Hydroxylamine Assimilation by Rhodobacter capsulatus E1F1

Abstract

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 His 6 -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 NH 2 OH and methyl viologen were 1 mM and 7 M, 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 NH 2 OH as nitrogen source, and up to 10 mM NH 2 OH 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 NH 2 OH. Also, E. coli cells overproducing HCP tolerate hydroxylamine better during anaerobic growth. These results suggest that HCP is involved in assimilation of NH 2 OH, a toxic product that could be formed during nitrate assimilation, probably in the nitrite reduction step.Bacteria use nitrate as a nitrogen source for growth, as a terminal electron acceptor for anaerobic respiration, or as an electron sink for redox balancing. Three different types of bacterial nitrate-reducing systems have been described: the cytoplasmic assimilatory Nas, the membrane-bound respiratory Nar, and the periplasmic dissimilatory Nap (1, 2). Nitrate assimilation is a key process of the nitrogen cycle that has been an object of biochemical and genetic research in higher plants, fungi, algae, and cyanobacteria, although it has only been scarcely studied in other bacteria. It is firmly established that the assimilatory nitrate-reducing system consists of a nitrate transport system and two metalloenzymes, the assimilatory nitrate and nitrite reductases, which catalyze the stepwise reduction of nitrate to nitrite and ammonium (1-4). Nitrate assimilation is usually regulated by nitrate/nitrite induction and ammonium repression. Genes coding for the assimilatory nitrate-reducing systems are normally clustered and have been cloned in several bacteria. These gene clusters code for regulatory and struct...

Dissimilatory Metabolism of Nitrogen Oxides in Bacteria: Comparative Reconstruction of Transcriptional Networks

“…The only exception seems to be R. capsulatus E1F1, whose genome contains an nsrR ortholog close to the hcp gene within the nitrate assimilation nas gene cluster [17]. However, in denitrifying species, including R. sphaeroides and B. japonicum, the FNR-like transcriptional factor NnrR activates expression of nitrite and NO reductases and of the nnrS gene [36–38].…”

“…In vitro studies demonstrated oxygen-sensitive hydroxylamine reductase activity of the E. coli Hcp protein, suggesting its possible role in detoxification of reactive by-products of nitrite reduction [16]. More recently, the requirement of the hcp gene for in vivo hydroxylamine reduction was observed in Rhodobacter capsulatus E1F1 [17]. …”

“…Co-localization of the nsrR ortholog and the hcp gene in R. capsulatus E1F1 suggested that NsrR and nitrite could be involved in the regulation of hydroxylamine assimilation in this α-proteobacterium [17]. NsrR is a member of the Rrf2 family of transcriptional regulators, which includes a putative Rrf2 regulator for a redox operon in D. vulgaris [25], the iron-responsive repressor RirA, which controls iron uptake in rhizobia [26], and the IscR repressor for the Fe-S cluster assembly operon in E. coli [27].…”

The Role of the Hybrid Cluster Protein in Oxidative Stress Defense

“…However, the optimal conditions of the oxygen-sensitive hydroxylamine reductase activity of R. capsulatus HCP (13), as well of the E. coli HCP (14), could only be found at pH 9. The anaerobically purified D. desulfuricans HCP also exhibited a very high K m value for hydroxylamine at neutral pH (15).…”

“…Moreno-Vivian and collaborators (13) reported the increase of anaerobic tolerance to hydroxylamine of E. coli cells overproducing Rhodobacter capsulatus HCP. However, the optimal conditions of the oxygen-sensitive hydroxylamine reductase activity of R. capsulatus HCP (13), as well of the E. coli HCP (14), could only be found at pH 9.…”