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J. Biol. Chem., Vol. 279, Issue 44, 45485-45494, October 29, 2004

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Hydroxylamine Assimilation by Rhodobacter capsulatus E1F1

REQUIREMENT OF THE hcp GENE (HYBRID CLUSTER PROTEIN) LOCATED IN THE NITRATE ASSIMILATION nas GENE REGION FOR HYDROXYLAMINE REDUCTION^*

Purificación Cabello, Carmen Pino¶, M. Francisca Olmo-Mira¶||, Francisco Castillo¶, M. Dolores Roldán¶**, and Conrado Moreno-Vivián¶

From the Departamento de Biología Vegetal, Área de Fisiología Vegetal, Edificio Celestino Mutis, 3^a planta, and the ^¶Departamento de Bioquímica y Biología Molecular, Edificio Severo Ochoa, 1^a planta, Campus Universitario de Rabanales, Universidad de Córdoba, 14071-Córdoba, Spain

Rhodobacter capsulatus E1F1 grows phototrophically with nitrate as nitrogen source. Using primers designed for conserved motifs in bacterial assimilatory nitrate reductases, a 450-bp DNA was amplified by PCR and used for the screening of a genomic library. A cosmid carrying an insert with four SalI fragments of 2.8, 4.1, 4.5, and 5.8 kb was isolated, and DNA sequencing revealed that it contains a nitrate assimilation (nas) gene region, including the hcp gene coding for a hybrid cluster protein (HCP). Expression of hcp is probably regulated by a nitrite-sensitive repressor encoded by the adjacent nsrR gene. A His₆-HCP was overproduced in Escherichia coli and purified. HCP contained about 6 iron and 4 labile sulfide atoms per molecule, in agreement with the presence of both [2Fe-2S] and [4Fe-2S-2O] clusters, and showed hydroxylamine reductase activity, forming ammonia in vitro with methyl viologen as reductant. The apparent K_m values for NH₂OH and methyl viologen were 1 mM and 7 µM, respectively, at the pH and temperature optima (9.3 and 40 °C). The activity was oxygen-sensitive and was inhibited by sulfide and iron reagents. R. capsulatus E1F1 grew phototrophically, but not heterotrophically, with 1 mM NH₂OH as nitrogen source, and up to 10 mM NH₂OH was taken up by anaerobic resting cells. Ammonium was transiently accumulated in the media, and its assimilation was prevented by L-methionine-D,L-sulfoximine, a glutamine synthetase inhibitor. In addition, hydroxylamine- or nitrite-grown cells showed the higher hydroxylamine reductase activities. However, R. capsulatus B10S, a strain lacking the whole hcp-nas region, did not grow with 1 mM NH₂OH. Also, E. coli cells overproducing HCP tolerate hydroxyl-amine better during anaerobic growth. These results suggest that HCP is involved in assimilation of NH₂OH, a toxic product that could be formed during nitrate assimilation, probably in the nitrite reduction step.

Received for publication, April 21, 2004 , and in revised form, August 16, 2004.

^* This work was supported by Ministerio de Ciencia y Tecnología Grant BMC2002-04126-CO3-03 and Junta de Andalucía Grant CVI 0117, Spain. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

^|| Recipient of a fellowship from Ministerio de Ciencia y Tecnología.

^** Supported by a postdoctoral contract from Junta de Andalucía, Spain.

To whom correspondence should be addressed. Tel. and Fax: 34-9-57-218588; E-mail: bb1movic{at}uco.es.

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