52NISR Research GRANT
2008 Young Investigator Research Grant

Analysis of tRNA modification system in halophilic archaea
Hiroki HIGASHIBATA
Department of Applied Biosciences, Faculty of Life Sciences, Toyo University

Research aims
Among over 80 modified nucleosides identified intRNAs, two remarkable nucleosides have an unusual 7deazaguanosine structure, namely, queuosine and archaeosine. Archaeosine is exclusively found in archaea, and present at position 15 in the D-loop of the tRNA. This position
plays a role in interacting with the D-loop and T-stem in thetertiary structure of the tRNA, suggesting that archaeosine
function in the stabilization of the tRNA structure. Queuosine is found in bacteria and eucarya, and present at position 34, the wobble position, in a subset of the tRNAs possessing a GUN anticodon (specific for amino acids Asp,Asn, His, and Tyr). The wobble position suggests a role inmodulating translational fidelity and/or efficiency. Disruption of a gene involved in queuosine biosynthetic pathwayof the pathogenic bacterium Shigella flexneri results in asignificantly reduced virulence1,2). This is due to the poor
expression of VirF, which is the most upstream regulator
protein of the virulence cascade. Therefore, the enzymes in
queuosine biosynthesis pathway are putative targets foranti-shigellosis drugs3). The preQo is an intermediate of archaeosine as well as queuosine, suggested that the biosynthetic pathway of archaeosine is closely related to that ofqueuosine. Biochemical and genetic studies in bacteria (Escherichia coli, Bacillus subtilis, and Streptomyces rimosus)
have shown that Guanosine 5-triphosphate (GTP) is converted to preQo in four steps4) (Fig. 1). In the biosynthetic
pathways to deazapurine-containing metabolites from GTP,
four enzymes (GCHI, QueD, QueE, and QueC) are necessary for production of preQo. Although the genes of these
enzymes involved in preQo biosynthesis are found in mostarchaea, the activity of each enzyme has not been confirmed yet. Moreover, nothing is presently known about thefinal step of biosynthesis of archaeosine. In order to clarify
the whole biosynthetic pathway of archaeosine and to infer
its function, gene disruption experiments were attempted.
Methods and Results
Halophiles grow in extremely saline environments, andtherefore, its intracellular KCl concentration is at high levels to maintain osmotic balance. The computational analysis suggests that archaeosine function in the stabilization of
the tRNA structure5). However, it is still unclear whether archaeosine stabilizes tRNA structure in high KCl concentration in vitro. Halophilic archaeon Halobacterium sp. NRC1 was utilized in this study because many genetic tools havebeen developed and its genome information is available.
Although ArcX protein concerned with the final step of archaeosine biosynthetic pathway should catalyze the uniquereaction converting nitrile [R–C≡ N] into amidine[R–C(NH)NH2], its gene has not been identified yet. Thegenome search for arcX gene was performed by using theamino acid sequences of the PUA and C2 domains intRNA-guanine transglycosylase (TGT), and the possible
candidate gene was found. Both of the PUA and C2 domains are present in ArcX. The PUA domain is highly conserved among tRNA modification enzymes, and the C2 domain supposedly stabilizes the l -formed tRNA in which
the D-loop is accessible to the catalytic domain6).
In order to obtain each DNA fragment encoding queC,queD, queE, tgt, and arcX gene involved in archaeosine
biosynthesis, polymerase chain reaction (PCR) was per-Fig. 1. The biosynthesis pathway of archaeosine. Abbreviation used: preQo, 7-cyano-7-deazaguanine; GCHI, GTP cyclohydrolase; TGT, tRNA-guaninetransglycosylase; ArcX, unknown enzyme(s); archaeosine, 7-formamidino-7-deazaguanosine.

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formed by using two primers based on the genome information of Halobacterium sp. NRC-1. A reaction mixture(25 m l) comprising 100 ng of genomic DNA, 7.5 pmol ofeach primer, and KOD plus ver. 2 DNA polymerase (Toyobo Co., Ltd., Japan) was subjected to PCR (98°C for
10 sec, 61°C for 30 sec, and 68°C for 3 min; 25 cycles), andthe amplified DNA fragment (approximately 2.5 kbp) was
gel-purified and successfully cloned into vector pUC19 digested with HincII. To construct the plasmids for gene disruption, the mevilolin (competitive inhibitor of HMG-CoA
reductase) resistant (Mevr) marker was inserted into theopen reading frame of each target gene. The Mevr marker
was obtained by PCR amplification using the shuttle vectorpWL1027) as a template. Transformations of Halobacterium sp. NRC-1 were performed by the PEG method described by Cline et al.8) The gene disruption mutants wereattempted to obtain by spreading transformation mixture on
the selective plate medium with simvastatin (a derivative ofmevinolin) at a final concentration of 25 m g/ml.

References
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Conclusion
Each DNA fragment encoding queC, queD, queE, tgt,and arcX gene involved in archaeosine biosynthesis wascloned into plasmid pUC19, and the plasmids for disruptionof target genes were successfully constructed by inserting
Mevr marker into the respective open reading frame ofthem.

7)

8)

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