Isolation and Taxonomic Characterization of Novel Haloarchaeal Isolates From Indian Solar Saltern: A Brief Review on Distribution of Bacteriorhodopsins and V-Type ATPases in Haloarchaea

Abstract

Haloarchaea inhabit high salinity environments worldwide. They are a potentially rich source of crucial biomolecules like carotenoids and industrially useful proteins. However, diversity in haloarchaea present in Indian high salinity environments is poorly studied. In the present study, we isolated 12 haloarchaeal strains from hypersaline Kottakuppam, Tamil Nadu solar saltern in India. 16S rRNA based taxonomic characterization of these isolates suggested that nine of them are novel strains that belong to genera Haloarcula, Halomicrobium, and Haloferax. Transmission electron microscopy suggests the polymorphic nature of these haloarchaeal isolates. Most of the haloarchaeal species are known to be high producers of carotenoids. We were able to isolate carotenoids from all these 12 isolates. The UV-Vis spectroscopy-based analysis suggests that bacterioruberin and lycopene are the major carotenoids produced by these isolates. Based on the visual inspection of the purified carotenoids, the isolates were classified into two broad categories i.e., yellow and orange, attributed to the differences in the ratio of bacterioruberin and lycopene as confirmed by the UV-Vis spectral analysis. Using a PCR-based screening assay, we were able to detect the presence of the bacteriorhodopsin gene (bop) in 11 isolates. We performed whole-genome sequencing for three bop positive and one bop negative haloarchaeal isolates. Whole-genome sequencing, followed by pan-genome analysis identified multiple unique genes involved in various biological functions. We also successfully cloned, expressed, and purified functional recombinant bacteriorhodopsin (BR) from one of the isolates using Escherichia coli as an expression host. BR has light-driven proton pumping activity resulting in the proton gradient across the membrane, which is utilized by V-Type ATPases to produce ATP. We analyzed the distribution of bop and other accessory genes involved in functional BR expression and ATP synthesis in all the representative haloarchaeal species. Our bioinformatics-based analysis of all the sequenced members of genus Haloarcula suggests that bop, if present, is usually inserted between the genes coding for B and D subunits of the V-type ATPases operon. This study provides new insights into the genomic variations in haloarchaea and reports expression of new BR variant having good expression in functional form in E. coli.

Assessment of 16S rRNA Gene-Based Phylogenetic Diversity of Archaeal Communities in Halite-Crystal Salts Processed from Natural Saharan Saline Systems of Southern Tunisia

“…The dominance of members of Halobacteria versus Nanohaloarchaea is in agreement with previous reports focused on the study of archaeal diversity in NaCl saturated samples like halite nodules, crystallizer ponds, crusts of halite and commercial salts, based on high- throughput sequencing methods [ 105 , 106 , 107 , 108 , 109 ]. It is interesting that Nanohaloarchaea was not reported in earlier studies as a component of archaeal community in hypersaline environment because of limitations of the approaches used before to evaluate the microbial diversity until the introduction of NGS technologies [ 110 ], citing the examples of the mismatches of primers used targeting the 16S rRNA gene copies with Nanohaloarchaea genes regions’ or the lack of their sequences in databases used for similarity searches [ 111 , 112 ]. In fact, Nanohaloarchaea , is a group of nanosized halophilic archaea lineage first recuperated from the soda lake Magadi in the east-African Rift Valley [ 113 ] and then from several hypersaline ecosystems where some complete genomes of uncultivated “ Candidatus Nanohaloarchaea ” have been assembled from metagenomes analysis [ 114 , 115 ].…”

Ion-pumping microbial rhodopsin protein classification by machine learning approach

“…Our group has recently published our findings on Initial 17 amino acids near the N-terminal rhodopsin sequences helps in the proper expression and folding of proton pumping rhodopsin [41]. Another published report on recombinant PWS-5 BR protein was expressed in E. coli with light driven proto pumping property by adding all trans retinal invitro [42]. This is the first detailed studied of Support vector machine based Proton pumping the recombinant bacteriorhodopsin protein expression by fishing it out bop gene using specific primers from these PWS isolates by choosing proper vector and host to demonstrate the light driven proton pumping property [43].…”

Cultivation of the Halobacterium salinarum biomass with high antioxidant activity for agricultural and food industry

“…The literature shows the characteristic spectrum of each carotenoids. Figure 2 shows the absorption spectrum of the extract of halobacteria, which shows the characteristic peaks of bacterioruberin (385,464, 490 and 522 nm), retinol (325 nm), β-carotin (453 nm) [8]. It can be seen that the main carotenoid contained in halobacteria is bacterioruberin.…”