Selective Phylogenetic Analysis Targeting 16S rRNA Genes of Hyperthermophilic Archaea in the Deep-Subsurface Hot Biosphere

Abstract

International drilling projects for the study of microbial communities in the deep-subsurface hot biosphere have been expanded. Core samples obtained by deep drilling are commonly contaminated with mesophilic microorganisms in the drilling fluid, making it difficult to examine the microbial community by 16S rRNA gene clone library analysis. To eliminate mesophilic organism contamination, we previously developed a new method (selective phylogenetic analysis [SePA]) based on the strong correlation between the guanine-plus-cytosine (G؉C) contents of the 16S rRNA genes and the optimal growth temperatures of prokaryotes, and we verified the method's effectiveness (H. Kimura, M. Sugihara, K. Kato, and S. Hanada, Appl. Environ. Microbiol. 72:21-27, 2006). In the present study we ascertained SePA's ability to eliminate contamination by archaeal rRNA genes, using deep-sea hydrothermal fluid (117°C) and surface seawater (29.9°C) as substitutes for deep-subsurface geothermal samples and drilling fluid, respectively. Archaeal 16S rRNA gene fragments, PCR amplified from the surface seawater, were denatured at 82°C and completely digested with exonuclease I (Exo I), while gene fragments from the deep-sea hydrothermal fluid remained intact after denaturation at 84°C because of their high G؉C contents. An examination using mixtures of DNAs from the two environmental samples showed that denaturation at 84°C and digestion with Exo I completely eliminated archaeal 16S rRNA genes from the surface seawater. Our method was quite useful for culture-independent community analysis of hyperthermophilic archaea in core samples recovered from deep-subsurface geothermal environments.

Culture-Independent Estimation of Optimal and Maximum Growth Temperatures of Archaea in Subsurface Habitats Based on the G+C Content in 16S rRNA Gene Sequences

“…However, we have previously reported that 16S rRNA gene sequences are naturally inscribed with the thermal features of their prokaryotic hosts (Kimura et al 2006(Kimura et al , 2007. Our observation was based on a high correlation between the growth temperature parameters of the prokaryotes and the guanine-plus-cytosine (G+C) content of their 16S rRNA sequences: 16S rRNA genes of hyperthermophiles and moderate thermophiles tend to have a high G+C content, whereas 16S rRNA genes of mesophiles and psychrophiles have relatively low G+C content (Galtier et al 1999;Khachane et al 2005).…”

“…In this study, we analyzed archaeal 16S rRNA genes in hot water samples collected from deep-sea hydrothermal vents and terrestrial hot springs, because the growth temperature parameters and the G+C content of 16S rRNA from archaea clearly have higher values than those from bacteria (Kimura et al 2007). The growth temperature parameters were determined by application of the MMT in order to estimate the in situ temperature of the subsurface environments, and the archaeal habitat of the hot subsurface environment was discussed in terms of our temperature determination.…”

Bhargavaea cecembensis gen. nov., sp. nov., isolated from the Chagos-Laccadive ridge system in the Indian Ocean

“…The majority of these taxa have been isolated from hydrothermal vents (Nakagawa et al, 2004;Prokofeva et al, 2005;Miroshnichenko & Bonch-Osmolovskaya, 2006;Naganuma et al, 2007;Reysenbach et al, 2006), deep-sea-sediments (Arakawa et al, 2006;Kaneko et al, 2007;Kimura et al, 2007;Shivaji et al, 2007), deep-sea carbonate crusts (Heijs et al, 2006) and seawater (Lauro & Bartlett, 2008). In the present study, we describe another novel bacterium belonging to a new genus, Bhargavaea gen. nov., a member of the Firmicutes.…”

GC-content of tRNA genes classifies archaea into two groups

“…Mesophilic bacteria, Escherichia coli and Bacillus subtilis, seem not so different from Euryarchaeota already sequenced. Recently, Kimura et al reported that there was a strong correlation between the optimum growth temperatures of archaeal species and the GC-contents of the 16S rRNA genes, and also that, with the exception of some species of halophilic archaea, 16S rRNA genes of mesophilic archaea had low GC-contents, and the 16S rRNA genes of thermophilic and hyperthermophilic archaea had much higher GC-contents than the 16S rRNA genes of mesophilic archaea (Kimura et al, 2007). Their result partially supports our inference: tRNA molecules which must take higher order structures to function should have higher GC-contents if they act in a higher temperature environment.…”