Jane I. Grove

Escherichia coli K-12 genes essential for the synthesis of c-type cytochromes and a third nitrate reductase located in the periplasm

The ‘aeg46.5 ’ operon was originally detected as an ‘anaerobically expressed gene’ located at minute 46.5 on the Escherichia coli linkage map. Subsequent results from the E. coli Genome Sequencing Project revealed that the ‘aeg46.5 ’ promoter was located in the centisome 49 (minute 47) region. Downstream from this promoter are 15 genes, seven of which are predicted to encode a periplasmic nitrate reductase and eight encode proteins homologous to proteins essential for cytochrome c assembly in other bacteria. All of these genes, together with the ‘aeg46.5 ’ promoter, have been subcloned on a 20 kb EcoRI fragment from Kohara phage 19D1. Evidence is presented that, as predicted, the region includes structural genes for two c‐type cytochromes of mass 16 kDa and 24 kDa, which are transcribed from the previously described ‘aeg46.5 ’ promoter, and that the first seven genes encode a functional nitrate reductase. We, therefore, propose that they should be designated nap (nitrate reductase in the periplasm) genes. Plasmids encoding the entire 20 kb region, or only the downstream eight genes, complemented five mutations resulting in total absence of all five known c‐type cytochromes in E. coli, providing biochemical evidence that these are ccm (for cytochrome c maturation) genes. The ccm region was transcribed both from the FNR‐dependent, NarL‐ and NarP‐regulated nap promoter (formerly the ‘aeg46.5 ’ promoter) and from constitutive or weakly regulated promoters apparently located within the downstream nap and ccm genes.

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.

Involvement of products of the nrfEFG genes in the covalent attachment of haem c to a novel cysteine-lysine motif in the cytochrome c552 nitrite reductase from Escherichia coli.

Cytochrome c552 is the terminal component of the formate‐dependent nitrite reduction pathway of Escherichia coli. In addition to four ‘typical’ haem‐binding motifs, CXXCH‐, characteristic of c‐type cytochromes, the N‐terminal region of NrfA includes a motif, CWSCK. Peptides generated by digesting the cytochrome from wild‐type bacteria with cyanogen bromide followed by trypsin were analysed by on‐line HPLC MS/MS in parent scanning mode. A strong signal at mass 619, corresponding to haem, was generated by fragmentation of a peptide of mass 1312 that included the sequence CWSCK. Neither this signal nor the haem‐containing peptide of mass 1312 was detected in parallel experiments with cytochrome that had been purified from a transformant unable to synthesize NrfE, NrfF and NrfG: this is consistent with our previous report that NrfE and NrfG (but not NrfF) are essential for formate‐dependent nitrite reduction. Redox titrations clearly revealed the presence of high and low mid‐point potential redox centres. The best fit to the experimental data is for three n = 1 components with mid‐point redox potentials (pH 7.0) of +45 mV (21% of the total absorbance change), −90 mV (36% of the total) and −210 mV (43% of the total). Plasmids in which the lysine codon of the cysteine–lysine motif, AAA, was changed to the histidine codon CAT (to create a fifth ‘typical’ haem c‐binding motif), or to the isoleucine and leucine codons, ATT and CTT, were unable to transform a Nrf− deletion mutant to Nrf+ or to restore formate‐dependent nitrite reduction to the transformants. The presence of a 50 kDa periplasmic c‐type cytochrome was confirmed by staining proteins separated by SDS–PAGE for covalently bound haem, but the methyl‐viologen‐dependent nitrite reductase activities associated with the mutated proteins, although still detectable, were far lower than that of the native protein. The combined data establish not only that there is a haem group bound covalently to the cysteine–lysine motif of cytochrome c552 but also that one or more products of the last three genes of the nrf operon are essential for the haem ligation to this motif.

The RsaI polymorphism of CYP2E1 and susceptibility to alcoholic liver disease in Caucasians : effect on age of presentation and dependence on alcohol dehydrogenase genotype

Twin studies in Caucasians suggest that susceptibility to alcoholic liver disease is, in part, genetically determined. Because most of the deleterious effects of alcohol are caused by its metabolism, attention has focused upon genes encoding ethanol metabolizing enzymes. Caucasians are polymorphic at only two of these gene loci--cytochrome P450 2E1 (CYP2E1) and alcohol dehydrogenase 3 (ADH3). We examined the frequency of the RsaI polymorphism of CYP2E1 and ADH3 genotype in 264 patients with alcoholic liver disease and 121 local control individuals. There was a non-significant excess of the rare c2 CYP2E1 allele in patients with advanced liver disease compared with control individuals/patients with steatosis only (0.029 versus 0.017/0.00). However, patients with the c2 allele presented at a younger age compared with those with the wild type c1 allele only (42.3 +/- 1.6 years versus 49.0 +/- 0.6 years; P = 0.001) with at least as advanced histology (93% cirrhotic versus 74%). Male patients had a higher frequency of the ADH3*2/*2 genotype (which encodes the less active gamma2 subunit) than control individuals [odds ratio (OR) 2.04 (1.11-3.76)], however, ADH3 genotype did not differ with histological stage or with age of presentation. Patients with advanced disease possessing the c2 allele had a significantly higher frequency of the ADH3*2/*2 genotype compared with c1 homozygotes [OR 3.71 (1.24-11.09)]. This study demonstrates that, although rare in Caucasians, possession of the mutant c2 allele of CYP2E1 increases the risk of alcoholic liver disease at a given level of cumulative alcohol consumption. This risk appears to be particularly manifest in individuals carrying the ADH3*2 allele, presumably reflecting increased metabolism of ethanol by CYP2E1. In the absence of the c2 allele, ADH3 genotype does not influence the risk of advanced alcoholic liver disease but, in males at least, may influence the risk of alcoholism.

Detection and characterization of novel polymorphisms in the CYP2E1 gene

To investigate whether interindividual variation in CYP2E1 levels can be explained by genetic polymorphism, we analysed DNA samples from 40 healthy individuals by single-strand conformational polymorphism analysis for polymorphisms in the CYP2E1 coding sequence and promoter region. DNA sequencing of samples showing mobility shifts on single-strand conformational polymorphism detected polymorphisms at positions -316 (A to G), -297 (T to A), -35 (G to T), 1107 (G to C; intron 1), 4804 (G to A Val179Ile; exon 4) and 10157 (C to T; exon 8). All individuals positive for either A(-316)G, G(-35)T, G(4804)A or the previously described RsaI polymorphism at -1019 were also positive for T(-297)A, which had the highest allele frequency of the observed polymorphisms (0.20). A(-316)G, G(-35)T and G(4804)A were detected at allele frequencies of 0.022, 0.052 and 0.013, respectively. The functional significance of the upstream polymorphisms was examined by preparing constructs of positions -549 to +3 of CYP2E1 containing the observed combinations of the polymorphisms fused to luciferase reporter genes and transfecting HepG2 cells. For the G(-35)T/T(-297)A construct, a 1.8-fold increase in luciferase activity compared with the wild-type sequence (P = 0.06) and 2.5-fold compared with T(-297)A only (P = 0.025) was observed. No significant difference in activity was observed between the other constructs. The significance of the predicted Val179Ile base change from G(4804)A was determined by expression of the wild-type and mutated full length cDNAs in lymphoblastoid cells. No significant difference in kinetic constants for chlorzoxazone hydroxylation between mutant and wild-type was observed. In summary, this study demonstrated six novel CYP2E1 polymorphisms, including three upstream of the promoter, but with the possible exception of G(-35)T, none appeared to be of functional significance.

RecN protein and transcription factor DksA combine to promote faithful recombinational repair of DNA double‐strand breaks

In rapidly dividing bacterial cells, the machinery for repair of DNA double‐strand breaks has to contend not only with the forces driving replication and transmission of the DNA but also its transcription. By exploiting I‐SceI homing endonuclease to break the Escherichia coli chromosome at one or more defined locations, we have been able to investigate how these processes are co‐ordinated and repair is accomplished. When breaks are induced at a single site, the SOS‐inducible RecN protein and the transcription factor DksA combine to promote efficient repair. When induced at two or more, distantly located sites, RecN becomes almost indispensable. Many cells that do survive have extensive deletions of sequences flanking the break, with end points often coinciding with imperfect repeat elements. These findings herald a much greater complexity for chromosome repair than suggested by current mechanistic models and reveal a role for RecN in protecting the chromosome from break‐induced chromosome rearrangements.

Regulation and sequence of the structural gene for cytochrome C552 from Escherichia coli: not a hexahaem but a 50kDa tetrahaem nitrite reductase

The structural gene, nrfA, for cytochrome C552, which is the terminal reductase of the formate‐dependent pathway for nitrite reduction to ammonia, has been located at co‐ordinate 4366 on the physical map of the Escherichia coli chromosome. The DNA sequence of nrfA encodes a tetrahaem c‐type cytochrome with a predicted Mr for the unprocessed product of 53788. Cleavage of the putative signal peptide at Ala‐26 would result in a mature, periplasmic cytochrome of Mr 50580 rather than a larger hexahaem cytochrome, as has been widely reported previously. A cytochrome of this size was detected by staining SDS‐polyacryla‐mide gels for covalently bound haem. This cytochrome was partially purified by anion exchange chromatography and confirmed to be cytochrome C552 by difference spectroscopy. Similar cytochromes were detected in five other E. coli strains including strain ST 249, which was used previously to purify and characterize the protein. A plasmid with an in‐phase deletion within nrfA directed the synthesis of a truncated haemoprotein of the predicted mass. In‐phase translational fusions to lacZ were used to locate the nrfA translation start, and the transcription start site was found by S1 mapping.

NITROREDUCTASE: A PRODRUG‐ACTIVATING ENZYME FOR CANCER GENE THERAPY

1. The prodrug CB1954 (5‐(aziridin‐1‐yl)‐2,4‐dinitrobenzamide) is activated by Escherichia coli nitroreductase (NTR) to a potent DNA‐crosslinking agent.

Heterozygotes for HFE mutations have no increased risk of advanced alcoholic liver disease

Background—Iron overload is common in the livers of alcoholics and may play a role in disease pathogenesis. An MHC like gene, HFE,has recently been identified that is mutated in most patients with hereditary haemochromatosis (C282Y in 90% and H63D in 45% of the remainder). Aim—To examine the hypothesis that these mutations determine hepatic iron status in alcoholics and play a role in predisposition to advanced alcoholic liver disease. Methods—TheHFE gene was genotyped in 257 patients with alcoholic liver disease and 117 locally matched healthy volunteers. In addition, iron staining was scored (0–4) on biopsy specimens from fibrotic/cirrhotic patients with and withoutHFE mutations matched for age and sex. Results—Some 15.7% of fibrotic/cirrhotic patients were C282Y heterozygotes compared with 13.7% of controls (p = 0.77). One control and three patients were C282Y homozygotes. Of chromosomes without the C282Y mutation, 68/442 (15.4%) of patients’ chromosomes carried the H63D mutation compared with 36/216 (16.6%) of control chromosomes (p = 0.91). Significant (>grade 1) hepatocyte iron staining was seen in 6/23 C282Y heterozygotes and 4/26 H63D heterozygotes compared with 4/23 controls. Conclusions—Possession of a single copy of either of the two HFEmutations influences neither liver iron content nor the risk of fibrotic disease in alcoholics.

Tumour necrosis factor-a promoter polymorphisms in primary biliary cirrhosis

Abstract Background/aims : The incidence of primary biliary cirrhosis (PBC) is increased in the close relatives of patients, suggesting that genetic factors play a role in disease susceptibility. Decreased in vitro production of tumour necrosis factor (TNF)-α has been reported in PBC patients, suggesting a potential aetiological role for this cytokine. The aim of this study was to examine two biallelic polymorphisms in the promoter region of the TNF-α gene, which may play a role in the control of TNF-α secretion, as candidate susceptibility loci in PBC. Methods : The polymorphisms at positions −238 and −308 in the TNF-α promoter region were analysed by polymerase chain reaction in 168 unrelated PBC patients and 145 local unrelated, geographically matched normal individuals. All PBC subjects were also genotyped for HLA DR8, a previously identified susceptibility locus in PBC. Results : The −308 TNF1/TNF1 genotype was seen in a similar proportion of PBC patients (66%) and controls (60%). However, this genotype was found significantly more frequently in the 95 PBC patients with more advanced disease (histological stage III/IV) (77%) than in either controls ( p p =0.001 OR 3.1 [1.6–5.9]). Linkage between TNF −308 and HLA DR8 was not seen. No association was found between PBC and the biallelic −238 TNF-α polymorphism, either in the whole PBC population or the histological Stage III/IV subgroup. Conclusions : Our study provides no evidence for involvement of the TNF-α −308 or −238 promoter polymorphisms in genetic predisposition to PBC. However, the significantly increased frequency of the −308 TNF1/TNF1 genotype seen in 95 patients with more advanced disease raises the possibility that this allele may be linked to disease progression rather than susceptibility. The finding of different allele frequencies in PBC patients in different disease subgroups emphasises the importance of clinical phenotype/case-mix in the design of disease association studies.