34NISR Research GRANT
2008 Research Grant

Development of halophilic protein expression system capableof creating a productive enzyme
Taketomo FUJIWARA
Department of Biological Science, Faculty of Science, Shizuoka University

Research aims
Haloarchaea are microorganisms that grow under extremely high saline conditions. Most of the proteins and enzymes in the haloarchaea requires a high concentration ofsalt for stability and catalytic activity1). This halophilic
property of the enzymes makes them useful for industrial
applications, such as food, drug, and fermentation products.
Recently, we have been trying to improve gene manipulation techniques in haloarchaea2). In this study, the regulatory system of haloarchaeal denitrification was analyzed
using the genetic method that we have been developing.
Haloferax volcanii grows by denitrification in an anaerobic environment3–5). The denitrifying enzymes of haloarchaea are induced only under the anaerobic condition, but
the mechanism of induction control is still unknown. As
shown in Fig. 1, a novel DNA-binding protein is encodedby the narR gene which is adjacent to the nitrate reductase
gene operon narBCGHD6). To elucidate the role of NarR in
the genetic control of denitrification in haloarchaea, we performed expression experiments using recombinant NarR inan narR-deletion mutant of Hfx. volcanii.
Methods
Deletion of the narR gene was performed by a doublecrossover method using the D pyrE2 mutant of Hfx. volcanii(strain H26)7). A pTAnarR plasmid for gene recombination
was constructed by insertion of the upstream (USR) anddownstream (DSR) regions of the narR gene into a pTA131
vector as shown in Fig. 2A. The pTAnarR plasmid was introduced into Hfx. volcanii H26, then a two-step selectionof homologous recombinants was performed based on thenonrequirement for uracil (1st step) and resistance to 5-fluoroorotate (2nd step). Deletion of the narR gene was demonstrated in strain SH01 by PCR amplification using USR-f
and DSR-r primers (Fig. 2B). Expression vectors for NarR

were constructed as follows: the narR gene was joined to a
Haloarcula marismortui katG gene promoter or a Hfx. volcanii nirK gene promoter. Here it is notable that KatG is aconstitutive enzyme while NirK is induced under the anaerobic condition2,4). The resulting DNA fragments were inserted into the Escherichia coli-Hfx. volcanii shuttle plasmid pMLH32S to construct expression vectors pHKNR andpHNNR, respectively (Fig. 2C)2,8). The pMLH32S,
pHKNR, and pHNNR vectors were introduced into SH01
to obtain SH02, SH03, and SH09, respectively. The mutantNarRs were also expressed by using the katG gene promoter.
Results
The effects of the NarR deletion on the growth of Hfx.
volcanii were analyzed using strains H26, SH01, SH02,
SH03, and SH09 as shown in panels A, B, C, D, and E, respectively, in Fig. 3. Under the aerobic condition, all fivestrains grew quickly and the growth rates were almost uniform. The strains that lacked NarR did not grow anaerobically in the presence of nitrate (B and C), whereas anaerobic growth was recovered by expression of the recombinant
NarR (D and E). The result clearly demonstrated that NarR
is essential for the denitrification-dependent anaerobic
growth of Hfx. volcanii.
NarR contains a helix-turn-helix DNA-binding motif inthe C-terminal end of the sequence. A novel Cys-rich motifthat involves four or five conserved cysteines was identified
in the NarR homologues (Fig. 4A). Point-mutagenesis ofthe narR gene was carried out by substitution of the four
conserved cysteines (Cys17, Cys81, Cys83, Cys91) and one
nonconserved cysteine (Cys100) with serine residues. The
five mutant NarRs were expressed in SH01 cells, and thenthe growth rate of each strain was measured. The result,shown in Fig. 4B, indicated that all four conserved cysteines in the Cys-rich motif were necessary for the regulatory function of NarR in denitrification.
Conclusion

Fig. 1. Gene structure of narR/narBCGHD. The novel DNA-binding
protein NarR is encoded next to the nitrate reductase gene narBCGHD inHfx. volcanii genome.

The novel DNA-binding protein NarR, which is characteristic of haloarchaea, controls denitrifying growth of themicrobe. It has been known that bacterial denitrification iscontrolled by the two-component FixLJ and/or FNR
system9), although these regulatory systems are not present

35Fig. 2. Experimental protocols. Deletion of the narR gene was performed by a double-crossover method (A, B). Haloarchaeal katG or nirK promoterswere utilized for expression of the recombinant NarR (C).

Fig. 3. Effect of NarR deletion on the growth of Hfx. volcanii. Growth rates of H26(A), SH01(B), SH02(C), SH03(D), and SH09(E) in the presence of
21% O2 (blue), 0.2% O2 supplemented with 50 mM KNO3 (red), or 0.2% O2 without KNO3 (green) were monitored.

in the archaea. Therefore, it is probable that the control ofdenitrification by the NarR system originally evolved inhaloarchaea. NarR homologues are present in six of thenine haloarchaeal species for which the total genome sequence is available. NarR-type DNA-binding proteins playa significant role in the regulation of several anaerobic metabolisms including denitrification in the haloarchaea10).

In this study, it was demonstrated that the four conserved
cysteines in the novel Cys-rich motif were essential for theNarR function. A proposed mechanism of denitrification
control by NarR is illustrated in Fig. 5. It is probable thatthe Cys-rich motif functions as an O2 or redox sensor. Further investigations of the NarR system (purification of therecombinant NarR, binding assays, etc.) will be performed

36Fig. 4. The Cys-rich motif of NarR. The putative amino acid sequence of the Hfx. Volcanii NarR (HVO_B0159) was aligned with those of the homologous
proteins(A). Mutant NarRs, in which four conserved cysteines (Cys17, Cys81, Cys83, Cys91) and one nonconserved cysteine (Cys100) were replaced withserine and expressed in SH01(B). Optical densities of each culture were measured after eight days cultivation (n3). Growth condition: 21% O2 (blue), 0.2%O2 supplemented with 50 mM KNO3 (red), and 0.2% O2 without KNO3 (green).

in the future.
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Fig. 5. Proposed mechanism of denitrification control by NarR. NarR isactivated in the anaerobic condition. Binding of the activated NarR withthe consensus sequence (CGAA(c/g)A(c/t)GTTC(a/g)) triggers transcription of the genes encoding denitrifying enzymes.