Regulation and Characterization of Two Nitroreductase Genes, nprA and nprB , of Rhodobacter capsulatus

Abstract

Among photosynthetic bacteria, strains B10 and E1F1 of Rhodobacter capsulatus photoreduce 2,4-dinitrophenol (DNP), which is stoichiometrically converted into 2-amino-4-nitrophenol by a nitroreductase activity. The reduction of DNP is inhibited in vivo by ammonium, which probably acts at the level of the DNP transport system and/or physiological electron transport to the nitroreductase, since this enzyme is not inhibited by ammonium in vitro. Using the complete genome sequence data for strain SB1003 of R. capsulatus, two putative genes coding for possible nitroreductases were isolated from R. capsulatus B10 and disrupted. The phenotypes of these mutant strains revealed that both genes are involved in the reduction of DNP and code for two major nitroreductases, NprA and NprB. Both enzymes use NAD(P)H as the main physiological electron donor. The nitroreductase NprA is under ammonium control, whereas the nitroreductase NprB is not. In addition, the expression of the nprB gene seems to be constitutive, whereas nprA gene expression is inducible by a wide range of nitroaromatic and heterocyclic compounds, including several dinitroaromatics, nitrofuran derivatives, CB1954, 2-aminofluorene, benzo[a]pyrene, salicylic acid, and paraquat. The identification of two putative mar/sox boxes in the possible promoter region of the nprA gene and the induction of nprA gene expression by salicylic acid and 2,4-dinitrophenol suggest a role in the control of the nprA gene for the two-component MarRA regulatory system, which in Escherichia coli controls the response to some antibiotics and environmental contaminants. In addition, upregulation of the nprA gene by paraquat indicates that this gene is probably a member of the SoxRS regulon, which is involved in the response to stress conditions in other bacteria.Nitroaromatics are released into the environment almost exclusively as a consequence of anthropogenic activities related to explosives, paints, dyes, and pharmacology industries (23). Microorganisms have developed many different strategies to remove and degrade these xenobiotic compounds, and oxidative or reductive pathways for the degradation of nitroaromatics have been widely studied (19,23,29). Polynitroaromatic compounds are usually degraded through the following two major reductive pathways: (i) reduction of the aromatic ring by the addition of hydride ions to produce hydride-Meisenheimer complexes and (ii) reduction of the nitro group(s) to a hydroxylamino or amino group(s) by nitroreductases (3, 10, 15, 25). The oxygen-sensitive nitroreductases catalyze one electron step reaction to produce a nitro radical anion that can be reoxidized by oxygen with the concomitant production of superoxide. However, the best-studied nitroreductases are oxygen insensitive since they do not produce radicals and they catalyze the sequential addition of pairs of electrons donated by NAD(P)H to convert the nitro groups into hydroxylamines or amines, often through nitroso derivatives (15, 23). These enzymes are usually homodimers of a 25-kDa pol...

Biotransformation of 4-chloro-2-nitrophenol into 5-chloro-2-methylbenzoxazole by a marine Bacillus sp. strain MW-1

“…Beunink and Rehm (1990) reported the formation of 4C2AP from reduction of 4C2NP under anaerobic conditions. The reduction of nitro group under aerobic conditions was initiated by an oxygen-insensitive nitro-reductase through the formation of hydroxylamines or amines (Reinado et al 2005;Peterson et al 1979;Spain 1995). Park et al (1999) reported formation of 4C2AP in the reductive pathway of degradation of 3-chloronitrobenzene under aerobic condition.…”

Reduction of polynitroaromatic compounds: the bacterial nitroreductases

“…The group A includes the major nitroreductase NfsA of Escherichia coli (Zenno et al , 1996a) and the homologous proteins SnrA of Salmonella typhimurium (Nokhbeh et al , 2002), PnrA of Pseudomonas putida JLR11 (Caballero et al , 2005b) and FRP of Vibrio harveyi , an NADPH‐flavin oxidoreductase (Lei et al , 1994). The group B includes two subgroups the B 1 subfamily with the Escherichia coli minor nitroreductase NfsB (Zenno et al , 1996b), the NR and RNR proteins of Enterobacter cloacae (Bryant et al , 1991; Koder et al , 2001), the classical nitroreductase Cnr of Salmonella typhimurium (Watanabe et al , 1990), the PnrB nitroreductase from P. putida JLR11 (Caballero et al , 2005b), the NAD(P)H‐flavin oxidoreductase FRaseI of Vibrio fischeri (Zenno et al , 1994), the Synechocystis DrgA quinone reductase (Matsuo et al , 1998; Takeda et al , 2007), and the major nitroreductase NprA of the phototrophic bacterium Rhodobacter capsulatus (Pérez‐Reinado et al , 2005), among other nitroreductase‐like proteins. The minor nitroreductase NprB of R. capsulatus (Pérez‐Reinado et al , 2005) and other putative uncharacterized nitroreductases of Escherichia coli and Salmonella (YdjA proteins) are included in the subfamily B 2 .…”

“…This flavoenzyme is rapidly inactivated by blue light, probably because the FMN prosthetic group produces an excited flavin that oxidizes some functional groups required for enzyme catalysis (Blasco et al , 1995). Recently, the nprA and nprB genes that code for two different nitroreductases have been isolated from R. capsulatus B10 (Pérez‐Reinado et al , 2005). Both NprA and NprB proteins share only 14% amino acid sequence identity with each other and belong to different subgroups within the group B of nitroreductases (Fig.…”

“…Expression of the nprA gene is induced by a wide range of nitroaromatic and nitroheterocyclic compounds, whereas nprB gene expression is constitutive. Mutational analysis indicates that both gene products are involved in 2,4‐dinitrophenol reduction, although a residual nitroreductase activity is found in the double mutant strain, suggesting the presence of additional nitroreductase(s) in this phototrophic bacterium (Pérez‐Reinado et al , 2005).…”

“…Also, the R. capsulatus NprA nitroreductase is induced by paraquat (Fig. 1), a superoxide‐generating agent, and at low NAD(P)H/NAD(P) + ratios (Pérez‐Reinado et al , 2005), and the NfrA nitroreductases from Staphylococcus aureus and Bacillus subtilis are induced under oxidative stress conditions (Mostertz et al , 2004; Streker et al , 2005). The Staphylococcus aureus NfrA protein also exhibits a weak disulfide reductase activity and it has been proposed that is involved in the control of the cellular redox balance and the thiol‐disulfide stress response (Streker et al , 2005).…”