Discovery of bacteriorhodopsins in Haloarchaeal species isolated from Indian solar salterns: deciphering the role of the N‐terminal residues in protein folding and functional expression

Abstract

Summary Interesting optical and photochemical properties make microbial rhodopsin a promising biological material suitable for various applications, but the cost‐prohibitive nature of production has limited its commercialization. The aim of this study was to explore the natural biodiversity of Indian solar salterns to isolate natural bacteriorhodopsin ( BR ) variants that can be functionally expressed in Escherichia coli . In this study, we report the isolation, functional expression and purification of BR s from three pigmented haloarchaea, wsp3 (water sample Pondicherry), wsp5 and K1 T isolated from two Indian solar salterns. The results of the 16S rRNA data analysis suggest that wsp3, wsp5 and K1 T are novel strains belonging to the genera Halogeometricum, Haloferax and Haloarcula respectively. Overall, the results of our study suggest that 17 N‐terminal residues, that were not included in the gene annotation of the close sequence homologues, are essential for functional expression of BR s. The primary sequence, secondary structural content, thermal stability and absorbance spectral properties of these recombinant BR s are similar to those of the previously reported Haloarcula marismortui Hm BRI . This study demonstrates the cost‐effective, functional expression of BR s isolated from haloarchaeal species using E. coli as an expression host and paves the way for feasibility studies for future applications.

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

“…The partial 16S rRNA sequences for pws2, pws4, and pws10 shared 100% identity score with Haloarcula salaria JCM 15759, Haloarcula japonica JCM7785 and Halomicrobium mukohataei JP60, respectively ( Table 1 ). All the isolates were further screened for the presence of the bop using degenerate primers (DegF and DegR) as reported earlier (Verma et al, 2019 ) ( Supplementary Table 1 ). The presence of an expected 450 bp PCR product on an agarose gel confirmed that all the strains except pws11 were positive for bop ( Supplementary Figure S1 ).…”

“…The protein sequence comparison shows that pws5-BR (locus tag -NLV14165.1) shares 92% sequence identity with H. marismortui BRI (HmBRI). Based on our previous study (Verma et al, 2019 ), we chose the pET22b expression vector to yield recombinant protein with C-terminal 6× His-tag for affinity purification. BR is a leaderless membrane protein and putting N-terminal affinity tag may interfere with the membrane localization and protein folding.…”

“…Our group has been working on isolation of extreme haloarchaeal strains from solar salterns to study the diversity and to identify novel BR sequences, if any, from the isolates. We reported the isolation of extremely halophilic archaea Halogeometricum borinquense strain wsp3 and Haloferax volcanii strain wsp5 from Marakkanam solar salterns Pondicherry and Haloarcul a strain K1 T from Thamaraikulam solar salterns Kanyakumari (Verma et al, 2019 ). In recent studies, our group has characterized several haloarchaeal strains isolated from Indian solar salterns.…”

Biochemical and Taxonomic Characterization of Novel Haloarchaeal Strains and Purification of the Recombinant Halotolerant α-Amylase Discovered in the Isolate

“…All three strains were isolated from solar saltern samples collected from Marakkanam solar salterns, India (12°11′13.0272″N and 79°55′40.4220″E) using the dilution-plate technique on the Halovibrio agar medium as described previously ( Verma et al, 2019 ). Briefly, on agar plates, the reddish-pink opaque convex colonies, 1–2 mm in diameter appeared in 7 days.…”

Data_Sheet_1.PDF

“…The protein sequence comparison shows that pws5-BR (locus tag -NLV14165.1) shares 92% sequence identity with H. marismortui BRI (HmBRI). Based on our previous study (Verma et al, 2019), we chose the pET22b expression vector to yield recombinant protein with Cterminal 6× His-tag for affinity purification. BR is a leaderless membrane protein and putting N-terminal affinity tag may interfere with the membrane localization and protein folding.…”