Gene duplication toward posttranscriptional advantage

Gene duplications occur as an evolutionary response in bacteria exposed to different selection pressures. Duplicated genes could persist through either the innovation of a novel beneficial function of one copy (neofunctionalization) or the accumulation of mutations reducing the functional capacity of individuals (subfunctionalization). However, gene duplications frequently encounter the reversible fate. One copy could be rapidly lost with the removal of the selective pressures. A variety of knowledge on gene duplications has been provided through significant in silico analysis, with which experimental approaches could lead to explanations on the important evolutional events.

Biological nitrogen fixation has evolved in the Early Archean world and the ability to fix atmospheric nitrogen is distributed across the bacterial and archaeal domains. Nitrogen fixation is conducted by a nitrogenase complex consisting of dinitrogenase and dinitrogenase reductase. Dinitrogenase is a heterotetramer of nifD and nifK gene products, while dinitrogenase reductase is a homodimer of the nifH gene product. The nif genes encoding the nitrogenase complex organize a conserved transcriptional unit, nifHDK. It is proposed that the distribution of the conserved cluster among bacterial and archeal domains would be performed through horizontal gene transfer from the last common ancestor. Subsequent intragenomic evolutions under specific selective pressures could rearrange the conserved gene cluster.

Azorhizobium caulinodans is a nitrogen fixing Rhizobium that induces symbiotic nodules on a tropical legume Sesbania rostrata. The nifH gene was reiterated in the chromosome. One copy constitutes a conserved nifHDK transcriptional unit, while the other positions upstream of nifQ gene. The phylogenetic analysis provided that the gene duplication would have occurred in the common ancestor of some symbiotic Rhizobia including A. caulinodans and Bradyrhizobium species.

Our experimental approach was initiated from the comparative analysis on the duplicated nifH genes. The analysis using nifH in-frame disruptants resulted in the possibility of the functional differentiation between the nifH1 and nifH2, which could suggest the persistence of the duplication (T. Iki et al., 2007a). However, the approach included the unexpected transcriptional activation on nifA gene encoding the transcriptional activator NifA, which might have disturbed the accurate comparison. Further analysis using nifH deletion mutants obtained the conclusion that the nifH2 gene, the additional copy, functioned primarily on the nitrogen fixation.

The parental nifH1 gene showed higher activities than the daughterly nifH2 copy in both the transcriptional and translational analysis using lacZ gene. Furthermore, the point mutation approach focusing on the 3' terminus nonsynonymous substitution showed that the NifH1 protein functions more efficiently than the NifH2 protein under the same expression level. These results contradicted the significance of the nifH2 gene on the nitrogen fixation. The contradiction motivated us to focus on the posttranscriptional regulation of the nif gene expression. Northern blot analysis showed that the monocistronic mRNA of nifH2 was significantly accumulated, while that of nifH1 was not, with the alternative accumulation the bicistronic mRNA of nifH1D. The significant accumulation of the monocistronic nifH2 mRNA was consistent with the severe decrease of nitrogen fixation activity caused by the deletion of nifH2 gene. It was supposed how the nifH2 gene could accumulate the monocistron in spite of the rather weak transcriptional activity compared with the nifH1 gene.

Bacterial polycistronic genomes contain a number of small extragenic palindromic sequences that may affect the expression of flanking genes. It is reported that the stem loops formed by the palindromic sequences stabilize the proximal mRNA against the 3' to 5' exonucleolytic degradation, while affect the distal gene expression either by the transcriptional termination or through the posttranscriptional processing on mRNA. The nifH1DK and nifH2Q polycistrons of A. caulinodans also contain small extragenic palindromic sequences, putatively form stem loops in mRNA, but there is no experimental evidence showing the actual effect on gene expression.

It was shown that the palindromic sequence between nifH1 and nifD attenuated the expression of distal genes as well as accelerated that of proximal genes. In consideration with the insufficient accumulation of monocistronic nifH1 mRNA, it was supposed that the dilemmatic functions of the palindromic sequence might be somehow inactivated within the native gene cluster in order to prevent the hazardous attenuation on the expression of nifDK genes on nitrogen fixation. In the subsequent analysis, it was found that the cloning of the palindromic sequence with the 5' coding sequence of the nifD gene suppressed the function of the palindromic sequence. This result supports the inactivation of the potentially active palindromic sequence within the nifHDK mRNA.

The duplication of nifH gene would have solved the dilemma, enabling the additional nifH2 gene to dedicate itself into the synthesis of the stable monocistronic nifH mRNA, which is stable and thus required for the efficient nitrogen fixation (Fig. 1). It was concluded that the gene duplication of nifH would be advantageous for A. caulinodans in the posttranscriptional level of the gene expression (T. Iki et al., 2007b, submitted).

B. japonicum have lost the parental copy which once constituted the conserved nifHDK cluster. The primal function of the nifH2 gene might confer a fatal gene loss of nifH1 and the consequent translocation of the nifH gene in A. caulinodans. In this sense, the putative gene translocation in a group of symbiotic Rhizobia would not the result of the reversible gene duplication in the transient selective pressures, but the progressive fate under the constant selective pressures purifying more efficient nitrogen fixers.

Characterization of a conserved GTPase, HflX, in bacterial mRNA decay

Through the experimental analysis on the prokaryotic gene duplication, it was recognized that the second structure of mRNA affected the gene expression considerably enough to confer impact on the gene evolution. The mRNA decay has been significantly studied in other bacteria such as Escherichia coli, while the current knowledge on the mRNA decay is not sufficient. One of the most important trans acting factors involving to the mRNA decay is the RNA chaperone, Hfq. The pleiotropic functions of the hexameric protein complex have been well documented. On the other hand, there is no report about hflX gene, consisting the conserved gene cluster with hfq in the prokaryotic genomes. Interestingly, the hflX gene is conserved in the eukaryotic and archaeal genomes (Fig. 2). In A. caulinodans, the hfq-hflX gene cluster positions adjacent to ntr genes which involved in the nitrogenous signal transduction, and it has been reported that the hfq is required for the expression of nifA in the posttranscriptional level. It was assumed that the hflX gene might play some significant role on the nif gene expression in A. caulinodans.

We challenged the venturous study to obtain several stimulating results indicating the involvement of HflX in the mRNA decay. The hflX mutant of A. caulinodans showed higher transcriptional activity of nifA, which was absent in the hfq mutant. Furthermore, it was found that the hflX mutant inhibited the stabilization of proximal mRNA by the extragenic palindromic sequence between nifH1 and nifD genes (T. Iki et al., preparing). Considering the distribution of hflX gene orthologs within the three kingdoms, it is possible that HflX might function in a variety of RNA maturations on both the prokaryotic and eukaryotic cells (Fig. 2). Our study could lead to the first detailed characterization of the highly conserved GTPase, HflX.

Fig. 1. The schematic mRNA synthesis during the nitrogen fixation by A. caulinodans.

Fig. 2. The distribution of HflX (left), and the possible function in bacteria (right) was shown.

近年、多くの細菌の全ゲノム塩基配列が解読されている。根粒菌についても約10種の菌株について全ゲノム塩基配列が報告された。ゲノム塩基配列情報から細菌の進化についてさまざまな解読が行われている。本論文では、根粒菌Azorhizobium caulinodans ORS571株のゲノム情報を用いて、窒素固定に関連する遺伝子の進化および機能の十分解明されていないGTPaseの1つhflXの機能解明を試みたものである。

本論文は2章よりなり、序論に続く第1章では根粒菌Azorhizobium caulinodans ORS571株のnifH遺伝子の倍化に焦点をあて、倍化後の機能分化などに関するさまざまな実験を行って進化について考察している。この根粒菌のnifHは倍化によってnifH1とnifH2の2つが存在しているが、アミノ酸配列レベルでは1アミノ酸が異なっている。しかしながら、このアミノ酸を置換させたタンパク質のアセチレン還元活性に違いは見られなかった。それぞれの遺伝子の破壊株を作製して、アセチレン還元能で窒素固定活性を調べたところ、窒素固定活性が高まる低酸素ガス濃度(酸素ガス3%)においてはNifH2が有意に高い活性を示した。遺伝子の発現をレポーターを用いて調べたところ、酸素ガスの濃度に拘わらず常にnifH1のほうがnifH2より多量に発現されていることが確認された。このことから、nifH1は遺伝子発現では高いが、実際の窒素固定能に対する寄与度は低いという矛盾が存在した。遺伝子発現されているmRNAに対してノザン分析を行ったところ、nifH1はオペロンの下流に存在するnifDおよびnifKとつながったmRNAで存在し、nifH2遺伝子は単独で切れた形で存在することが分かった。機能解析の結果、nifH1の下流には1個、nifH2の下流には2個のパリンドローム様構造が機能的に存在することが判明した。これらのことから、nifH1のパリンドロームは機能的であるものの、窒素固定細胞では機能していないと結論付けられた。倍化したnifH遺伝子における解析により、パリンドローム構造に依存したmRNAの安定性が遺伝子発現レベルを決定していることを明らかとした。

第2章では、機能がほとんど解明されていないGTPaseの1つであるHflXについて、Azorhizobium caulinodans ORS571を用いて解明を試みた。nifH1の転写について、レポーターを用いて調べたところ、hflXが存在する株に対してhflX破壊株では3'側にパリンドローム構造が存在する構造のnifH1の量を減少させた。このことから、hflXはnifH1の転写後の安定性に関与する可能性を示唆した。hflXのとなりにはhfqが存在し、Hfqタンパク質はRNAシャペロンとしてmRNAのポリA付加と関与しているとされている。細菌におけるポリA付加はmRNAの分解を促進するためのもので、hfq遺伝子のとなりに存在するhflxがmRNAの分解の制御に関与する可能性は高い。

以上、本論文では根粒菌の窒素固定に関連する倍化した遺伝子の進化および機能が不明なGTPaseの1つの機能の解明を試みたものであり、審査委員一同は学術上、応用上価値あるものと認め、博士(農学)の学位論文として十分な内容を含むものと認めた。