestrospongia sp.
O’Keefe et al. 1998 had isolated Adociavirin from Adocia sp. sponge at Bay water area, New Zealand; extract that dissolved in distillation water potential
as antisitopatic inside of CEM-SS cell which infection from HIV-1. Matsunaga et al. 1992 had been isolated 1-acid carboxymethylnicotinic from sponge
Antosigmella raromicroscera which can be used as protease inhibitor. Li et al. 2006a had been isolated 399 bacteria from the sponges Stelletta tenuis,
Halichondria rugosa, Dysidea avara, and Craniella australiensis in the South China Sea, among which, 13 isolates from S. tenuis, 42 from H. rugosa, and 20
from D. avara showed pronounced broad-spectrum antimicrobial activities and enzymatic potentials. Many of the pharmacologically most promising natural
products from sponges are complex polyketides. The fact that polyketide synthases PKSs are almost absent in metazoans suggests a microbial origin. PKSs are
therefore a particularly good study object to investigate the role of symbionts in the chemistry of marine sponges Castoe et al. 2007.
Polyketide Synthas e and Nonribos omal Peptide Synthetas e
The structural characteristics of marine natural products have revealed that they mainly belong to two important chemical families, namely, polyketides and
cyclopeptides, and are synthesized by multifunctional enzymes called polyketide synthases PKSs and nonribosomal peptide synthetase NRPSs. Polyketides are a
group of secondary metabolites, exhibiting remarkable diversity in their structures and functions. Polyketide natural prod ucts are known for their wide range of
pharmacologically impor tant activities, including antimicrobial, antifungal, antiparasitic and antitumor properties Hill 2005.
Nonribosomal peptides are part of a family of complex natural products built from simple amino acid monomers. They can be found in bacteria and fungi
where they are synthesized by nonribosomal peptide synthetase NRPS which are large multimodular and multifunc tional proteins. Nonribosomal peptides as well as
the hybrid products are of much interest because of their pharmaceutical properties such as the immunosuppressant cyclosporine Schwartzer et al. 2003.
Schirmer et al. 2005 had already characterized PKS gene cluster in metagenomic libraries from Discordemia dissolute. The PKS gene cluster is 110 kb
and contain of three open reading frames ORF. The first PKS ORF codes for a remarkably large protein of 25.572 amino acids with a predicted molecular mass of
2.7 MDa. The most remarkable features of this large PKS gene cluster are the presence of a complete set of reductive domains ketoreductase, dehydratase, and
enoylreductase in all except one module, which lacks the ER, and C- methyltransferase domains in 8 of the 14 modules. The products of the PKS gene
clusters have more similarity with fatty acids Figure 1.
Figure 1 Organization of the multimodular PKS gene cluster in metagenomic
libraries from D. dissolute Schirmer et al. 2005. Nguyen 2009 had already investigated the polyketides biosynthetic
pathway in the spo nge Theonella swinhoei and the beetle Paederus fuscipes. The adenylation A domain of an NRPS module is responsible for specific selection
and activation of a defined amino acid. Expression of A domains should help us gain insights into the biosynthetic pathways of pederin and onnamides. There were
two A do mains on the module PedF2 and PedH6 of the ped gene cluster and also two A domains on the module OnnI2 and OnnJ4 of the onn gene cluster. In
agreement with the structure of onnamide B, the prediction results showed that glycine and arginine were the specific amino acids of NRPS modules on the onn
gene cluster Figure 2.
Figure 2 Two A domains inside the biosynthetic gene cluster of Onnamide B Nguyen 2009.
MATERIALS AND METHODS
Duration and Place of Study
This research was carried out from July 2011 to March 2012. Extraction, fractionation and purification of antimicrobial compounds were carried out in the
laboratory of Microbiology, Department of Biology and Biopharmaca Research Center, IPB, Indonesia. Molecular genetic analysis was carried out in Yohda
Laboratory, Department of Biotechnology and Life Sciences, Tokyo University of Agriculture and Technology TUAT, Japan. The flowchart of method s that used in
this study is given in F igure 3.
Materials
Three bacterial isolates coded as SAB E-31, SAB E-41 and SAB E-57 had been isolated from sponge Jaspis sp. at Waigeo Island, Raja Ampat District, West
Papua Province by Abubakar 2009. These bacteria were used for the extraction, fractionation and purification of antimicrobial compounds. Specific primer 63 f 5-
CAGGCCTAACACATGCAAGTC-3 and 1387r 5-GGGCGGWGTGTACAAG GC-3 was used for analysis of 16S rDNA Marchesi et al. 1998. Degenerate
pr imer f: 5-GCSATGGAYCCSCARCARCGSVT-3; r: 5-GTSCCSGTSCCRTG SSCYTCSAC-3 for KS domain and degenerate primer f: 5-AARDSIGGIGSIG
SITAYBICC-3; r: 5-CKRWAICCICKIAIYTTIAYYTG-3 for A domain Schirmer et al. 2005.
Extraction of Antimicrobial Compounds
Each of those three isolates was sub-cultured in 500 ml Seawater Complete Broth media bacto peptone 2.5 g, yeast extract 0.5 g, glycerol 1.5 ml, seawater 375
ml and distilled water 125 ml and incubated in fluctuate incubator with 100 rpm at 30
C until the culture reached the stationary phase. After that, 10 liquid bacterial inocula from previously incubation were cultured in 500 ml SWC broth and
incubated in the same condition until reached the stationary phase for secondary metabolite production Muller et al. 2004.
Extraction of antimicrobial compo unds was done by modifying the method from Sunaryanto et al. 2010. As much 500 ml of liquid bacterial culture were
mixed with 500 ml of ethyl acetate solvent, incubated at room temperature for 24 hours and stirred for 2 hours with 250 rpm. These mixtures were separated and the
ethyl acetate layers were evaporated with rotary evaporator until the drying residue was obtained as crude extract. The crude extract of bacterial cells were dissolved
with ethyl acetate pro analyze to get 100 mgml concentration.
Figure 3 Flowchart of procedural steps used in this study.
Antimicrobial Activity Test
Antibacterial and antifungal activities from crude extracts were tested by using a gar diffusion method a gainst microbial test strains. As much 100 µl bacterial
crude extracts dissolved in ethyl acetate pro analyze were applied carefully into 6 mm paper disks Whatman and at the same time, the disks were dried up using a
Antimicrobial Activity Test Extraction of Antimicrobial
Compounds
Fractionation of Bacterial Crude Extract
Purification of Antimicrobial Compounds
Detection of Antimicrobial Compounds
DNA Extraction
PCR Amplification of Ketos ynthase KS and Adenylation A Domain
Cloning of DNA Fragments Encod ing KS and A Domain
Sequencing and Bioinformatics Analysis of KS and A Domain
Morphological and Molecular Identification
hairdryer at 40 C. After that, the disks were sterilized under UV light for 2 hours
and put into agar plate that have been seeded with 1 vv of microbial test strains conc entration 1x10
6
CFUml, OD
620
0.45. The plate was incubated at 4 C for 3
hours to optimize the diffusion of bacterial crude extract into media. This assay was carried out in triplicate. The diameters of inhibition zones were measured in
millimeters after incubation for 24 hours at 37 C. Control disks were soaked with
ethyl acetate solvent and prepared in the same manner Sudirman 2010.
Detection of Antimicrobial Compounds
Antimicrobial compounds in each of the bacterial crude extract were detected using bioautography method. As much 10 µl of crude extracts were
spotted on TLC plates MERCK Silica Gel 60 F
254
and eluted with vertical chromatography using n-butanol : ethyl acetate solvent mixture with the ratio of
3:7 vv. The spots on TLC plate were detected under UV light at 254 nm and 366 nm wave- length. After that, the retardation factor Rf values were calculated. The
spots on TLC plate were cut off and dried up in room temperature. The developed TLC plates were sterilized under UV light for 1 hour before covered by 15 ml of
melting SWC 45 C containing test strains, and incubated at 37
C for 24 hours. Diameter of inhibition zone around the chromatogram indicated that the spo t was
an active fraction Sudirman 2005 .
Fractionation of Bacterial Crude Extract
Bacterial crude extract of isolate SAB E-41 was fractionated using semi automated flash chromatography Buchi Pump Controller C-610. As much 3 g of
crude extract was dissolved with chloroform- methanol solvent mixture 90-10 and injected into silica gel-column chromatography column dimension 0.40 x 150
mm, particle size of silica gel 40 x 63 µm. Chloroform- methanol solvent mixture 90-10 was flowed into silica gel-column chromatography with the flow rate
of 3.5 mlminutes. The polarity level in the column was increased slowly by changing the methanol concentration from 20, 30, 50, 70 until 90. Two
hundred and five fractions were collected 5 mleach fraction from fraction collector and combined into thirty fractions based on the same chromatogram.
These fractions were dried up and dissolved with chloroform- methanol-ethyl acetate solvent 1:1:1 to test their antimicrobial activity.
Purification of Antimicrobial Compounds
Fifteen active fractions were further purified using preparative thin layer chromatography PTLC technique. Each of the active fractions was spotted onto
silica gel plate MERCK Silica Gel 60 F
254
; 0.1 mm thickness and eluted with n- butanol-ethyl acetate solvent mixture 3:7. The active spots on the silica gel plate
were detected under UV light at 254 nm and 366 nm wave-length. The active spots were extracted directly from the silica gel plate and dissolved with chloroform-
methanol-ethyl acetate solvent mixture 1:1:1. The active fractions that were purified by preparative TLC were tested for their antimicrobial activity.
Morpholog ical and Molecular Identification
Morphological characterization of those three isolates was performed using Gram staining p rocedure. Molecular analysis of 16S rDNA was done using specific
primer, 63f 5-CAGGCCTAACACATGCAAGTC-3 and 1387r 5-GGG CGGWGTGTACAAGGC-3 Marchesi et al. 1998. The PCR cycling condition
for 16S rDNA was carried out under the following condition such as initial denaturation at 94
C for 5 min, followed by 30 cycles of denaturation at 94 C for 1
min, annealing at 55 C for 1 min, elongation at 72
C for 1 min and post PCR at 72
C for 10 min. PCR products of 16S rDNA were purified using GENECLEAN
®
II Kit. These PCR products were sub-cloned into T-Vector pMD20 and transformed into competent E. coli DH5-
α using heat shock method Sambrook Russell 2001. Afterwards, several steps such as PCR colony, isolation and
restriction of recombinant plasmid, PCR sequencing and purification of PCR products were done before the 16S rDNA sequence analysis.
DNA Extraction
Each of bacterial isolates were sub-cultured into SWC broth media and incubated at room temperature for 24 hours. After that, 1.5 ml bacteria isolate was
drawn into microtube and centrifuged 18.000xg for 10 min to obtain bacteria
pellet that were used for DNA extraction. The supernatant was discarded and 250 µl Tris-EDTA TE buffer was added and centrifuged at 8000 rpm for 10 min. The
supernatant was discarded and pellet was re-suspended three times in TE buffer. As much 250 µl TE and 5 µl lysozyme were added together and microtube slowly
inverted to allow mixing and incubated at 37 C for 30 min. After incubation, the
solution was added with 500 µl SDS 10 and 10 µl proteinase K and incubated again at 37
C for 60 min. Afterwards, as much 80 µl NaCl was added together with 100 µl CTAB 10 and incubated at 65
C for 20 min. After incubation, added again the solution with 650 µl PCI and shake n strongl y then centrifuged at 14.000 rpm
for 10 min. The upper solution was transferred into a new microtube then 650 µl CI was added and centrifuged again in same condition. DNA was precipitated using
absolute ethanol 2x vol and Na acetate 3 M 0.1 vol and incubated overnight in freezer. After that, 1 ml ethanol 70 was added for final washing and centrifuged
at 12.000 rpm for 10 min. The supernatant was discarded and pellet was air dried overnight. After this step, 20 µl of TE was added and the extracted DNA was
stored at -20 C for further applications Sambrook Russel 2001.
PCR Amplification of KS and A Domain
KS domain of PKS and A do main of NRPS genes from those three isolates were amplified using PCR primers such as degenerate KS domain f: 5-GCSATG
GAYCCSCARCARCGSVT-3; r: 5-GTSCCSGTSCCRTGSSCYTCSAC-3 and degenerate A domain f: 5-AARDSIGGIGSIGSITAYBICC-3; r: 5-CKRWAICC
ICKIAIYTTIAYYTG-3 Schirmer et al. 2005. The PCR cycling condition for KS domain was carried out in three steps such as initial denaturation at 94
C for 5 min, followed by 35 cycles of denaturation at 94
C for 1 min, annealing at 50 C for 1
min, elongation at 72 C for 1 min 10 sec and post PCR at 72
C for 10 min. The PCR cycling condition for A domain was the same as for KS domain except for
annealing which was carried out at 55 In all cases, the reaction mixtures contained 4 µl dNTP mix 2.5 mM, 5 µl
10X Ex Taq buffer, primer forward and reverse 10 µM; each of 5 µl, 2 µl DNA template 500 ngµl, 1 µl TaKaRa Ex Taq
C for 1 min.
TM
5 unitsµl and 28 µl milli Q. The
total volume of reaction mixtures was 50 µl. PCR products were analyzed using
agarose gel electrophoresis 1 bv.
Cloning o f DNA Frag me nts Encoding KS and A Domain
Purification of PCR products from KS domain of PKS gene 700 bp and A domain of NRPS gene 1000 bp were carried out using GEN ECLEAN
®
II Kit. Each of the purification prod ucts was sub-cloned into T-Vector pMD20 TaKaRa
and transformed into competent E. coli DH5α using heat shock method Sambrook
Russell 2001. Isolation of recombinant plasmid was performed using Mag Extractor
T M
Quick Plasmid Miniprep kit Toyobo, Japan. Restriction of recombinant plasmid was conducted using the combination of restriction enzymes
such as BamHI BioLabs and XbaI TaKaRa. The reaction mixtures contained 0.2 µl BamHI 5 unitsµl, 0.2 µl XbaI 5 units µl, 1 µl 10X NE buffer 4, 0.5 µl DNA
template 500 ngµl and 8.1 µl milli Q. The total volume of reaction mixtures was 10 µl. The reaction mixtures were incubated at 37
C for 24 hours and the restriction product was analyzed using
agarose gel electrophoresis 1 bv.
Sequencing a nd Bioinformatics Analysis of KS and A Domain
DNA fragments that were inserted into plasmid T-Vector pMD20 named pMD20-KS domain and pMD20-A domain were used for the sequencing process.
M13 primer RV and M13 primer M4 were used for PCR sequencing. The PCR cycling condition was carried in three steps such as initial denaturation at 96
C for 5 min, followed by 25 cycles of denaturation at 96
C for 1 min, annealing at 50 C
for 30 sec, elongation at 60 C for 1 min and post PCR at 4
C for an unlimited time. The Big Dye
®
X Terminator
T M
purification kit was used to purify the PCR product before DNA sequencing. The DNA was run in an automated DNA sequencer using
ABI 3130 XL Genetic Analyzer. The DNA sequences were compared to the database available at NCBI using BlastN program for 16S rDNA and BlastX
program for KS and A domain. Construction of phylogenetic tree was carried out using MEGA5 program with neighbor-joining method.
RESULTS
Antimicrobial Activity o f Bacterial Crude Extracts
Antimicrobial compounds of three bacterial isolates can be extracted using ethyl acetate solvent. During the extraction process, the ethyl acetate solvent will
be formed two layers. First layer was aqueous phase that contained the antimicrobial compounds which have been extracted by ethyl acetate solvent.
Second layer was organic phase that contained the bacterial cultures. The first layer was obtained and separated from bacterial cultures then evaporated with rotary
evaporator until the drying residue was obtained as crude extract. Crude extracts of isolates SAB E-31, SAB E-41 and SAB E-57 showed
different antimicrobial activity against non-pathogenic and pathogenic microbes. The bacterial crude extract of isolate SAB E-41 showed better antimicrobial
activity than bacterial crude extracts of isolates SAB E-31 and SAB E-57. Crude extracts of those three isolates demonstrated the best activity against S. aureus
Table 3.
Table 3 Diameter average of inhibition zone mm from three bacterial crude extracts 100 mgml produced by sponge-associated bacteria
Bacte rial Isolates Diame te r Ave rage of Inhibition Zone mm
BS SA EC PA EPEC K1-1 CA CT
SAB E-31 8.3
10.5 5.3
4.8 5.6
2.5 2.5
SAB E-41 10.5
14.5 7.5
6.7 8.7
5.7 4.5
SAB E-57 9.5
13.2 6.4
5.8 6.5
3.8 2.8
Positive Control Ampicilin 100 mgml
Negative Control 14
30 9
27 -
- -
Ethyl acetate -
- -
- -
- -
BS = B. subtilis; SA = S. aureus; EC = E. coli; PA = P. aeruginosa; EPEC K1-1 = Enteropa thogenic E. coli K1-1; CA = C. albicans; CT = C. tropicalis; = non-
pathogen; = pa thogen.
Crude extracts of those three isolates showed different antimicrobial
activity against EPEC K1-1, C. albicans and C. tropicalis whereas for positive control with ampicilin 100 mgml, there was no inhibition a gainst these pathogenic
microbe s Figure 4. Ethyl acetate solvent that used as a negative control didn’t inhibit the growth of non-pathogenic or pathogenic microbes.
Figure 4 Antimicrobial activity of three bacterial crude extracts isolates SAB E-31, SAB E-41 and SAB E-57 using agar diffusion method; C+ = positive
control ampicilin 100 mgml; C- = negative control ethyl acetate.
Thin Layer Chromatography and Bioautography
TLC analysis for three bacterial crude extracts showed that there were many spots in silica gel plate with many k inds of retardation factor Rf values Table 4.
The solvent system, n-butanol-ethyl acetate with the ratio of 3:7 had successfully separated the component of bacterial crude extract. Six spots and many kinds of Rf
values were obtained from this solvent system Figure 5. Table 4 Variation of Rf values from three bacterial crude extracts eluted with
different solvent systems
Solve nt Syste ms Crude
Extracts Numbe r of Spots
Rf Value s λ 254
nm λ 366
nm λ 254
nm λ 366
nm
n-but : CH
3
COOH : ddH
2
3:1:1 O
SAB E-31 SAB E-41
SAB E-57 3
3 3
2 2
2 0.80; 0.61; 0.31
0.91; 0.72; 0.35 0.88; 0.72; 0.35
0.80; 0.61 0.91; 0.72
0.88; 0.72
n-but : EtOAc : ddH
2
2:3:1 O
SAB E-31 SAB E-41
SAB E-57 3
4 4
2 2
2 0.91; 0.53; 0.44
0.91; 0.83; 0.67; 0.55 0.92; 0.85; 0.61; 0.50
0.91; 0.53 0.91; 0.83
0.92; 0.85
n-but : EtOAc 3:7
SAB E-31 SAB E-41
SAB E-57 6
6 6
3 3
3 0.88; 0.72; 0.62;
0.55; 0.44; 0.23 0.92; 0.77; 0.66;
0.60; 0.46; 0.27 0.91; 0.75; 0.65;
0.56; 0.45; 0.31 0.88; 0.55; 0.23
0.92; 0.60; 0.27 0.91; 0.56; 0.31
Figure 5 Profile of each bacterial crude extract on silica gel plate merck 60 F
254
eluted with n-butanol and ethyl acetate mixture 3:7; Red box: active spotsfractions.
The spotsfractions which were detected under UV light at 254 and 366 nm wave- length were further tested for their antimicrobial activity using bioautography
method. This method was able to quickly detect which spotsfractions were the active fractions or pollutant compounds in bacterial crude extract. Part of the silica
gel plate was cut based on the separated spotsfractions and the different of Rf values in order to make the visualization of inhibition zones clearer. Diameter of
inhibition zone around the chromatogram indicated that the spot was an active fraction Figure 6. Bioautography detection resulted that at least 4 active
spotsfractions showed antimicrobial activity against P. aeruginosa and 2 active spotsfractions which inhibited the growth of S. aureus Table 5.
Figure 6 Antimicrobial activity of active spo tsfractions using bioautography
method.
Table 5 Active spotsfractions of three bacterial crude extracts detected using bioautography method
Bacte rial Isolates
Rf Value s Microbial Test Strains
SA PA
CA
SAB E-31 Rf
1
- = 0.88
- -
Rf
2
- = 0.72
- -
Rf
3
- = 0.62
+ -
Rf
4
- = 0.55
++ -
Rf
5
- = 0.44
++ -
Rf
6
= 0.23 -
++ -
SAB E-41 Rf
1
- = 0.92
- -
Rf
2
+++ = 0.77
- -
Rf
3
+++ = 0.66
++ -
Rf
4
- = 0.60
+++ -
Rf
5
- = 0.46
+++ -
Rf
6
= 0.27 -
++ -
SAB E-57 Rf
1
- = 0.91
- -
Rf
2
+++ = 0.75
- -
Rf
3
- = 0.65
++ -
Rf
4
+ = 0.56
+++ -
Rf
5
- = 0.45
+++ -
Rf
6
- = 0.31
++ -
+ = Weak inhibition; ++ = Medium inhibition; +++ = Strong inhibition; SA = S. aureus; PA = P. aeruginosa; CA = C. albicans; = pathogen.
Fractionation of Bacterial Crude Extract from Isolate SAB E-41
Two hundred and five fractions were successfully collected from the fractionation process and combined into thirty composite fractions based on the
same chromatogram. These composite fractions were evaporated until the drying residue was obtained as crude extract. These crude extracts were dissolved with
chloroform- methanol-ethyl acetate mixture 1:1:1 in concentration of 100 mgml. Antimicrobial activity of thirty compos ite fractions were tested to S. aureus, P.
aeruginosa, EPEC K1-1 ,
C. albicans and C. tropicalis. Fifteen composite fractions coded as BA-1, BA-2, BA-3, BA-4, BA-5, BA-
6, BA-7, BA-8, BA-11, BA-12, BA-13, BA-14, BA-15, BA-17 and BA-18 showed different antimicrobial activity against S. aureus, P. aeruginosa, EPEC K1-1 and
C. albicans Table 6. Fraction BA-2 that was eluted by chloroform- methanol 90-10 solvent system showed antifunga l activity against C. albicans whereas
no active fractions showed antimicrobial activity against C. tropicalis.
Table 6 Antimicrobial activity of thirty composite fractions collected from silica gel-column chromatography
Solve nt Syste ms and Fractions Diame te r of Inhibition zone mm
SA PA EPEC K1-1 CA CT
Chloroform-Methanol 90-10 Fraction BA-1
Fraction BA-2 Fraction BA-3
Fraction BA-4 Fraction BA-5
Fraction BA-6 Fraction BA-7
7 8
7 3
5 4
4 2
6 4
3 4
4 2
7 10
3 10
3 2
3 -
3 -
- -
- -
- -
- -
- -
-
Chloroform-Methanol 80-20 Fraction BA-8
Fraction BA-9 Fraction BA-10
2 -
- -
- -
- -
- -
- -
- -
- Chloroform-Methanol 70-30
Fraction BA-11 Fraction BA-12
3 10
3 3
- -
- -
- -
Chloroform-Methanol 50-50 Fraction BA-13
Fraction BA-14 Fraction BA-15
Fraction BA-16 Fraction BA-17
Fraction BA-18 14
2 2
- 12
3 2
- -
- -
- 7
- -
- -
- -
- -
- -
- -
- -
- -
-
Chloroform-Methanol 30-70 Fraction BA-19
Fraction BA-20 Fraction BA-21
Fraction BA-22 Fraction BA-23
- -
- -
- -
- -
- -
- -
- -
- -
- -
- -
- -
- -
-
Chloroform-Methanol 20-80 Fraction BA-24
Fraction BA-25 Fraction BA-26
Fraction BA-27 -
- -
- -
- -
- -
- -
- -
- -
- -
- -
-
Chloroform-Methanol 10-90 Fraction BA-28
Fraction BA-29 Fraction BA-30
- -
- -
- -
- -
- -
- -
- -
- Negative Control Chloroform-Methanol
- -
- -
- Positive Control Ampicilin 100 mgml
30 27
- -
-
SA = S. aureus; PA = P. aeruginosa; EPEC K1-1 = Enteropathogenic E. coli K1-1; CA = C. albicans; CT = C. tropicalis; = pathogen.
Fraction BA-13 that was eluted by chloroform- methanol 50 -50 solvent system demonstrated the highest inhibition against S. aureus followed by
fraction BA-17. The diameter of inhibition zone that formed by these two active
fractions were about 12 mm and 14 mm Figure 7. Fraction BA-2 and BA-4 that was eluted by chloroform- methanol 90 -10 showed the best activity against
EPEC K1-1. The diameter of inhibition zone that formed by these two active fractions were about 10 mm Figure 7.
Figure 7 Antimicrobial activity of thirty compos ite fractions; C+ = positive control
ampicilin 100 mgml; C- = negative control chloroform- methanol.
Purificat ion of Antimicrobial Compounds using Preparative TLC
Fifteen composite fractions that had an active fraction were further purified using PTLC technique. Antimicrobial compounds in active fractions could be
extracted directly from silica gel plate and dissolved with chloroform- methanol- ethyl acetate 1:1:1. Antimicrobial compounds of active fractions, obtained from
PTLC technique were tested to S. aureus, EPEC K1-1 and C. albicans. Fraction BA-13 has demonstrated as the most antimicrobial compounds
compared to the other fractions Table 7. This fraction resulted four different of active compounds with Rf values of 0.87; 0.50; 0.41 and 0.12 Figure 8. These
four active compounds displayed significant activity against S. aureus and EPEC K1-1. Fractions BA-17 and BA-18 carried two kinds of active compounds with Rf
values of 0.93; 0.12 for the BA-17 and 0.93; 0.25 for the BA-18 Figure 8. Both of these active compounds showed activity against S. aureus. Fraction BA-2 carried
one active compound Rf 0.77 that showed activity against C. albicans Figure 9.
Table 7 Rf values of active compounds from fifteen composite fractions
Active Compounds
Rf Value s Diame te r of Inhibition Zone mm
SA EPEC K1-1
CA BA-1
Rf
1
4 = 0.81
4 -
BA-2 Rf
1
8 = 0.77
14 2
BA-3 Rf
1
2 = 0.78
2 -
BA-4 Rf
1
Rf = 0.87
2
6 = 0.66
- 4
- -
- BA-5
Rf
1
Rf = 0.81
2
10 = 0.53
- 2
- -
- BA-6
Rf
1
Rf = 0.87
2
Rf = 0.65
3
10 = 0.35
- -
2 -
- -
- -
BA-7 Rf
1
Rf = 0.87
2
Rf = 0.62
3
6 = 0.38
- -
4 -
- -
- -
BA-8 Rf
1
Rf = 0.90
2
Rf = 0.71
3
8 = 0.35
- -
- -
- -
- -
BA-11 Rf
1
Rf = 0.87
2
3 = 0.68
- -
- -
- BA-12
Rf
1
Rf = 0.90
2
Rf = 0.71
3
Rf = 0.62
4
Rf = 0.41
5
10
= 0.12 -
- -
- -
- -
- -
- -
- -
-
BA-13 Rf
1
Rf = 0.87
2
Rf = 0.72
3
Rf = 0.60
4
Rf = 0.50
5
Rf = 0.41
6
3
= 0.12 -
- 4
8 12
10 -
- 6
4 2
- -
- -
- -
BA-14 Rf
1
Rf = 0.90
2
Rf = 0.75
3
Rf = 0.68
4
2 = 0.58
- -
- -
- -
- -
- -
-
BA-15 Rf
1
Rf = 0.93
2
Rf = 0.78
3
Rf = 0.68
4
2 = 0.33
- -
- -
- -
- -
- -
-
BA-17 Rf
1
Rf = 0.93
2
Rf = 0.71
3
Rf = 0.68
4
Rf = 0.33
5
14
= 0.12 -
- -
2 -
- -
- -
- -
- -
-
BA-18 Rf
1
Rf = 0.93
2
Rf = 0.41
3
Rf = 0.25
4
2 = 0.12
- 2
- -
- -
- -
- -
-
SA = S. aureus; EPEC K1-1 = Enteropathogenic E. coli K1-1; CA = C. albicans; = pathogen.
Figure 8 Profile of active compounds from fraction BA-13, BA-17 and BA-18 on
silica gel plate merck 60 F
254
Arrow head.
Figure 9 Antimicrobial activity of fifteen compos ite fractions purified using PTLC technique; C- = negative control ethyl acetate.
Morphological and Molecular Identification Bas ed on 16S rDNA Analysis
Morphological characterization of isolates SAB E-31, SAB E-41 and SAB E-57 was carried out using Gram staining bacteria. Gram staining results showed
that three marine bacterial isolates were rod-shaped, motile, formed spores and Gram-pos itive bacteria. These isolates have the same characteristics with the genus
of Bacillus Figure 10.
Figure 10 Gram staining of three bacterial isolates coded as: A. SAB E-31; B. SAB E-41 and C. SAB E-57.
