database (http://camera.calit2.net) includes environmental metagenomic and genomic sequence data, associated environmental parameters ('metadata'), ...
Bioresources for Rural Livelihood: Volume-I, Pages 107–111 Edited by : G.K. Kulkarni, B.N. Pandey and B.D. Joshi Copyright © 2010, Narendra Publishing House
MOLECULAR CHARACTERIZATION OF SALT TOLERANCE GENES FROM AQUATIC MICROBES: CONCEPTS AND APPLICATION IN TRANSGENIC RESEARCH Mitali Dhiman, Prinyanka Das, Agniswar Sarkar, Mousumi Saha, B.K. Behera, and S.S. Mishra
ABSTRACT As inhabitants of natural and artificial aqueous environments, bacteria survive dramatic changes in extracellular osmolality. Soil bacteria survive periods of low and high rainfall, uropathogens survive urine concentration and dilution, and industrial organisms tolerate concentrated nutrient solutions as well as the extracellular accumulation of metabolic products (Wood, 1999). Bacteria respond both passively and actively to changes in the osmolality of their environment. It has been observed that bacteria respond to osmotic upshifts in three overlapping phases: dehydration (loss of some .cell water) (phase I), adjustment of cytoplasmic solvent composition and rehydration (phase II), and cellular remodeling (phase III) (Wood, 1999). Some of the candidate genes from microbes for salinity tolerance and draws together the hypotheses about the functions of these genes. 1. monocytogenes can survive a variety of environmental stresses, growth having been reported at NaCI concentrations as high as 10% (30) and at temperatures as low as 20.1°C (Boscari et aI., 2002). The ability of the organism to survive both high salt concentrations and low temperatures is attributed mainly to the accumulation of the compatible solute glycine betaine (Boscari et aI., 2002). The genetic basis of glycine betaine uptake in other Gram-positive bacteria has been studied extensively. Bacillus subtilis has been shown to possess three transport systems for glycine betaine: the secondary uptake system opuD and two binding-protein-dependent transport systems, opuA and opuC (proU). The secondary transport system betP, is involved in glycine betaine accumulation in Corynebacterium glutamicum. Sleator et aI., (1999) described characterization and disruption of betL, a gene which plays an important role in glycine betaine uptake in 1. monocytogenes and which exhibits high homologies to the secondary glycine betaine uptake systems of other Gram-positive bacteria. It is felt that detailed molecular studies are required to be made in identifying these genes that are important for salinity tolerance, so that these can be suitably utilized in developing salt tolerant varieties for transgenic research. The details of salt
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tolerance genes, their mechanism of salt tolerance and prospects of application has been elaborated in the present paper.
Keywords : Salt tolerance genes, Microbes, Osmoregulation, Glycine Betaine.
INTRODUCTION Over two-thirds of the surface of the earth is covered by oceans, which have an average depth of about 3800 m. As each drop of ocean water contains > 105 cells, the > 1030 microbial cells in the ocean represent the largest reservoir of microbes on earth. Communities of bacteria, archaea, protists and unicellular fungi account for most of the oceanic biomass and metabolism. Marine microbes are known to play an essential role in the global cycling of nitrogen, carbon, oxygen, phosphorous, iron, sulfur and trace elements (Karl, 2007). Salinity tolerance comes from genes that limit the rate of salt uptake from the soil or water and the transport of salt throughout the plant, adjust the is ionic and osmotic balance of cells in roots and shoots and regulate leaf development and the onset of senescence (Munns, 2005). However very little progress has been made in this regard so far as the gene expression pattern and analysis has been difficult. Most of the sequenced culturable microorganisms from the deep-sea are Alteromonadales from the Gammaproteobacteria. Unique properties of sequenced deep-sea microbes are that they all have a high ratio of rRNA operon copies per genome size, and that their intergenic regions are larger than average (Lauro and Bartlett, 2008). These properties are characteristic of bacteria with an opportunistic lifestyle and a high degree of gene regulation to respond rapidly to environmental changes when searching for food.
OSMOREGULATION IN BACTERIA Adaptation of bacteria to high solute concentrations involves intracellular accumulation of organic compounds called osmolytes. Osmolytes (often referred to as compatible solutes because they can be accumulated to high intracellular concentrations without adversely affecting cellular processesm can be either taken up from the environment or synthesized de novo, and they act by counterbalancing external osmotic strength, thus preventing water loss from the cell and plasmolysis. Since the water permeability of the cytoplasmic membrane is high, imposed imbalances between turgor pressure and the osmolality gradient across the bacterial cell wall are short in duration. Osmoregulation is a fundamental phenomenon developed by bacteria, fungi, plants, and animals to overcome osmotic stress. The most widely distributed strategy of response to hyperosmotic stress is the accumulation of compatible solutes, which protects the cells and allows growth. One of the most effective compatible solutes widely used by bacteria is glycine betaine, the N-trimethyl derivative of glycine, which can be accumulated intracellularly at high concentration through either synthesis, uptake, or both. Bacteria respond to osmotic upshifts in three overlapping phases: dehydration (loss of some cell water) (phase I), adjustment of
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cytoplasmic solvent composition and rehydration (phase II), and cellular remodeling (phase III). Responses to osmotic downshifts are not yet well characterized, but they are also likely to proceed in three phases: water uptake (phase I), extrusion of water and co solvents (phase II), and cytoplasmic cosolvent reaccumulation and cellular remodeling (phase III).
GLYCINE BETAINE TRANSPORT SYSTEM IN MICROBES Some of the candidate genes from microbes for salinity tolerance and draws together the hypotheses about the functions of these genes. L. monocytogenes can survive a variety of environmental stresses, growth having been reported at NaCI concentrations as high as 10% and at temperatures as low as 20.1 °C . The ability of the organism to withstand hostile environments is illustrated by an outbreak of listeric septicemia which was linked to consumption of salted mushrooms (7.5% NaCl) stored at low temperatures (Boscari et aI. 2002). The ability of the organism to survive both high salt concentrations and low temperatures is attributed mainly to the accumulation of the compatible solute glycine betaine (Boscari et aI., 2002). The genetic basis of glycine betaine uptake in other grampositive bacteria has been studied extensively. Bacillus subtilis has been shown to possess three transport systems for glycine betaine: the secondary uptake system opuD and two binding-protein-dependent transport systems, opuA and opuC (proV). The secondary transport system betP, isolated by Peter et aI., is involved in glycine betaine accumulation in Corynebacterium glutamicum. Sleator et aI., 1999)described characterization and disruption of betl, a gene which plays an important role in glycine betaine uptake in 1. monocytogenes and which exhibits high homologies to the secondary glycine betaine uptake systems of other Gram-positive bacteria. Boscari et aI., (2002) eported that the molecular characterization and disruption of betS, a gene which plays an important role in high affinity Na_-coupled glycine betaine and proline betaine transport in S. meliloti. Furthermore, they showed that betS is constitutively expressed, whereas BetS activity depends on posttranslational activation by high osmolarity and is most likely the emergency system transporting betaines for immediate osmotic protection. Many microorganisms possess two or more glycine betaine transport systems. Salmonella typhimurium, for example, possesses two genetically distinct pathways, a constitutive lowaffinity system (ProP) and an osmotically induced high-affinity system (ProU), while B. subtilis has three glycine betaine transport systems, OpuD, OpuA, and OpuC. Generally these transport systems can be divided into two groups. The first of these are the multicomponent, binding-protein dependent transport systems which belong to the super-family of prokaryotic and eucaryotic ATP-binding cassette transporters or traffic A TPases. Members of this family, including OpuA and OpuC of B. subtilis and ProU of E. coli, couple hydrolysis of A TP to substrate translocation across biological membranes. The second group belongs to a family of secondary transporters involved in the uptake of trimethylammonium compounds. Members of this family, including OpuD of B. subtilis and BetP of C. glutamicum, form single-component me-chanisms which couple proton
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motive force to solute transport across the membrane. Transporters for glycine betaine have been extensively investigated at the molecular level in the gram-negative enteric bacteria Escherichia coli and Salmonella enterica serovar Typhimurium and in the gram-positive soil bacteria Bacillus subtilis and Corynebacterium glutamicum. In E. coli and serovar Typhimurium, two transport systems, ProP and ProU, are primarily responsible for the uptake of glycine betaine. ProP, a secondary transporter, functions as an H- symporter and is regulated mainly at the activity level. ProU is a binding proteindependent transporter, a member of the ABC super family, and is regulated at the level of both transcription and activity. Previous works have demonstrated the crucial role of glycine betaine for osmotic stress resistance in the gram-negative soil bacterium Sinorhizobium meliloti, the alfalfa (Medicago sativa L.)-symbiotic species, and have led to investigations of glycine betaine synthesis and transport. The biosynthetic pathway, the enzymatic oxidation of choline or choline-O-sulfate to glycine betaine, has been well characterized at the molecular level and involves four genes, betICBA, organized in one operon. In addition to the genes encoding a presumed regulatory protein (betl), the betaine aldehyde dehydrogenase (betB), and the choline dehydrogenase (betA), enzymes also found in E. coli, S. meliloti, unlike other bacteria, possesses an additional gene (bete) which encodes a choline sulfatase catalyzing the conversion of choline-O-sulfate and, to a lesser extent, phosphorylcholine into choline. In B. subtilis, three high-affinity effective glycine betaine transporters have been characterized so far. Two systems, OpuA and OpuC, are members of the ABC superfamily of transporters and one, OpuD, is a secondary transporter. OpuA and OpuC present identity to the periplasmic binding protein ProU transporter from E. coli, but as a grampositive bacterium, B. subtilis lacks the periplasm, and the binding proteins are anchored in the cytoplasm membrane to prevent their loss in the surrounding medium. Whereas OpuC can transport a wide variety of compatible solutes such as glycine betaine, choline, ectoine, and carnitine, OpuA and OpuD exhibit a restricted substrate specificity for glycine betaine. With respect to osmotic adaptation, C. glutamicum is another well-studied gram-positive soil bacterium. In this organism, two secondary carriers for the uptake of glycine betaine have been characterized: the high-affinity, Na_-coupled glycine betaine uptake system BetP, and EctP, which prefers ectoine to glycine betaine. Both systems are regulated by the external osmolarity on the level of activity. BetP and EctP are closely related to each other and to other prokaryotic carriers for compatible solutes, such as the glycine betaine transporter OpuD from B. subtilis, the choline transporter BetT and the carnitine transporter CaiT from E. coli, the glycine betaine transporter BetL from Listeria monocytogenes, and the putative BetP proteins from Mycobacterium tuberculosis and from Haemophilus injluenzae.
TIMELINE OF OSMOSENSING Osmoregulators are devices that implement the response of an organism to changing environmental osmolality. An individual device may both detect and respond to solvent changes (i.e., osmosensors may also be osmoregulators). Alternatively, osmosensors and
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osmoregulators may be separate and communication between or among them may require additional signal transduction machinery. The high water permeability and solute selectivity of biomembranes ensure that changes in extracellular osmolality imposed with membraneimpermeant cosolvents trigger extensive changes in cell structure and chemistry. Thus, in principle osmosensors may detect changes in extracellular water activity (direct osmosensing) or they may detect and elicit responses tailored to address secondary consequences of osmotic shifts (indirect osmosensing). It is therefore expected that an array of osmosensors may detect and control a temporal cascade of cellular changes and osmoregulatory responses. As a result, the “osmotic history” of each cell will determine its response to new osmotic stimuli.
DATABASES AND COMPUTING TOOLS Marine microbe researchers are highly organized with respect to storing and sharing their data. The Community Cyberinfrastructure for Advanced Marine Microbial Ecology Research and Analysis (CAMERA) aims to develop global methods for monitoring microbial communities in the ocean and their response to environmental changes. The CAMERA’s database (http://camera.calit2.net) includes environmental metagenomic and genomic sequence data, associated environmental parameters (‘metadata’), pre-computed search results, and software tools to support powerful cross-analysis of environmental samples (Seshadri et al., 2007). Most of the sequenced culturable microorganisms from the deep-sea are Alteromonadales from the Gammaproteobacteria. Unique properties of sequenced deepsea microbes are that they all have a high ratio of rRNA operon copies per genome size, and that their intergenic regions are larger than average (Lauro and Bartlett, 2008). These properties are characteristic of bacteria with an opportunistic.
