Cloning And Expression Of Bop Gene From Halobiforma Sp. AJ5 And Analysis Of Evolution Of Halophilic Genomes

A novel bop gene was described from Halobiforma lacisalsi strain AJ5~T, an extremely halophilic archaeon isolated from Ayakekum Lake, China. Following six rounds of PCR amplification based on the conserved fragment of the bop gene, the complete sequence of the bop gene, including the 5' and 3' flanking regions of the conserved fragment, were obtained by the Ligation-mediated PCR Amplification (LPA) approach. The data presented provides us with further insight into the distribution of bop-like genes in the family Halobacteriaceae. This is the first example of a bop-like gene in halophiles found in the high-pH environment. Alignment and hydropathy analysis of the deduced amino acid sequence identified the conserved functional sites as well as some variations compared to other bacterio-opsins. Molecular phylogenetic analysis revealed the position of the bacterio-opsin of strain AJ5, which is closest to that of Haloterrigena sp. arg-4 with 85% identity. In the presence of all-trans retinal, recombinant Escherichia coli cells expressing the gene turned dark purple. The purple membrane from the recombinant E. coli showed maximal absorption at 540 nm.The ultraviolet could do harm to the cell, while the haloarchaea make use of the visable light by bop gene. We have investigated the strategy of Halobacterium sp. NRC-1 and other members of the family Halobacteriaceae to survive UV irradiation, based on an integrated analysis of various genomic and proteomic features such as dinucleotide composition and distribution of tetranucleotides in the genome and amino acid composition of the proteins. The low dipyrimidine content may help Halobacterium reduce formation of photoproducts in its genome. The usage of residues susceptible to reactive oxygen species attack is reduced significantly in Halobacterium, which helps the organism to minimize protein damage. We then correlated the expression of the zim gene with the genomic structure to reexamine the importance of the putative mismatch repair pathway proposed previously. Our results showed that Halobacterium sp. NRC-1 and other haloarchaea (Haloarcula marismortui, Haloquadratum walsbyi) have optimized their genomic and proteomic structures to reduce damage induced by UV irradiation, often present at high levels in habitats where these organisms thrive.In the types of mutations, frameshift could change the coding sequence dramatically. By employing BLASTX and an algorithm developed by us, we have detected eight frameshifts within genomes of Escherichia coli to facilitate further mining of the frameshift in halophiles. Two frameshifts involved toxin genes, one occurred in a stress response gene, one in a putative endonuclease, one in a gene involved in maintaining the growth rate when cell density is high, and the functions of the remaining three were unknown. Combined with previous transcriptomic and proteomic data, the functions of the ORFs involved in the frameshifts were investigated. Frameshift may provide rapid evolution of microbial populations. We postulate that E. coli uses frameshift as a mechanism for rapid adaptation. The algorithms and methods developed in this study may be applied to analyze frameshifts in high throughput prokaryotic genomic sequences and help to understand how organisms adapt to multiple environmental factors within their habitats.