isolation and molecular characterization of salt tolerant genes from marine bacteria nethravathi ah

44 Pages·2017·1.34 MB·English

ISOLATION AND MOLECULAR CHARACTERIZATION OF
SALT TOLERANT GENES FROM MARINE BACTERIA

Thesis Submitted To
University of Agricultural Sciences, Dharwad
in partial fulfillment of the requirements for the

Degree of

Master of Science (Agriculture)
in
Molecular Biology and Biotechnology

By
NETHRAVATHI A.H.

DEPARTMENT OF BIOTECHNOLOGY
COLLEGE OF AGRICULTURE, DHARWAD
UNIVERSITY OF AGRICULTURAL SCIENCES
DHARWAD-580 005

July, 2014
ADVISORY COMMITTEE

DHARWAD
July, 2014 (NARAYAN MOGER)
MAJOR ADVISOR

Approved by
Chairman: _____________________________
(Dr. NARAYAN MOGER)

Members:
1).____________________________
(Dr.B. FAKRUDIN)

2). ___________________________
(Dr.V.P. SAVALGI)

3).____________________________
(Dr. NAGARATHNA UPPINAL)
CONTENTS

Chapter No. Title
1. INTRODUCTION
2. REVIEW OF LITERATURE
3. MATERIAL AND METHODS
4. EXPERIMENTAL RESULTS
5. DISCUSSION
6. SUMMARY
7 REFERENCES
8. APPENDIX
LIST OF TABLES

Table
Title
No.
Isolated salt tolerant bacterial cultures from the marine water sample of the
1.
West-Costal area of India used in this study
Screening of isolates with ectA and ectB degenerate primers and their
2.
salinity tolerance (%)
3. BLASTn result of ectB
4. BLASTp result of ectB

LIST OF FIGURES

Figure No. Title
1. Vector map of pTZ57R/T
2. Vector map of pET32C+
3. Vector map of pNHNM1(pTZ57R/T + ectB)
4. Vector map of pNHNM11(pET32C+ + ectB)
5. Complete nucleotide and amino acid sequence of ectB
LIST OF PLATES

Plate No. Title
1. Screening the isolates for salt tolerance activity
2. PCR amplification of ectA from the genomic DNA
3. PCR amplification of ectB from the genomic DNA
4. Colony PCR confirmation of ectB gene in pTZ R/T
5. Restriction confirmation of ectB clones in pTZ R/T with EcoRI and HindIII
6. Colony PCR confirmation of ectB gene in pET32C+
Restriction confirmation of ectB clones in pET32C+ with EcoRI and
7.
HindIII
8. SDS-PAGE analysis for expression of ectB

LIST OF APPENDICES

Appendix
Title
No.
I. Media for bacterial culture
II. DNA isolation
III. Agarose gel electrophoresis
IV. Selection plate preparation
V. Selection plate preparation
VI. Buffers and solutions for SDS PAGE
ABBREVIATIONS

Abbreviations Description

APS : Ammonium per sulphate
SDS : Sodium dodecyl sulphate
DTT : Dithiothritol
TEMED : Tri ethyl methyl diammine
X- gal : 5-Bromo-4chloro-3- indo- -D-galactopyranoside
IPTG : Isopropyl B-D thiogalactopyranoside
NCBI : National centre for Biotechnological information
BLAST : Basic Local Alignment Search tool
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) and can severely limit the productivity of approximately 6% of the world’s land and 20% of the
irrigated land negatively impacts agricultural yield worldwide (Munns, 2005)
Water is a unique component of the Earth’s surface and water abundance is precisely the reason
why our world is called the “Blue planet”. Among the three major habitats of biosphere, the marine water
covers 70 per cent and fresh water environment covers 2-3 per cent of total earth surface to which soil is
no exception. Different water bodies all over the planet provide the largest inhabitable space for living
organisms particularly the microbes (Dhevendaran, 2008).
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).
Among halophilic microorganisms, variety of heterotrophic and methanogenic archaea, photosynthetic,
lithotrophic and heterotrophic bacteria, photosynthetic and heterotrophic eukaryotes has been reported.
Being the reservoir of the salt tolerant species, marine environment is the promising source for salt
tolerant genes (Rajan, 2010).
Salinity presents a multifold challenge to all organisms in terms of perturbed osmotic balance,
ionic disequilibria and generation of toxic metabolites. All organisms exhibit characteristic responses
towards such abiotic stress and possess means to combat these. 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. The ability to adapt to altered
osmolarity of the surrounding growth medium is of fundamental importance for uni- and multi-cellular
organisms. A dynamic and important property of cells is their ability to rapidly adapt to changes in
external media, for example, increasing NaCl. As general rule of thumb, bacteria and eukaryotes usually
accumulate neutral compatible solutes whereas archaea prefer negatively charged solutes. Organic
osmolytes fall into three general chemical categories: (i) Zwitterionic solutes (ii) non-charged solutes and
(iii) anionic solutes. Intertwined with these organic solutes are K and Na, which also contribute to osmotic
balance in cell (Roberts, 2005).
Abiotic stress indeed is a complex process, which signals cell to adapt themselves at the
molecular, biochemical and physiological levels. Plants follow a number of salt tolerant mechanisms such
as synthesis of osmolytes and polyamines, reducing reactive oxygen species, the antioxidant defense
mechanism, ion transport and compartmentalization (Agarwal and Jha, 2010).
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 Cl¯ are accumulated in
the cytoplasm, until the internal salt concentration is similar to the extracellular one. This mechanism is
observed in extremely halophilic bacteria and archaea. On the other hand ‘organic-osmolyte strategy’ is
adopted by majority of prokaryotes by uptake or synthesis of compatible solutes (Roebler and Muller,
2001). These mechanisms help to re-establish the turgor pressure and circumvent the detrimental
consequences of water loss when exposed to increasing osmolality. The products obtained from
extremophiles such as proteins, enzymes (extremozymes) and compatible solutes like ectoine, glycine
betain, trehalose etc are of great interest of biotechnology (Satyanarayana et al., 2005).
Enormous scientific interest in oceanic organisms has given birth to the discipline called marine
biotechnology, it is a bridge between traditional and molecular biology and mainly focuses on new
products from sea (Dhevendaran, 2008). Advances in molecular biology and genetic engineering have
made possible the use of marine resources for development in the field of aquaculture, agriculture and
medicine.
Genetic engineering has been used as a relatively fast and precise way of improving salt
tolerance in crop plants to enhance and sustain agricultural productivity. Much effort has been made
globally to identify and isolate genes from variety of eukaryotic and prokaryotic sources, useful for
transferring to plants. Osmolyte biosynthesis is one of the well-studied and efficient system for
development of transgenic plants (Rajan, 2010). The successful examples include the engineering of
ectoine synthesis with genes from Halomonas elongate in tobacco plant (Nakayama et al., 2000). Also an
attempt to enhance of glycine betain and trehalose synthesis and their use in metabolic engineering of
stress tolerance has been reported (Storm, 1998). In the light of above background, the present study
was undertaken with following objectives:
1. Isolation and Molecular Characterization of salt tolerant genes from Marine bacteria.
REVIEW OF LITERATURE
2.1 Abiotic stress
Numerous stresses caused by complex environmental conditions, e.g. bright light, UV, to high
and low temperatures, freezing, drought, salinity, heavy metals and hypoxia, leads to substantial crop
losses worldwide (Mahajan and Tuteja, 2005 and Mittler, 2006). Among the abiotic factors that are
shaping plant evolution, water availability is the most important (Kijne, 2006). Water stress in its
broadest sense encompasses both drought and salt stress.
Salinity is one of the major abiotic factors limiting global agricultural productivity, rendering an
estimated one-third of the world’s irrigated land unsuitable for crops (Frommer et al., 1999). Salt
stress in plant cells is primarily caused by a combination of osmotic and ionic stress resulting from
high Na+ concentration in the soil (Hasegawa et al., 2000).
Stress can be detrimental for sensitive microorganisms and decrease the activity of surviving
cells, due to the metabolic load imposed by the need for stress tolerance mechanisms. Soil salinity is
a stress factor relating to microbial selection process and can reduce bacterial diversity and control
microbial abundance, composition and functions (Ibekwe et al., 2010). The salt requirement and
tolerance properties of the bacteria are highly variable and may vary according to the growth
temperature and the nature of the nutrients available (Quillaguaman et al., 2004).
Several classifications have been proposed; one that is very well accepted considers the
optimum growth of the microorganisms at different salt concentrations. Kushner in 1978 defined
several categories of microorganisms on the basis of their optimal growth: (1) Non halophiles are
those 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 optimal growth in media containing 2.5 to 5.2 M NaCl
(15- 32% salt) (Ventosa, 2006). (Ibekwe et al., 2010) reported that salinity negatively affects biological
activity by high osmotic strength (low water potential), which can be attributed to the toxic effect on
microbial growth, except tolerant halophilic bacteria (Yildirim et al., 2008).
2.2 Abiotic resistance (osmotic adaptation)
Exposure of microorganisms to high osmolality environments triggers rapid fluxes of cell
water along the osmotic gradient out of the cell, thus causing a reduction in turgor and dehydration of
the cytoplasm. Microorganisms actively respond to variations in the osmolality of their habitat (Miller
and Wood, 1996). To counteract the outflow of water, microorganisms increases their intracellular
solute pool by accumulation of very high intercellular concentrations of ions or by amassing large
amounts of very high intracellular concentrations of ions or by amassing large amounts of organic
osmolytes. This stress reaction, generally referred to as osmorregulation or osmoadaption, is aimed at
maintaining turgor and volume within boundaries acceptable for normal cellular physiology (Record et
al., 1998). Bacterial adaptation to increase in salinities in the environment not just involves turgor
adjustment but is much broader on a cellular basis. It involves structural changes in the cell envelope,
in the cytoplasmic membrane and a major reprogramming of the cell metabolism that is only poorly
understood.
The ability of microbial cells to adapt to fluctuations of external osmolarity (osmoadaptation)
via activation of specific mechanisms (osmoregulation) is essential for their survival. Halophilic and
halotolerant microorganisms have evolved a number of metabolic, enzymatic and regulatory
mechanisms that prevent cell dehydration. Osmoadaptation based on accumulation of inorganic ions,
mostly K+ (salt type of osmoadaptation), is employed in extremely halophilic members of
Halobacteriaceae and Halanaerobiales, as well as anaerobic acetogenic species of Halobacteroides
(Khmelenina et al., 2010).
A different strategy of osmotic adaptation in halophilic and halotolerant microorganisms is the
accumulation of organic osmotic solutes (glycine betaine, ectoine, glycerol, simple sugars, etc., often
termed “compatible solutes”). Such molecules are generally uncharged or zwitterionic, and their
presence in high intracellular concentrations does not require far-going adaptation of the proteins. The
intracellular solute concentrations can be rapidly adjusted according to the outside salinity, so that
microorganisms using this “low-salt-in” strategy can often adapt to life at a wide range of salinities
(Galinski, 1995; Oren, 2002a, 2002b, 2011, 2013b).
Metabolic acclimation via the accumulation of compatible solutes is often regarded as a basic
strategy for the protection and survival of plants under abiotic stress (Hanson and Hitz, 1982; Bohnert
and Jensen, 1996; Sakamoto and Murata, 2000; Shabala and Cuin, 2006). Multiple functions for
these compounds have been suggested. In addition to the conventional role of these compatible
solutes in cell osmotic adjustment (Yancey et al., 1982 and Bray, 1993), they are also suggested to
act as lowmolecular- weight chaperones, stabilizing the photosystem II complex, protecting the
structure of enzymes and proteins, maintaining membrane integrity and scavenging of reactive
oxygen species (ROS) (Robinson and Jones, 1986; Smirnoff and Cumbes, 1989). Recently, it was
also shown that some of these compatible solutes are very efficient in reducing the extent of K+ loss in
response to both salinity (Cuin and Shabala, 2005, 2007a) and oxidative stress (Cuin and Shabala,
2007b) in barley and Arabidopsis roots.
2.2.1 Bacterial accumulation of intracellular concentrations of ions
Under salinity stress, growth depression results from water deficiency and ion imbalance
(Wyn Jones, 1981). In response to hyper osmotic stress, bacteria adjust their cell turgor by controlling
fluxes of ions across cellular membranes. The cytoplasm is exposed to high ionic strength to achieve
osmotic equilibrium by maintaining a cytoplasmic salt concentration similar to that of the surrounding
media. The most rapid response to osmotic up-shock, both in gram-positive and gram-negitive
bacteria, is a stimulation of potassium (K) uptake (Whatmore et al., 1990). Potassium acts as second
messenger for stress signaling and as regulatory element for transcription control as well as counter
ion for glutamate and other compounds during osmotic stress response. Among inorganic ions,
increased potassium influx is belived to be a hallmark of bacterial reponse to hyper osmatic stress, K+
being the most abundant inorganic ion in the cell cytosol (Balaji et al., 2000). The impact of potassium
for bacterial life is indicated by the diversity of transport system present for this particular ion.
According to Shabala (2009) bacterial exposure to hyper osmotic stress, though result in an increased
cellular accumulation of K+ it is the severity and a nature of hyper osmotic stress that determines the
direction of net K+ efflux across the membrane and, ultimately, the intercellular K+ content suggested
that the process is, at least partially, explained by the voltage gating of K+ permeable channels is not
available yet, it has been reported in Eshcherichia coli that the K+ efflux is mediated by glutathione
activated kefB/kefC proteins (Miller et al., 2000). The occurrence of active potassium transport
systems correlates potassium dependent bioenergetic homeostasis and survival of bacterial cells
under stress conditions. In many enteric bacteria primary active P-type ATPases of the Kdp-type are
present in addition to secondary Kup-type transporter. A comparison of potassium transport systems
in certain bacterial strains revealed that in contrast to Corynebacterium glumtamicum (harboring
KtrAB), Mycobacteria tuberculosis harbored genes encoding the high affinity potassium ATPase Kdp
while Corynebacterium diphtheriae carried a KtrAB transport system in addition to a CglK-type
channel (Ochrombel et al., 2011).
2.2.2 Intracellular biosynthesis of osmotically active compounds (Compatible
solutes)
An adaptation to counter balance the osmotic difference is accumulation of low molecular
weight hydrophilic molecules, which do not interfere with cell metabolism. Furthermore, bacteria take
up suitable compounds from their surrounding (da Costa et al., 1998). Compatible solutes are low-
molecular weight osmoregulatory compounds that are highly water-soluble sugars, alcohols, amino
acids, betaines, ectoines or their derivatives. In addition to their stabilizing effects, they are used as
salt antagonists, stress protective agents, moisturizers and therapeutics. They stabilize enzymes,
DNA and whole cells against stresses such as freezing, drying and heating. They increase freshness
of foods by stabilizing components. Induction of osmolytes in cells can increase protein folding and
thereby improve salt tolerance, which could be useful in agriculture and xeriscaping (Roberts, 2005
and Detkova and Boltyanskaya, 2007).
Compatible solutes have protein-stabilizing properties that help in the proper folding of
polypeptide chains (Arakawa and Timasheff, 1985). Due to their stabilizing effect on protein
molecules they are also sometimes referred to as chemical chaperones (Chattopadhyay et al., 2004).
Conformational shift of protein towards folded, nativelike states induced by preferential exclusion of
the solute is responsible for the chaperone-like effects (Kolp et al., 2006). Compatible solutes exert
their effect through changes in solvent structure and/or subtle changes in the dynamic properties of
the protein rather than by changing the structure of the protein itself (Lamosa et al., 2003).
Compatible solutes also interact with nucleic acids and can influence protein-DNA interactions (Pul et
al., 2007 and Kurz, 2008).