The phototrophic bacterium Rhodobacter sphaeroidesDSM 158 is able to reduce nitrate to nitrite by means of a periplasmic nitrate reductase which is induced by nitrate and is not repressed by ammonium or oxygen. Recently, a 6.8 kb PstI DNA fragment carrying the napABCgenes coding for this periplasmic nitrate-reducing system was cloned [Reyes, Roldán, Klipp, Castillo and Moreno-Vivián (1996) Mol. Microbiol. 19, 1307–1318]. Further sequence and genetic analyses of the DNA region upstream from the napABCgenes reveal the presence of four additional napgenes. All these R. sphaeroidesgenes seem to be organized into a napKEFDABCtranscriptional unit. In addition, a partial open reading frame similar to the Azorhizobium caulinodans yntCgene and the Escherichia coli yjcCand yhjKgenes is present upstream from this napgene cluster. The R. sphaeroides napKgene codes for a putative 6.3 kDa transmembrane protein which is not similar to known proteins and the napEgene codes for a 6.7 kDa transmembrane protein similar to the Thiosphaera pantotrophaNapE. The R. sphaeroides napFgene product is a 16.4 kDa protein with four cysteine clusters that probably bind four [4Fe-4S] centres. This iron–sulphur protein shows similarity to the NapF and NapG proteins of E. coliand Haemophilus influenzae.Finally, the napDgene product is a 9.4 kDa soluble protein which is also found in E. coliand T. pantotropha. The 5´ end of the naptranscript has been determined by primer extension, and a δ⁷⁰-like promoter has been identified upstream from the napKgene. The same transcriptional start site is found for cells growing aerobically or anaerobically with nitrate. Different mutant strains carrying defined polar and non-polar insertions in each napgene were constructed. Characterization of these mutant strains demonstrates the participation of the napgene products in the periplasmic nitrate reduction in R. sphaeroides.

Bacterial periplasmic nitrate reductases (Nap) can play different physiological roles and are expressed under different conditions depending on the organism.Rhodobacter sphaeroidesDSM158 has a Nap system, encoded by thenapKEFDABCgene cluster, but nitrite formed is not further reduced because this strain lacks nitrite reductase. Nap activity increases in the presence of nitrate and oxygen but is unaffected by ammonium. Reverse transcription-PCR and Northern blots demonstrated that thenapKEFDABCgenes constitute an operon transcribed as a single 5.5-kb product. Northern blots andnap-lacZfusions revealed thatnapexpression is threefold higher under aerobic conditions but is regulated by neither nitrate nor ammonium, although it is weakly induced by nitrite. On the other hand, nitrate but not nitrite causes a rapid enzyme activation, explaining the higher Nap activity found in nitrate-grown cells. Translationalnap′-′lacZfusions reveal that thenapKandnapDgenes are not efficiently translated, probably due to mRNA secondary structures occluding the translation initiation sites of these genes. Neither butyrate nor caproate increasesnapexpression, although cells growing phototrophically on these reduced substrates show a very high Nap activity in vivo (nitrite accumulation is sevenfold higher than in medium with malate). Phototrophic growth on butyrate or caproate medium is severely reduced in the NapA⁻mutants. Taken together, these results indicate that nitrate reduction inR. sphaeroidesis mainly regulated at the level of enzyme activity by both nitrate and electron supply and confirm that the Nap system is involved in redox balancing using nitrate as an ancillary oxidant to dissipate excess reductant.

Cyanide is a highly toxic compound that is found in wastewaters generated from different industrial activities, such as mining or jewellery. These residues usually contain high concentrations of other toxic pollutants like arsenic and heavy metals that may form different complexes with cyanide. To develop bioremediation strategies, it is necessary to know the metabolic processes involved in the tolerance and detoxification of these pollutants, but most of the current studies are focused on the characterization of the microbial responses to each one of these environmental hazards individually, and the effect of co‐contaminated wastes on microbial metabolism has been hardly addressed. This work summarizes the main strategies developed by bacteria to alleviate the effects of cyanide, arsenic and heavy metals, analysing interactions among these toxic chemicals. Additionally, it is discussed the role of systems biology and synthetic biology as tools for the development of bioremediation strategies of complex industrial wastes and co‐contaminated sites, emphasizing the importance and progress derived from meta‐omic studies.

Cyanide is one of the most toxic chemicals produced by anthropogenic activities like mining and jewelry industries, which generate wastewater residues with high concentrations of this compound. Pseudomonas pseudoalcaligenes CECT5344 is a model microorganism to be used in detoxification of industrial wastewaters containing not only free cyanide (CN−) but also cyano-derivatives, such as cyanate, nitriles and metal-cyanide complexes. Previous in silico analyses suggested the existence of genes putatively involved in metabolism of short chain length (scl-) and medium chain length (mcl-) polyhydroxyalkanoates (PHAs) located in three different clusters in the genome of this bacterium. PHAs are polyesters considered as an alternative of petroleum-based plastics. Strategies to optimize the bioremediation process in terms of reducing the cost of the production medium are required. In this work, a biological treatment of the jewelry industry cyanide-rich wastewater coupled to PHAs production as by-product has been considered. The functionality of the pha genes from P. pseudoalcaligenes CECT5344 has been demonstrated. Mutant strains defective in each proposed PHA synthases coding genes (Mpha−, deleted in putative mcl-PHA synthases; Spha−, deleted in the putative scl-PHA synthase) were generated. The accumulation and monomer composition of scl- or mcl-PHAs in wild type and mutant strains were confirmed by gas chromatography-mass spectrometry (GC–MS). The production of PHAs as by-product while degrading cyanide from the jewelry industry wastewater was analyzed in batch reactor in each strain. The wild type and the mutant strains grew at similar rates when using octanoate as the carbon source and cyanide as the sole nitrogen source. When cyanide was depleted from the medium, both scl-PHAs and mcl-PHAs were detected in the wild-type strain, whereas scl-PHAs or mcl-PHAs were accumulated in Mpha− and Spha−, respectively. The scl-PHAs were identified as homopolymers of 3-hydroxybutyrate and the mcl-PHAs were composed of 3-hydroxyoctanoate and 3-hydroxyhexanoate monomers. These results demonstrated, as proof of concept, that talented strains such as P. pseudoalcaligenes might be applied in bioremediation of industrial residues containing cyanide, while concomitantly generate by-products like polyhydroxyalkanoates. A customized optimization of the target bioremediation process is required to gain benefits of this type of approaches.