Nethravathi A. H1, Narayan Moger2, Amruta 3 And P. U. Krishnaraj 4
1,2,4
Department of Biotechnology University of Agricultural Sciences, Dharwad-580 005,(Karnataka),
3
National center for Cell Science, Pune (Maharashtra).
ABSTRACT
A moderately halophilic bacterium produces ectoine capable of living in marine water. Ectoine is the compatible solute which accumulates in the cell membrane, are known to be involved in salt tolerance activity in most the halophiles. In the present study, an attempt was made to isolate the genes responsible for ectoine synthesis from the marine bacterial isolates. Nine isolates obtained from the cultures collection of the Department of Biotechnology, UAS, Dharwad were screened to salt tolerance activity on nutrient agar supplemented with different concentrations of sodium chloride from 0%, 5%, 10%, 15%, which revealed that only the four isolates AUDI8, AUDI18, AUDI34 and AUDI144 were showing tolerance up to 15%. The PCR analysis revealed that these four isolates AUDI8, AUDI18, AUDI34 and AUDI144 were positive for the presence of ectA and ectB. AUDI34 ectB was cloned in pTZ57R/T transformed then maintained in E. coli DH5α. The pNHNM1 and pNHNM3 was sub cloned into prokaryotic expression vector pET32C+ then transformed into E. coli BL21 and confirmed clones pNHNM11 and pNHNM13 were induced with IPTG and the SDS-PAGE analysis showed that the 46.1 kDa proteins.
Key words: Compatible solute;Ectoine genes; Salt tolerance; Halophilic bacteria; osmotic adoption.
I. INTRODUCTION
Adverse environmental factors of multifarious types affect plant growth, metabolism and ultimately the final yield of crop plants. An increased incidence of abiotic and biotic stresses that impact productivity of principal crops is being witnessed all over the world. Among the different environmental factors, salinity, drought, temperature, flooding, pollutants and radiation are the major abiotic factors
limiting the productivity of crops. Salinity in soil or water is one of the major abiotic stresses (Liu et al.,
2011). Halophiles are salt loving microorganisms that grow over an extended range of salt concentration, (3-15% NaCl, w/v and above) and inhabit hyper saline environments (Shivananda and Mugeraya 2011). Salinity presents a multifold challenge to all organisms in terms of perturbed osmotic balance, ionic disequilibria and generation of toxic metabolites. Depending upon the genetic make-up of the organism and the environment it normally dwells in, these responses vary greatly although some reactions seem to be universal for steady survival in salt environment (Roberts, 2005). Microbes existing
in saline environments have developed two principle mechanisms of adaptation viz; ‘Salt-in-cytoplasm
strategy’ which means that inorganic ions, mainly K+
and Clˉ are accumulated in the cytoplasm, until the
(extremozymes) and compatible solutes like ectoine, glycine betain, trehalose etc are of great interest of
biotechnology (Satyanarayana et al., 2005).
II. MATERIALS AND METHODS
Bacterial samples
During this study, previously isolated salt tolerant bacterial cultures from the marine water sample of the West-Costal area of India were used (Alone, 2012), to isolate and study molecular characterization of the salt tolerant genes in marine bacterial isolates through PCR based method. This study was carried out in Department of Biotechnology, University of Agricultural Science Dharwad.
Nine isolates (AUDI8, AUDI13, AUDI18, AUDI34, AUDI144, AUDI69, AUDI80, AUDI228, AUDI360) were screened to check their salt tolerance activity on nutrient agar supplemented with different concentrations of sodium chloride from 0% (256 mM), 5% (855 mM), 10% (1711 mM) and
15% (2566 mM) incubated at room temperature (280C) and observed for growth from next day of
inoculation.
Primer designing
The Coding sequence of ectA and ectB genes in Halomonas sp. was retrieved from NCBI database.
Using Bio-edit tool degenerate primers were designed by considering the starting codon of ectB for
forward primer and for reverse primer designing starting sequence of ectC were used in such a way that
to amplify a 622-720 bp region of the ectA and 1359-1407 bp region of ectB gene. The designed primers
were synthesized at Sigma-Aldrich chemicals Pvt. Ltd, Bangalore. The sequences for the forward and the reverse primers are as follows: ectAF
5’ATGASSMCKMCAAYMRWRMMYTT-3’ ectA R
5’-CGTTCRAGMRTYTGGGTCTGCAT-3’ ectB F
5’-ATGCAGACCCARAYKCTYGAACG-3’ ectB R
5’-CTTCVAGGTTACGAACGATCAT-3’
S= G/C M=A/C R=A/G V=A/G/C W=A/T Y=C/T K= G/T
DNA isolation and PCR amplification of ectA and ectB genes
Bacterial genomic DNA was isolated using modification of existing techniques as described by
(Khandeparkar et al., 2011). DNA used for PCR was prepared as follows: A 20 μl volume of reaction
mixture contained 10x Taq assay buffer, 2 mM dNTPs, 5pM of each forward and reverse primer and one
unit of Taq DNA polymerase, 100 ng/µl template and the final volume was adjusted to 20 μl by adding water. Amplification was carried out in a thermal cycler. After an initial denaturation at 94ºC for 5 min, 35 cycles of the three- step PCR amplification were completed, each consisting of denaturation at 94ºC
for 1min, primer annealing at 59.9ºC and 55.8 ºC for 45sec for ectA and ectB respectively. Primer
extension at 72ºC for 2 min final extension at 72ºC for 10 min. at the end of amplification cycles to complete then hold at 4ºC.
Separation of amplified products
PCR products were separated by gel electrophoresis using a horizontal 1% agarose gel. The master mix (100 µl) was prepared and distributed in to tubes containing 100 ng/µl of template. The PCR
was eluted by using Qiagen gel extraction kit as per user manual. The eluted ectB PCR fragment was sent for sequencing to SciGenom Labs Pvt. Ltd, Cochin, India. Using universal M13 forward and reverse primers sequencing was undertaken. Further, the homology search was done with BLAST
algorithm available at http://www.ncbi.nih.gov.
Cloning of ectB gene
The gel purified 1359-1407 bp amplicon was ligated into pTZ57R/T. The ligation reaction mixture was prepared for final volume of 30 µl as per the directions given in InsTAclone PCR cloning kit
manual (Fermentas Life Sciences, EU). The ligated products were used for E. coli DH5α transformation.
Transformants were screened through blue and white colony assay. Then further, the clones were
confirmed by PCR amplification by using degenerate primer and also by restriction analysis using EcoRI
and HindIII. Then these constructs were named as pNHNM1 and pNHNM3. In order to assess the
expression of cloned ectB, it was sub cloned into prokaryotic expression vector pET32C+ transformed
into E. coli BL21 and the constructs were named as pNHNM11 and pNHNM13 and the confirmed
clones were induced with IPTG and analysed on the SDS-PAGE.
III. RESULTS AND DISCUSSION
The bacterial isolates AUDI8, AUDI18, AUDI34, AUDI44 were found to be very effective salt tolerant organisms when grown on nutrient agar supplemented with different concentrations of sodium chloride were ranging from 0% to 15%. Kushner in 1978 defined several categories of microorganisms on the basis of their optimal growth: (1) Non halophiles are that grow best in media containing less than 0.2 M NaCl (1% salt); (2) Slight halophiles grow best in media with 0.2 to 0.5 M NaCl (1-3% salt); (3) Moderate halophiles grow best with 0.5 to 2.5 M Nacl (3-15%) salt and extream halophiles show optiumal growth in media containing 2.5 to 5.2 M NaCl (15- 32% salt) (Ventosa, 2006).
Plate 1. Screening the isolates for salt tolerance activity A=0%, B=5%, C=10%, D=15%
1. AUDI180, 2. AUDI13, 3. AUDI8, 4. AUDI34, 5. AUDI144, 6. AUDI69, 7. AUDI8, 8. AUDI228, 9. AUDI360
Hence, the isolates (AUDI8, AUDI13, AUDI18, AUDI34, AUDI144, AUDI69, AUDI80, AUDI228, AUDI360) were employed for understanding the diversity of presence of ectoine genes as
well as in identifying the presence, cloning and expression of ectB. About 622-720 bp and 1359-1407 bp
amplicons of ectA and ectB were amplified from the genomic DNA of 4 bacterial isolates only and were
Plate 2. PCR amplification of ectA from genomic DNA
L. λ-DNA/ EcoRI +HindIII marker (GeNei)
1. AUDI8, 2. AUD18, 3. AUDI34, 4. AUDI44
Plate 3. PCR amplification of ectB from the genomic DNA L.1 Kb ladder (GeNei)
1. AUDI8, 2. AUD18, 3. AUDI34, 4. AUDI44.
The gene cluster ectABC coding for diaminobutyric acid (DABA) acetyltransferase (EctA),
DABA aminotransferase (EctB) and ectoine synthase (EctC) was isolated and characterized by several
researchers (Canovas et al., 1998; Ono et al., 1999; Kuhlman and Bremer, 2002) and reported to have
role in salt tolerance of bacteria.
The nucleotide sequences of ectB were analysed using BLAST algorithm available at
http://www.ncbi.nlm.nih.gov. Cloned ectB had 85% homology with Chromohalobacter sp. NJS-2
ectABC operon (EF486239.1), Chromohalobacter salexigens DSM 3043 (CP000285.1) for
diaminobutyrate aminotransferase apoenzyme and Halomonas elongate (AJ011103.2) for
diaminobutyrate 2-oxoglutarateaminotransferase.
1 ATG CAG ACC CAG ATT CTC GAA CGC ATG GAA TCC GAA GTC CGG ACC 45 1 M Q T Q I L E R M E S E V R T 15
46 TAT TCA CGT TCT TTT CCT ACC GTT TTC ACT GAA GCC AAG GGC GCG 90 16 Y S R S F P T V F T E A K G A 30
91 CGC CTG CAT GCC GAG GAC GGC AAC CAG TAC ATC GAT TTT CTC GCC 135 31 R L H A E D G N Q Y I D F L A 45
136 GGC GCC GGC ACG CTC AAC TAC GGT CAC AAC CAC CCC AAG CTC AAG 180 46 G A G T L N Y G H N H P K L K 60
181 CAG GCA CTG GCC GAT TAC ATC GCC TCC GAT GGC ATC GTC CAT GGT 225 61 Q A L A D Y I A S D G I V H G 75
226 CTG GAC ATG TGG AGC GCG GCC AAG CGC GAC TAT CTG GAA ACC CTC 270 76 L D M W S A A K R D Y L E T L 90
271 GAA GAG GTG ATC CTC AAG CCG CGT GGC CTG GAT TAC AAG GTT CAT 315 91 E E V I L K P R G L D Y K V H 105
316 CTG CCG GGC CCG ACG GGC ACC AAT GCC GTG GAA GCC GCC ATT CGA 360 106 L P G P T G T N A V E A A I R 120
361 CTG GCG CGC AAC GCC AAG GGT CGT CAC AAC ATC GTC ACC TTC ACC 405 121 L A R N A K G R H N I V T F T 135
406 AAC GGA TTC CAT GGC GTG ACC ATG GGC GCG CTG GCC ACC ACC GGC 450 136 N G F H G V T M G A L A T T G 150
451 AAT CGC AAG TTC CGT GAA GCC ACC GGC GGT ATC CCG ACC CAG GGC 495 151 N R K F R E A T G G I P T Q G 165
496 GCC AGC TTC ATG CCG TTC GAT GGC TAC ATG GGC GAG GGC GTC GAC 540 166 A S F M P F D G Y M G E G V D 180
541 ACC CTG AGC TAC TTC GAG AAA CTG CTC GGC GAC AAC TCC GGT GGT 585 181 T L S Y F E K L L G D N S G G 195
586 CTC GAC GTT CCC GCG GCC GTG ATC ATC GAG ACG GTG CAG GGC GAG 630 196 L D V P A A V I I E T V Q G E 210
631 GGC GGT ATC AAT CCG GCC GGC ATC CCG TGG CTG CAG CGC CTG GAA 675 211 G G I N P A G I P W L Q R L E 225
676 AAG ATC TGC CGC GAT CAC GAC ATG CTG CTG ATC GTC GAC GAC ATT 720 226 K I C R D H D M L L I V D D I 240
721 CAG GCC GGC TGC GGT CGT ACG GGC AAG TTC TTC AGC TTC GAG CAT 765 241 Q A G C G R T G K F F S F E H 255
766 GCC GGC ATC ACG CCG GAC ATC GTC ACC AAC TCC AAG TCC CTG TCG 810 256 A G I T P D I V T N S K S L S 270
811 GGT TTC GGC CTG CCG TTC GCG CAT GTG CTG ATG CGC CCG GAA CTG 855 271 G F G L P F A H V L M R P E L 285
856 GAT ATC TGG AAG CCC GGC CAG TAC AAC GGC ACG TTC CGT GGT TTC 900 286 D I W K P G Q Y N G T F R G F 300
946 AGC GAC GAC ACC TTC GAG CGC GAC GTT CAG CGC AAG GGC CGT GTG 990 316 S D D T F E R D V Q R K G R V 330
991 GTC GAG GAT CGC TTC CAG AAG CTT GCC AGC TTC ATG ACC GAG AAA 1035 331 V E D R F Q K L A S F M T E K 345
1036 GGG CAT CCG GCC AGC GAG CGT GGC CGT GGC CTG ATG CGT GGC CTG 1080 346 G H P A S E R G R G L M R G L 360
1081 GAC GTC GGT GAC GGC GAC ATG GCC GAC AAG ATC ACC GCA CAA GCG 1125 361 D V G D G D M A D K I T A Q A 375
1126 TTC AAG AAC GGG CTG ATC ATC GAG ACA TCC GGC CAT TCA GGC CAG 1170 376 F K N G L I I E T S G H S G Q 390
1171 GTG ATC AAG TGC CTT TGC CCG TTG ACC ATT ACC GAC GAA GAC CTC 1215 391 V I K C L C P L T I T D E D L 405
1216 GTC GGC GGC CTG GAC ATC CTC GAG CAG AGC GTC AAG GAA GTC TTC 1260 406 V G G L D I L E Q S V K E V F 420
1261 GGT CAA GCC TAA GTC CAT TGT TCG TTA GTC CAC TAA ATT GTC ATT 1305 421 G Q A * V H C S L V H * I V I 435
1306 CGC AAA TGT GTT TAC AGT GGG CAC CGC CTG CGG CCA CGA GGT CGC 1350 436 R K C V Y S G H R L R P R G R 450
1351 GGG CTT CTT ACT CAG ATC TGC AGA GGA TTG CGC 1383 451 G L L T Q I C R G L R
Fig 2. Complete nucleotide and amino acid sequence of
ect
B
Fig3.Vector map of pNHNM11 (pET32C+containing ectB)
EctB was further sub cloned into prokaryotic expression vector pET32C+, to study the expression
of cloned ectB in E. coli BL21. E. coli BL21 containing pNHNM11 construct harboring ectB from
AUDI34 were subjected to SDS-PAGE. The protein bands corresponding to ~46.1 kDa was observed
for ectB in pNHNM11 indicating the expression of the cloned gene.
Plate 4. SDS-PAGE analysis for expression of ectB L. PPMW116632 GeNei (protein marker)
1. pNHNM11 uninduced, 2. pNHNM11 induced
The BLASTp results of amino acid sequence of cloned ectB showed 90 % homology with reported
ectB of Halomonas sp. SMT L15 (BAD99615.1), 89 % homology with diadenosine tetraphosphatase of
Halomonas elongata (WP013332345.1) and ectB-T7 (synthetic construct) (AGJ01122.1).
IV. CONCLUSION
As marine microbes are the major sources for ectoine genes, the current study encourages one to
use PCR amplicon to identify variants of ectB and explore the probability of discovery of novel ectoine
genes. Thus, it is necessary to clone large number of such genes from different sources, study their
effectiveness against salt tolerance, identify variants of ectB, bio safety and deploy them in agricultural
crop plants.
V. AKNOWLEDGEMENT We are grateful to Dr. Narayan moger for his assistance.
BIBLIOGRAPHY
1. Alone, A., 2012, Functional and molecular characterization of marine and freshwater microorganisms. MSc(Agri)
Thesis, Institute of Agri Biotechnology, dharwad.
2. Canovas, D., Vargas, C., Calderon, M. I., Ventosa, A. and Nieto, J. J., 1998, Characterization of the Genes for the
Biosynthesis of the Compatible Solute Ectoine in the Moderately Halophilic Bacterium Halomonas elongata DSM
3043. System. Appl. Microbiol., 21: 487-497.
3. Liu, W. Y., Wang, J. and Yuan, M., 2011, Halomonas aidingensis sp.nov., a moderately halophilic bacterium isolated
from aiding salt lake in Xinijiang, China. Antonie van Leewenhoek., 99(3): 663-670.
4. Khandeparkar, R., Verma, P. and Deobagkar. D., 2011, A novel halotolerant Xylanase from marine isolated Bacillus
subtilis cho40: gene cloning and sequencing. New Biotechnol., Vol.28: 814–821.
5. Kuhlman, A. U. and Bremer, E., 2002, Osmotically regulated synthesis of the compatible solute ectoine in Bacillus
pasteurii and related Bacillus spp. Appl. Env. Microbiol.,68: 772-783.
6. Kushner, D. J., 1978, Life in high salt and solute concentration. In: Kushner DJ (ed) Microbial life in Extreme
Environments. Academic Press, London, 317-368.
7. Ono, H., Sawada, K., Khunajakr, N., Toa, T., Yamamoto, M., Hiramoto, M., Shinmyo, A., Takano, M. and Murooka,
Y., 1999, characterization of biosynthetic enzymes for ectoine as a compatible solute in a moarately halophilic
eubacterium, halomonas elongate.J. Bacteriol., 181: 91-99.
8. Roberts, M. F., 2005, Organic compatible solutes of halotolerant and halophilic microorganisms. Saline Syst., 1: 1–30.
9. Roebler, M. and Muller, V., 2001, Osmoadaptation in bacteria and archae: common priniciples and differences.
Environ. Microbiol., 3(12): 743-754.
10. Sambrook, J. and Russell, D. W., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory,
Cold Spring Harbour, New York.
11. Satyanarayana, T., Raghukumar, C. and Shivaji, S., 2005, Extremophilic microbes: Diversity and perspectives. Curr.
12. Shivananda, P. and Mugeraya, G., 2011, Halophilic bacteria and their compatible solutes, osmoregulation and potential
applications. Curr. Sci., 100(10): 1516-1521.
13. Ventosa, A., 2006, Unusual micro-organisms from unusual habitats: hypersaline environments. In Logan NA,
Lappin-scott HM, Oyston PCF (edu). SGM Symp. 66: Prokaryotic diversity- mechanisms and significance.
