Jeffrey A. Cole

Nitrate, bacteria and human health

Nitrate is generally considered a water pollutant and an undesirable fertilizer residue in the food chain. Research in the 1970s indicated that, by reducing nitrate to nitrite, commensal bacteria might be involved in the pathogenesis of gastric cancers and other malignancies, as nitrite can enhance the generation of carcinogenic N-nitrosamines. More recent studies indicate that the bacterial metabolism of nitrate to nitrite and the subsequent formation of biologically active nitrogen oxides could be beneficial. Here, we will consider the evidence that nitrate-reducing commensals have a true symbiotic role in mammals and facilitate a previously unrecognized but potentially important aspect of the nitrogen cycle.

A Reassessment of the FNR Regulon and Transcriptomic Analysis of the Effects of Nitrate, Nitrite, NarXL, and NarQP as Escherichia coli K12 Adapts from Aerobic to Anaerobic Growth

The transcription factor FNR, the regulator of fumarate and nitrate reduction, regulates major changes as Escherichia coli adapts from aerobic to anaerobic growth. In an anaerobic glycerol/trimethylamine N-oxide/fumarate medium, the fnr mutant grew as well as the parental strain, E. coli K12 MG1655, enabling us to reveal the response to oxygen, nitrate, and nitrite in the absence of glucose repression or artifacts because of variations in growth rate. Hence, many of the discrepancies between previous microarray studies of the E. coli FNR regulon were resolved. The current microarray data confirmed 31 of the previously characterized FNR-regulated operons. Forty four operons not previously known to be included in the FNR regulon were activated by FNR, and a further 28 operons appeared to be repressed. For each of these operons, a match to the consensus FNR-binding site sequence was identified. The FNR regulon therefore minimally includes at least 103, and possibly as many as 115, operons. Comparison of transcripts in the parental strain and a narXL deletion mutant revealed that transcription of 51 operons is activated, directly or indirectly, by NarL, and a further 41 operons are repressed. The narP gene was also deleted from the narXL mutant to reveal the extent of regulation by phosphorylated NarP. Fourteen promoters were more active in the narP+ strain than in the mutant, and a further 37 were strongly repressed. This is the first report that NarP might function as a global repressor as well as a transcription activator. The data also revealed possible new defense mechanisms against reactive nitrogen species.

Respiratory detoxification of nitric oxide by the cytochrome c nitrite reductase of Escherichia coli

Nitric oxide is a key element in host defense against invasive pathogens. The periplasmic cytochrome cnitrite reductase (NrfA) of Escherichia coli catalyzes the respiratory reduction of nitrite, but in vitro studies have shown that it can also reduce nitric oxide. The physiological significance of the latter reaction in vivo has never been assessed. In this study the reduction of nitric oxide byEscherichia coli was measured in strains active or deficient in periplasmic nitrite reduction. Nrf+ cells, harvested from cultures grown anaerobically, possessed a nitric-oxide reductase activity with physiological electron donation of 60 nmol min−1· mg dry wt−1, and an in vivo turnover number of NrfA of 390 NO⋅ s−1was calculated. Nitric-oxide reductase activity could not be detected in Nrf− strains. Comparison of the anaerobic growth of Nrf+ and Nrf− strains revealed a higher sensitivity to nitric oxide in the NrfA− strains. A higher sensitivity to the nitrosating agentS-nitroso-N-acetyl penicillamine (SNAP) was also observed in agar plate disk-diffusion assays. Oxygen respiration by E. coli was also more sensitive to nitric oxide in the Nrf− strains compared with the Nrf+ parent strain. The results demonstrate that active periplasmic cytochromec nitrite reductase can confer the capacity for nitric oxide reduction and detoxification on E. coli. Genomic analysis of many pathogenic enteric bacteria reveals the presence ofnrf genes. The present study raises the possibility that this reflects an important role for the cytochrome cnitrite reductase in nitric oxide management in oxygen-limited environments.

A seven-gene operon essential for formate-dependent nitrite reduction to ammonia by enteric bacteria

The DNA sequence of the regulatory region and the structural gene, nrfA, for cytochrome C552 of Escherichia coli K‐12 have been reported. We have now established that nrfA is the first gene in a seven‐gene operon, designated the nrf operon, at least five of which are essential for formate‐dependent nitrite reduction to ammonia. This operon terminates just upstream of the previously sequenced gltP gene encoding a sodium‐independent, glutamate and aspartate transporter. Expression of lac fused to nrfA, nrfE or nrfG is regulated by oxygen repression, FNR‐dependent anaerobic induction, nitrite induction and nitrate repression during anaerobic growth, exactly as previously reported for the nrfA promoter, in contrast, expression of the gltP‐lac fusion was FNR‐independent.

Nitrate reduction in the periplasm of gram-negative bacteria

In contrast to the bacterial assimilatory and membrane-associated, respiratory nitrate reductases that have been studied for many years, it is only recently that periplasmic nitrate reductases have attracted growing interest. Recent research has shown that these soluble proteins are widely distributed, but vary greatly between species. All of those so far studied include four essential components: the periplasmic molybdoprotein, NapA, which is associated with a small, di-haem cytochrome, NapB; a putative quinol oxidase, NapC; and a possible pathway-specific chaperone, NapD. At least five other components have been found in different species. Other variations between species include the location of the nap genes on chromosomal or extrachromosomal DNA, and the environmental factors that regulate their expression. Despite the relatively small number of bacteria so far screened, striking correlations are beginning to emerge between the organization of the nap genes, the physiology of the host, the conditions under which the nap genes are expressed, and even the fate of nitrite, the product of Nap activity. Evidence is emerging that Nap fulfills a novel role in nitrate scavenging by some pathogenic bacteria.

The biogenesis of c‐type cytochromes in Escherichia coli requires a membrane‐bound protein, DipZ, with a protein disulphide isomerase‐like domain

A mutant of Escherichia coli K‐12, JCB606, which lacks all five c‐type cytochromes synthesized during anaerobic growth in the presence of nitrite or tri‐methylamine‐N‐oxide (TMAO), was totally defective in Nrf activity and also partially defective in TMAO reductase activity. The mutation in strain JCB606 was shown to affect expression of the tor operon, which contributes almost equally with the products of the dms operon to the rate of TMAO reduction by bacteria during anaerobic growth in the presence of TMAO. The mutation in strain JCB606, dipZ, was mapped by P1 transduction close to the mel operon at co‐ordinate 4425 on the E. coli chromosome, the gene order being nrf–fdhF–mel–dipZ–ampC. Recombinant plasmids that restored Nrf activity to test‐tube cultures of the mutant were isolated from a cosmid library. A 2.7 kb EcoRV–Smal fragment (co‐ordinates 4443 to 4446 kb on the physical map of the E. coli chromosome) was found potentially to encode three genes arranged in at least two operons. The second gene, dipZ, was sufficient to complement the JCB606 mutation. The translated DNA sequence predicts that DipZ is a 53kDa integral membrane protein with a 37kDa N‐terminal domain including at least six membrane‐spanning helices and a 16kDa carboxy‐terminal hydrophilic domain which includes a protein disulphide isomerase‐like motif. It is suggested that DipZ is essential for maintaining cytochrome c apoproteins in the correct conformations for the covalent attachment of haem groups to the appropriate pairs of cysteine residues.

Different physiological roles of two independent pathways for nitrite reduction to ammonia by enteric bacteria

Operon fusion strains and mutants of Escherichia coli K-12 lacking the NADH-dependent nitrite reductase have been used to determine the regulation and physiological roles of two independent pathways for nitrite reduction to ammonia. Both the formate-and NADH-dependent pathways (Nrf and Nir, respectively) were totally repressed during aerobic growth, partially active during anaerobic growth in the absence of nitrite and further induced anaerobically by nitrite. Both were dependent upon a functional Fnr protein (a transcription activator of genes for anaerobic respiration). During anaerobic growth in the presence of nitrate, the Nir pathway was fully induced but Nrf was strongly repressed. Mutants defective in the NarL protein, which induces transcription of nitrate reductase genes but represses fumarate reductase genes in the presence of nitrate, were derepressed for Nrf activity during growth with nitrate, but the Nir enzyme was less active. The synthesis of Nrf components was also sensitive to glucose repression and weak activation by NarL during growth in the absence of nitrate. These data indicate that the Nir pathway provides a mechanism for detoxifying nitrite formed in the cytoplasm as a product of nitrate reduction. In contrast, the electrogenic reduction of nitrite by the Nrf pathway provides a secondary source of energy during anaerobic growth and is consequently repressed by the NarL protein when the thermodynamically more favourable electron acceptor, nitrate, is available. Two short DNA sequences, 5′-TACCAT-3′ and 5′-CTCCTT-3′, were found in the promoters of operons known to be activated or repressed by the NarL protein. It is proposed that NarL activates nir B transcription by binding to one or both of these sequences located 5′ to the RNA polymerase binding site, but represses other operons, including nrf, by binding close to the transcription start.

The NsrR Regulon of Escherichia coli K-12 Includes Genes Encoding the Hybrid Cluster Protein and the Periplasmic, Respiratory Nitrite Reductase

Successful pathogens must be able to protect themselves against reactive nitrogen species generated either as part of host defense mechanisms or as products of their own metabolism. The regulatory protein NsrR (a member of the Rrf2 family of transcription factors) plays key roles in this stress response. Microarray analysis revealed that NsrR represses nine operons encoding 20 genes in Escherichia coli MG1655, including the hmpA, ytfE, and ygbA genes that were previously shown to be regulated by NsrR. Novel NsrR targets revealed by this study include hcp-hcr (which were predicted in a recent bioinformatic study to be NsrR regulated) and the well-studied nrfA promoter that directs the expression of the periplasmic respiratory nitrite reductase. Conversely, transcription from the ydbC promoter is strongly activated by NsrR. Regulation of the nrf operon by NsrR is consistent with the ability of the periplasmic nitrite reductase to reduce nitric oxide and hence protect against reactive nitrogen species. Gel retardation assays were used to show that both FNR and NarL bind to the hcp promoter. The expression of hcp and the contiguous gene hcr is not induced by hydroxylamine. As hmpA and ytfE encode a nitric oxide reductase and a mechanism to repair iron-sulfur centers damaged by nitric oxide, the demonstration that hcp-hcr, hmpA, and ytfE are the three transcripts most tightly regulated by NsrR highlights the possibility that the hybrid cluster protein, HCP, might also be part of a defense mechanism against reactive nitrogen stress.

The roles of the polytopic membrane proteins NarK, NarU and NirC in Escherichia coli K-12: two nitrate and three nitrite transporters

Two polytopic membrane proteins, NarK and NarU, are assumed to transport nitrite out of the Escherichia coli cytoplasm, but how nitrate enters enteric bacteria is unknown. We report the construction and use of four isogenic strains that lack nitrate reductase Z and the periplasmic nitrate reductase, but express all combinations of narK and narU. The active site of the only functional nitrate reductase, nitrate reductase A, is located in the cytoplasm, so nitrate reduction by these four strains is totally dependent upon a mechanism for importing nitrate. These strains were exploited to determine the roles of NarK and NarU in both nitrate and nitrite transport. Single mutants that lack either NarK or NarU were competent for nitrate‐dependent anaerobic growth on a non‐fermentable carbon source, glycerol. They transported and reduced nitrate almost as rapidly as the parental strain. In contrast, the narK–narU double mutant was defective in nitrate‐dependent growth unless nitrate transport was facilitated by the nitrate ionophore, reduced benzyl viologen (BV). It was also unable to catalyse nitrate reduction in the presence of physiological electron donors. Synthesis of active nitrate reductase A and the cytoplasmic, NADH‐dependent nitrite reductase were unaffected by the narK and narU mutations. The rate of nitrite reduction catalysed by the cytoplasmic, NADH‐dependent nitrite reductase by the double mutant was almost as rapid as that of the NarK+‐NarU+ strain, indicating that there is a mechanism for nitrite uptake by E. coli that is in‐dependent of either NarK or NarU. The nir operon encodes a soluble, cytoplasmic nitrite reductase that catalyses NADH‐dependent reduction of nitrite to ammonia. One additional component that contributes to nitrite uptake was shown to be NirC, the hydrophobic product of the third gene of the nir operon, which is predicted to be a polytopic membrane protein with six membrane‐spanning helices. Deletion of both NarK and NirC decreased nitrite uptake and reduction to a basal rate that was fully restored by a single chromosomal copy of either narK or nirC. A multicopy plasmid encoding NarU complemented a narK mutation for nitrite excretion, but not for nitrite uptake. We conclude that, in contrast to NirC, which transports only nitrite, NarK and NarU provide alternative mechanisms for both nitrate and nitrite transport. However, NarU might selectively promote nitrite ex‐cretion, not nitrite uptake.

Location and sequence of the promoter of the gene for the NADH-dependent nitrite reductase of Escherichia coli and its regulation by oxygen, the Fnr protein and nitrite

The DNA sequence containing the start of the Escherichia coli nirB gene is reported. The N-terminal amino acid sequence of purified NADH-dependent nitrite reductase coincided with that predicted from the DNA sequence, confirming that nirB is the structural gene for nitrite reductase apoprotein and identifying the translation start point. Using nuclease S1 mapping, the sole transcription startpoint for the nirB gene was found 23 or 24 base-pairs upstream from the ATG initiation codon. By subcloning successively smaller DNA fragments into a beta-galactosidase expression vector plasmid, we located the promoter within a sequence bounded by a TaqI site at +14 with respect to the transcription startpoint and a HpaII site at -208. Measurements in vivo of beta-galactosidase expression and RNA levels due to nirB promoter activity showed that this promoter was activated during anaerobic growth. Optimal activity was found only after anaerobic growth in the presence of nitrite. The sequence of the nirB promoter is compared with sequences found at other anaerobically activated promoters.