Functional characterization of a major compatible solute in Deep Sea halophilic eubacteria of active volcanic Barren Island, Andaman and Nicobar Islands, India

Highlights

• Ectoine biosynthesis genes from deep sea eubacteria, Bacillus clausii NIOT-DSB04 was PCR amplified, cloned and expressed.

• Recombinant diaminobutyric acid aminotransferase and ectoine synthase has the molecular mass of 46 kDa and 15 kDa.

• Recombinant ectoine synthase activity of the expressed cells was higher than that of uninduced cells.

• Sequence and phylogenetic analysis revealed that the ectA, B and C sequences were conserved in many eubacteria.

Abstract

Ectoine, a cyclic tetrahydropyrimidine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) is a compatible solute, serves as a protective compound in many halophilic eubacterial cells under stress. In this study, the ectoine biosynthesis genes (ectA, B and C) from the genomic DNA of a deep sea eubacteria, Bacillus clausii NIOT-DSB04 was PCR amplified, cloned into the expression vector pQE30 with a 6 × histidine tag and expressed in M15 cells. The lysates of induced cells with diaminobutyric acid aminotransferase and ectoine synthase disclosed two clear expressed bands with molecular masses of 46 kDa and 15 kDa as estimated by SDS-PAGE. The recombinant ectoine synthase activity of the expressed cells was at higher level than that of uninduced cells. In silico sequence and phylogenetic analysis of nucleotides and amino acids revealed that the ectA, B and C sequences of Bacillus clausii NIOT-DSB04 were conserved in many eubacteria.

Introduction

Most halophilic and halotolerant bacteria adapt to various environmental stress by accumulating low-molecular weight organic compounds called compatible solutes. These compatible solutes equilibrate the external osmotic pressure and support intracellular turgor which is higher than that of surrounding environment. Among halophilic microorganism, variety of heterotrophic and methanogenic archaea, photosynthetic, lithotrophic and heterotrophic bacteria have been reported to biosynthesis compatible solutes (Oren et al., 1997). Compatible solutes also play a major role in protection of cells and its components from freezing, desiccation and temperature stress (Muller et al., 2005). Among the compatible solutes, ectoine is the most extensively studied and has been found to be distributed in many chemoheterotrophic halophilic eubacteria (Vargas et al., 2006). The cyclic amino acid ectoine was originally discovered as an osmoprotectant in oxygenic phototrophs of the Ectothiorhodospira group (Galinski et al., 1985) and subsequently found in many other Gram-negative and Gram-positive bacteria (Kempf and Bremer, 1998).

Ectoine protects the halophilic bacterial cells bio membranes, proteins, enzymes and nucleic acids against high or low temperature, salt concentration and low water activity. The organic osmolyte ectoine are amphoteric, water-binding organic molecules compatible with the cellular metabolism without adversely affecting the physiological processes of the bacterial cells (Galinski and Truper, 1994). Ectoine have been used in cosmetics as stabilizers, since it protects the skin cells against different damaging factors such as heating, freezing, desiccation and UV radiation (Graf et al., 2008). Various investigations underline the outstanding antiaging properties and immunoprotective potential of ectoine on skin (Pfluecker et al., 2005).

The biosynthetic pathways of ectoine have been elucidated in many halophilic eubacteria and usually occur in four enzymatic steps (Peters et al., 1990). First being the conversion of aspartate semialdehyde into diaminobutyric acid by diaminobutyrate transaminase (ectB), which is subsequently acetylated to N-γ-acetyldiaminobutyrate by diaminobutyrate acetyl transferase (ectA). The cyclic condensation of this compound by ectoine synthase (ectC) leads to the formation of ectoine (Prabhu et al., 2004). Since ectoine have gained more importance in biotechnology research, the necessity in elucidation of the genes responsible for ectoine biosynthesis in halophilic and halotolerant eubacteria isolated from extreme environment is of great demand for the development of new technologies for ectoine production. To the best of our knowledge, no report on functional characterization of ectoine biosynthesis genes from deep sea eubacteria, Bacillus clausii. In this study, ectoine biosynthesis gene cluster (ectABC) from Bacillus clausii NIOT-DSB04 was characterized and determined the diversity and phylogenetic relationship of ectA, B and C genes with other eubacteria.