The heterotrophic eubacterial and archaeal co-inhabitants of the halophilic Dunaliella salina in solar salterns fed by Bay of Bengal along south eastern coast of India

Halophilic microbes are studied to understand the metabolic pathways adopted by organisms in such extreme environment and for their biotechnological exploitation. In thallosohaline environments worldwide, the autotrophic alga Dunaliella salina Teodoresco is omnipresent, but it is being recently realised that the heterotrophic components vary in different regions. The unexplored eastern coastline of India abutted by Bay of Bengal was investigated for the heterotrophic halophilic microbes in this region. The waters in the salterns – replicas of natural hyper-saline water bodies of that region, were collected at four sites along 650 km of the coastal belt. In cultures set up from these waters, green and pink colonies were observed. The green colonies were found to be those of D. salina while the pink colonies were of heterotrophs. To identify the heterotrophic microbes, light microscopy, 16S rRNA typing and pigment profiling through spectrophotometry and HPLC were done. The cells in pink colonies were rod shaped. 16S rRNA typing of cells in these colonies detected the presence of Halomonas sp. – a eubacterium. The pigment profile of cells in pink cultures matched that of the archaea – Halobacterium; bacterioruberin derivatives were found. Thus, it was concluded that Halomonas and Halobacterium spp. are among the co-inhabitant heterotrophs of D. salina. Cultures of D. salina established from these salterns showed the typical three colours seen in the ponds of different sub-plots of salterns. They were green until 30 days, turning dark orange by 60 days and pink when 90 day old. In the 90 day old cultures, innumerable rod shaped cells were found. These cells were similar to the cells of the waters from the ponds of pink sub-plots of salterns and the pink colonies established from saltern waters in the laboratory. In the old (90 days) laboratory cultures of D. salina, the glycerol and proteins released from degenerating cells and the increase in salt concentration to super saturation levels due to evaporation of water in the medium led to the gregarious appearance of the heterotrophs – the co-inhabitants in natural environment.     

Adaptive response of Haloferax mediterranei to low concentrations of NaCl (< 20%) in the growth medium

Halobacteria require 20–25% NaCl for optimal growth and lyse when the salt concentration falls below 10%. The response of Haloferax mediterranei cells to low concentrations of NaCl (< 20%) in the medium was studied. The cells adapted to and grew in concentrations of NaCl as low as 10% and survived in concentrations lower than 5%. The cells synthesised a red pigment, bacterioruberin, in response to stress caused by a low concentration of NaCl (< 20%). Received: 3 January 1997 / Accepted: 18 April 1997     

A halorhodopsin-overproducing mutant isolated from an extremely haloalkaliphilic archaeon Natronomonas pharaonis

The halorhodopsin (hR)-overproducing mutant strain KM-1 was isolated from the extremely haloalkaliphilic archaeon Natronomonas pharaonis type strain DSM2160(T). hR-enriched membranes were easily obtained by washing the cells with distilled water. The membranes were claret colored owing to two pigments: hR and bacterioruberin. The hR component in the absorption spectra changed from blue to purple upon the addition of Cl(-) and had a K(m) value of 1.7mM. Overexpression of hR in strain KM-1 might be caused by the point mutation Asp324-->Asn in the bacteriorhodopsin activator homologues of N. pharaonis. The mutation changed the hR-expression pattern from inducible to constitutive in the late exponential phase.     

Pigments of Rubrobacter radiotolerans

The highly radioresistant Rubrobacter radiotolerans, contains red pigments. Since the pigments could not be extracted by usual methods, a new method was developed in which the pigments were extracted with organic solvents after addition of 10 N KOH to the intact cells, followed by neutralization. These pigments were also extracted after treatment with achromopeptidase, but not with lysozyme. The extracted pigments separated into two main spots by TLC (48.6% and 22.6%), and were confirmed to be carotenoids by chemical tests. The two major pigments had 13 conjugated double bonds as determined from the main maximum wavelength of the light absorption spectra. Their molecular weights were determined to be 740 and 722 by mass spectrometry. The mass spectra of their TMS-derivatives revealed that they contained four and three tertiary OH groups, respectively. Confirming their identical light and IR spectra, these pigments were determined to be bacterioruberin and monoanhydrobacterioruberin, respectively, the characteristic carotenoids of halophilic bacteria. The existence of these pigments in bacteria other than halobacteria provides interesting new evidence on the distribution of these compounds.