Gene orders in the upstream of 16S rRNA genes divide genera of the family Halobacteriaceae into two groups

Abstract

In many prokaryotic species, 16S rRNA genes are present in multiple copies, and their sequences in general do not differ significantly owing to concerted evolution. At the time of writing, the genus Haloarcula of the family Halobacteriaceae comprises nine species with validly published names, all of which possess two to four highly heterogeneous 16S rRNA genes. Existence of multiple heterogeneous 16S rRNA genes makes it difficult to reconstruct a biological phylogenetic tree using their sequence data. If the orthologous gene is able to be discriminated from paralogous genes, a tree reconstructed from orthologous genes will reflect a simple biological phylogenetic relationship. At present, however, we have no means to distinguish the orthologous rRNA operon from paralogous ones in the members of the family Halobacteriaceae. In this study, we found that the dihydroorotate oxidase gene, pyrD, was present in the immediate upstream of one 16S rRNA gene in each of ten strains of the family Halobacteriaceae whose genome sequences have been determined, and the direction of the pyrD gene was opposite to that of the 16S rRNA genes. In two other strains whose genome sequences have been determined, the pyrD gene was present in far separated positions. We designed PCR primer sets to amplify DNA fragments encompassing a region from the conserved region of the pyrD gene to a conserved region of the tRNA-Ala gene or the 23S rRNA gene to determine the 16S rRNA gene sequences preceded by the pyrD gene, and to see if the pyrD gene is conserved in the immediate upstream of rRNA operon(s) in the type strains of the type species of 28 genera of the family Halobacteriaceae. Seventeen type strains, including the ten strains mentioned above, gave amplified DNA fragments of approximately 4000 bp, while eleven type strains, including the two strains mentioned above, did not give any PCR products. These eleven strains are members of the Clade I haloarchaea, originally defined by Walsh et al. (2004) and expanded by Minegishi et al. (2010). Analysis of contig sequences of three strains belonging to the Clade I haloarchaea also revealed the absence of the pyrD gene in the immediate upstream of any 16S rRNA genes. It may be scientifically sound to hypothesize that during the evolution of members of the family Halobacteriaceae, a pyrD gene transposition event happened in one group and this was followed by subsequent speciation processes in each group, yielding species/genera of the Clade I group and 'the rest' of the present family Halobacteriaceae.

Draft Genome Sequence of the Extremely Halophilic Archaeon Halogranum salarium B-1 T

“…salarium B-1 T harbored the genes encoding, in order, nonhistone chromosomal protein MD1, dihydroorotate oxidase, valyl-tRNA synthetase, phenylalanyl-tRNA synthetase subunit ␣ and ␤, tryptophanyl-tRNA synthetase, tRNA intron endonuclease, DNA topoisomerase I, haloacid dehalogenase-like hydrolase, DEAD/DEAH box helicase, lipoic acid synthase, pyruvate dehydrogenase E1 component subunit ␣ and ␤, pyruvate dehydrogenase E2 component, and dihydrolipoamide dehydrogenase in the upstream of the 16S rRNA gene. This gene order was consistent with those of Haloferax volcanii DS2 and Halogeometricum borinquense DSM 11551 T (8), which supported their close relationship in the complete rpoB= gene-based phylogeny (5). More detailed analysis of the genome will provide further insight into the genomic differences with respect to other haloarchaea and metabolism of the extreme halophiles.…”

Halomicroarcula pellucida gen. nov., sp. nov., a non-pigmented, transparent-colony-forming, halophilic archaeon isolated from solar salt

“…mukohataei Hmuk_R0017 (orthologue) and Hmuk_R0036 (Tindall et al, 2009), which makes it difficult to discuss phylogeny from a tree reconstructed from those heterogeneous sequence data. Recently, Minegishi et al (2012) found that the dihydroorotate oxidase gene, pyrD, was present immediately upstream of one 16S rRNA gene in each of ten members of the family Halobacteriaceae whose genome sequences have been determined, including species of the genera Haloarcula and Halomicrobium, as well as species of genera that possess a single rRNA operon. Minegishi et al (2012) suggested that the 16S rRNA genes were orthologous, and designed a PCR primer set to amplify DNA fragments encompassing a region from the conserved region of the pyrD gene to a conserved region of the 23S rRNA gene.…”

The Family Halobacteriaceae

“…Other genetic markers have been proposed in recent years for phylogenetic tree reconstruction for the members of the family by multilocus sequence analysis (Dennis and Shimmin 1997;Enache et al 2007a;Minegishi et al 2010aMinegishi et al , 2012aPapke et al 2007). Use of the markers atpB, EF-2, radA, rpoB 0 , and secY enabled differentiation of individual strains within species, as well as the delineation of species and genera, including the identification of potential novel species and even family-like relationships (Papke et al 2011).…”