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.In several recent studies, genome-wide transcription data have been analyzed to reveal the extent of the biochemical changes as Escherichia coli K12 adapts from aerobic to anaerobic growth. Salmon et al. (1) compared RNA isolated from cultures of strain MC4100 that had been grown aerobically or anaerobically and also from an anaerobic culture of an fnr mutant that lacks FNR, 2 the regulator of fumarate and nitrate reduction, which is a global regulator of many oxygen-regulated genes. In similar experiments, Kang et al. (2) grew both strain MG1655, for which the complete genome sequence is available, and an isogenic fnr mutant aerobically and anaerobically in a minimal salts medium, and compared their transcriptome data with those from the previous study. In both groups of experiments, glucose was used as the carbon source for growth, and in both studies rigorous statistical methods and cluster analysis were used to analyze the data. In the former study, expression levels of 1,445 genes changed in response to the availability of oxygen. Although the corresponding figure in the study of Kang et al. (2) was 962, only 334 genes were common to both data sets, and of those, 123 appeared to be regulated in opposite directions. Thus only 211 genes showed similar responses, 10% of the 2073 genes for which changes were observed.Both of the previous studies were valuable in revealing the far greater extent of chan...

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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.

Neisseria gonorrhoeae survives anaerobically by reducing nitrite to nitrous oxide catalyzed by the nitrite and nitric oxide reductases, AniA and NorB. P aniA is activated by FNR (regulator of fumarate and nitrate reduction), the two-component regulatory system NarQ-NarP, and induced by nitrite; P norB is induced by NO independently of FNR by an uncharacterized mechanism. We report the results of microarray analysis, bioinformatic analysis, and chromatin immunoprecipitation, which revealed that only five genes with readily identified NarP-binding sites are differentially expressed in narP ؉ and narP strains. These include three genes implicated in the truncated gonococcal denitrification pathway: aniA, norB, and narQ. We also report that (i) nitrite induces aniA transcription in a narP mutant; (ii) nitrite induction involves indirect inactivation by nitric oxide of a gonococcal repressor, NsrR, identified from a multigenome bioinformatic study; (iii) in an nsrR mutant, aniA, norB, and dnrN (encoding a putative reactive nitrogen species response protein) were expressed constitutively in the absence of nitrite, suggesting that NsrR is the only NO-sensing transcription factor in N. gonorrhoeae; and (iv) NO rather than nitrite is the ligand to which NsrR responds. When expressed in Escherichia coli, gonococcal NarQ and chimaeras of E. coli and gonococcal NarQ are ligand-insensitive and constitutively active: a "locked-on" phenotype. We conclude that genes involved in the truncated denitrification pathway of N. gonorrhoeae are key components of the small NarQP regulon, that NarP indirectly regulates P norB by stimulating NO production by AniA, and that NsrR plays a critical role in enabling gonococci to evade NO generated as a host defense mechanism.In contrast to Escherichia coli that can inhabit a variety of environments and utilize numerous carbon sources and electron acceptors, some niche dwellers such as the obligate human pathogen Neisseria gonorrhoeae are far less versatile. The gonococcus can grow aerobically using glucose, lactate, or pyruvate as carbon sources and electron donors, and for many years it was thought to be an obligate aerobe. However, following the isolation of gonococci from patients alongside obligate anaerobes, it became clear that they could survive in the absence of oxygen in vivo using nitrite as an alternative electron acceptor (1, 2). Although gonococci express both a copper-containing nitrite reductase, AniA (NGO1276), and a single subunit nitric oxide reductase, NorB (NGO1275), which reduce nitrite via nitric oxide to nitrous oxide (2-5), denitrification is incomplete, because they lack genes for nitrate reduction, and there is a premature stop codon in the nitrous oxide reductase gene (nosZ, XNG1300), and the putative regulator of the nitrous oxide reduction genes, nosR (XNG1301), is also degenerate (see Fig. 1A). During oxygen-limited or anaerobic growth, AniA is the major anaerobically induced outer membrane protein (6). It is expressed by bacteria infecting patients, confirming th...

Expression of two genes of unknown function, Staphylococcus aureus scdA and Neisseria gonorrhoeae dnrN, is induced by exposure to oxidative or nitrosative stress. We show that DnrN and ScdA are di-iron proteins that protect their hosts from damage caused by exposure to nitric oxide and to hydrogen peroxide. Loss of FNR-dependent activation of aniA expression and NsrR-dependent repression of norB and dnrN expression on exposure to NO was restored in the gonococcal parent strain but not in a dnrN mutant, suggesting that DnrN is necessary for the repair of NO damage to the gonococcal transcription factors, FNR and NsrR. Restoration of aconitase activity destroyed by exposure of S. aureus to NO or H 2 O 2 required a functional scdA gene. Electron paramagnetic resonance spectra of recombinant ScdA purified from Escherichia coli confirmed the presence of a di-iron center. The recombinant scdA plasmid, but not recombinant plasmids encoding the complete Escherichia coli sufABCDSE or iscRSUAhscBAfdx operons, complemented repair defects of an E. coli ytfE mutant. Analysis of the protein sequence database revealed the importance of the two proteins based on the widespread distribution of highly conserved homologues in both gram-positive and gram-negative bacteria that are human pathogens. We provide in vivo and in vitro evidence that Fe-S clusters damaged by exposure to NO and H 2 O 2 can be repaired by this new protein family, for which we propose the name repair of iron centers, or RIC, proteins.

Analysis of the Neisseria gonorrhoeae DNA sequence database revealed the presence of two genes, one encoding a protein predicted to be 37.5% identical (50% similar) in amino acid sequence to the Escherichia coli FNR protein and the other encoding a protein 41% and 42% identical (54 and 51% sequence similarity) to the E. coli NarL and NarP proteins respectively. Both genes have been cloned into E. coli and insertionally inactivated in vitro. The mutated genes have been transformed into gonococci and recombined into the chromosome. The fnr mutation totally abolished and the narP mutation severely diminished the ability of gonococci to: (i) grow anaerobically; (ii) adapt to oxygen‐limited growth; (iii) initiate transcription from the aniA promoter (which directs the expression of a copper‐containing nitrite reductase, AniA, in response to the presence of nitrite); and (iv) reduce nitrite during growth in oxygen‐limited media. The product of nitrite reduction was identified to be nitrous oxide. Immediately upstream of the narL/narP gene is an open reading frame that, if translated, would encode a homologue of the E. coli nitrate‐ and nitrite‐sensing proteins NarX and NarQ. As transcription from the aniA promoter was not activated during oxygen‐limited growth in the presence of nitrate, the gonococcal two‐component regulatory system is designated NarQ–NarP rather than NarX–NarL. As far as we are aware, this is the first well‐documented example of a two‐component regulatory system working in partnership with a transcription activator in pathogenic neisseria. A 45 kDa c‐type cytochrome that was synthesized during oxygen‐limited, but not during oxygen sufficient, growth was identified as a homologue of cytochrome c peroxidases (CCP) of other bacteria. The gene for this cytochrome, designated ccp, was located, and its regulatory region was cloned into the promoter probe vector pLES94. Transcription from the ccp promoter was repressed during aerobic growth and induced during oxygen‐limited growth and was totally FNR dependent, suggesting that the gonococcal FNR protein is a transcription activator of at least two genes. However, unlike AniA, synthesis of the CCP homologue was insensitive to the presence of nitrite during oxygen‐limited growth.

Escherichia coli MelR protein is a transcription activator that is essential for melibiose-dependent expression of the melAB genes. We have used chromatin immunoprecipitation to study the binding of MelR and RNA polymerase to the melAB promoter in vivo. Our results show that MelR is associated with promoter DNA, both in the absence and presence of the inducer melibiose. In contrast, RNA polymerase is recruited to the melAB promoter only in the presence of inducer. The MelR DK261 positive control mutant binds to the melAB promoter but cannot recruit RNA polymerase. Further analysis of immunoprecipitated DNA, by using an Affymetrix GeneChip array, showed that the melAB promoter is the major, if not the sole, target in E. coli for MelR. This was confirmed by a transcriptomics experiment to analyze RNA in cells either with or without melR.Expression of the Escherichia coli melAB genes, which encode proteins necessary for transport and metabolism of the disaccharide melibiose, is dependent on the transcription activator, MelR, encoded by the adjacent melR gene (13). Previous studies have shown that transcription from the melAB promoter is activated by MelR and have focused on using biochemistry to understand the mechanism of activation (1,4,7,12). Recent work has shown that MelR activates transcription by direct interaction with the RNA polymerase subunit via residue D261 (5). Although activation requires the inducer melibiose, in vitro studies have shown that MelR can bind to the melAB promoter both in the presence and absence of melibiose (1). In the experiments presented here, we have exploited novel chromatin immunoprecipitation (ChIP) and microarray technologies to study the interactions of MelR in vivo. ChIP has been used to investigate MelR and RNA polymerase binding to the melAB regulatory region in vivo, while microarrays have been used to show that the melAB promoter is the principal target for MelR in E. coli. MATERIALS AND METHODSE. coli strains, plasmids, and oligonucleotides. Bacterial strains, plasmids, and oligonucleotides used in this work are listed in Table 1. In all experiments, E. coli strains WAM131, WAM132, or MG1655, carrying plasmids as appropriate, were grown to mid-exponential phase (optical density at 650 nm of 0.4 to 0.6) in minimal M63 medium, supplemented with fructose and amino acids, either with or without melibiose, according to the same method used previously in studies of the regulation of the E. coli mel operon (13).ChIP. In all experiments, in vivo cross-linking of bacterial nucleoprotein was initiated by the addition of formaldehyde (final concentration of 1%) to cultures. After 20 min, cross-linking was quenched by the addition of glycine (final concentration of 0.5 M). Typically, cells were then harvested from 10 ml of culture by centrifugation, washed twice with Tris-buffered-saline (pH 7.5), resuspended in 1 ml of lysis buffer (10 mM Tris [pH 8.0], 20% sucrose, 50 mM NaCl, 10 mM EDTA, 10 mg of lysozyme per ml) and incubated at 37°C for 30 min. Following lysis, 4 ml of immuno...

A C-terminal green fluorescent protein (GFP) fusion to a model target protein, Escherichia coli CheY, was exploited both as a reporter of the accumulation of soluble recombinant protein, and to develop a generic approach to optimize protein yields. The rapid accumulation of CheY∷GFP expressed from a pET20 vector under the control of an isopropyl-β-d-thiogalactoside (IPTG)-inducible T7 RNA polymerase resulted not only in the well-documented growth arrest but also loss of culturability and overgrowth of the productive population using plasmid-deficient bacteria. The highest yields of soluble CheY∷GFP as judged from the fluorescence levels were achieved using very low concentrations of IPTG, which avoid growth arrest and loss of culturability postinduction. Optimal product yields were obtained with 8 μM IPTG, a concentration so low that insufficient T7 RNA polymerase accumulated to be detectable by Western blot analysis. The improved protocol was shown to be suitable for process scale-up and intensification. It is also applicable to the accumulation of an untagged heterologous protein, cytochrome c(2) from Neisseria gonorrhoeae, which requires both secretion and extensive post-translational modification.