Roles and regulation of the iron-sulphur proteins, HCP, NapG and NapH, induced during anaerobic growth of E. coli

The periplasmic nitrate reductase (Nap) has been shown to support anaerobic growth of Escherichia coli K-12 under nitrate-limiting conditions. Two of the Nap proteins, NapG and NapH, are predicted to contain four and two [4Fe-4S] clusters, respectively. In this thesis it is reported that, during fermentative growth. Nap plays a role in redox balancing. This role is most pronounced in a strain that lacks menaquinol and tiierefore cannot use the menaquinol-dependent ftunarate reductase to fulfil a redox balancing role during glucose fermentation. Nitrate stimulated the growth of both a AmenBC AnapGH and an isogenic AmenBC nap^ strain to the same extent, even although the Nap activity was extremely low. This showed that the residual 1% electron flow in the strain deleted for NapG and NapH was sufficient to fulfil this redox balancing function. Using artificial quinones, NapG and NapH were shown to be linked to oxidation of quinones with high midpoint redox potentials. NapF^ and NapF' strains were grown anaerobically after either aerobic or anaerobic growth and NapF was shown to be involved in adaptation from aerobic to anaerobic growth. The hybrid cluster protein (HCP) contains two Fe-S clusters, one of which is a hybrid [4Fe-2S-20] cluster. Despite intensive study, its physiological function is unclear. E. coli HCP is detected after anaerobic growth with nitrate or nitrite, so a possible role for it in some stage of the nitrogen cycle has been proposed. To study the regulation of HCP, an hcprlacZ fusion was constructed and transformed into_^r, arcA and norR mutant strains of E. coli. Transcription from the hep promoter was induced during anaerobic growth. Only the jhr mutant was defective in hep expression, suggesting that transcription from the promoter in response to anaerobiosis is dependent on FNR. Nitrate and nitrite fiirther induced transcription from the hep promoter. The parental strain and the narL, narP and narLmrP mutants were grown anaerobically in medium supplemented with nitrite or nitrate. The nitrite and nitrate response of the hep promoter was mediated by both of the response regulator proteins, NarL and NarP. It is argued that NarL plays a dual role at the hep promoter acting as an activator during growth in the presence of a low concentration of nitrite or nitrate and as both repressor and activator in the presence of high nitrite or nitrate concentrations. Gel retardation assays were used to show that FNR and NarL form a complex with the hep promoter, thus confirming that their effect on transcription is direct. A technique involving the rapid amplification of cDNA ends (RACE) was used to demonstrate that transcription of the hcp~hcr operon initiates at a thymine nucleotide located 31 bp upstream of the translation-initiation codon. A A/icp strain was constructed by homologous recombination. When grown in medium supplemented with nitrate, the growth rate and yield of the parental strain and the hhep mutant were the same, suggesting that HCP is not involved in nitrate-dependent growth. Both HCP^ and HCP' strains were equally sensitive to nitric oxide and hydroxylamine. It was concluded therefore that HCP is unable to protect bacteria against nitric oxide or hydroxylamine toxicity in vivo. HCP was overexpressed from a recombinant plasmid and subsequently purified on a nickel column for biochemical studies. A qualitative method using reduced methyl viologen as an electron donor was developed for use in attempts to identify a possible substrate of HCP in vitro. Nitrite, nitrate and hydroxylamine were tested, but no evidence was presented that any of them can be used as an electron acceptor

The Role of Histone H4 Biotinylation in the Structure of Nucleosomes

Background: Post-translational modifications of histones play important roles in regulating nucleosome structure and gene transcription. It has been shown that biotinylation of histone H4 at lysine-12 in histone H4 (K12Bio-H4) is associated with repression of a number of genes. We hypothesized that biotinylation modifies the physical structure of nucleosomes, and that biotin-induced conformational changes contribute to gene silencing associated with histone biotinylation. Methodology/Principal Findings: To test this hypothesis we used atomic force microscopy to directly analyze structures of nucleosomes formed with biotin-modified and non-modified H4. The analysis of the AFM images revealed a 13% increase in the length of DNA wrapped around the histone core in nucleosomes with biotinylated H4. This statistically significant (p,0.001) difference between native and biotinylated nucleosomes corresponds to adding approximately 20 bp to the classical 147 bp length of nucleosomal DNA. Conclusions/Significance: The increase in nucleosomal DNA length is predicted to stabilize the association of DNA with histones and therefore to prevent nucleosomes from unwrapping. This provides a mechanistic explanation for the gene silencing associated with K12Bio-H4. The proposed single-molecule AFM approach will be instrumental for studying the effects of various epigenetic modifications of nucleosomes, in addition to biotinylation

NapGH components of the periplasmic nitrate reductase of Escherichia coli K-12: location, topology and physiological roles in quinol oxidation and redox balancing.

Nap (periplasmic nitrate reductase) operons of many bacteria include four common, essential components, napD, napA, napB and napC (or a homologue of napC ). In Escherichia coli there are three additional genes, napF, napG and napH, none of which are essential for Nap activity. We now show that deletion of either napG or napH almost abolished Nap-dependent nitrate reduction by strains defective in naphthoquinone synthesis. The residual rate of nitrate reduction (approx. 1% of that of napG+ H+ strains) is sufficient to replace fumarate reduction in a redox-balancing role during growth by glucose fermentation. Western blotting combined with beta-galactosidase and alkaline phosphatase fusion experiments established that NapH is an integral membrane protein with four transmembrane helices. Both the N- and C-termini as well as the two non-haem iron-sulphur centres are located in the cytoplasm. An N-terminal twin arginine motif was shown to be essential for NapG function, consistent with the expectation that NapG is secreted into the periplasm by the twin arginine translocation pathway. A bacterial two-hybrid system was used to show that NapH interacts, presumably on the cytoplasmic side of, or within, the membrane, with NapC. As expected for a periplasmic protein, no NapG interactions with NapC or NapH were detected in the cytoplasm. An in vitro quinol dehydrogenase assay was developed to show that both NapG and NapH are essential for rapid electron transfer from menadiol to the terminal NapAB complex. These new in vivo and in vitro results establish that NapG and NapH form a quinol dehydrogenase that couples electron transfer from the high midpoint redox potential ubiquinone-ubiquinol couple via NapC and NapB to NapA

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

Molecular Basis of Acute Cystitis Reveals Susceptibility Genes and Immunotherapeutic Targets

Tissue damage is usually regarded as a necessary price to pay for successful elimination of pathogens by the innate immune defense. Yet, it is possible to distinguish protective from destructive effects of innate immune activation and selectively attenuate molecular nodes that create pathology. Here, we identify acute cystitis as an Interleukin-1 beta (IL-1β)-driven, hyper-inflammatory condition of the infected urinary bladder and IL-1 receptor blockade as a novel therapeutic strategy. Disease severity was controlled by the mechanism of IL-1β processing and mice with intact inflammasome function developed a moderate, self-limiting form of cystitis. The most severe form of acute cystitis was detected in mice lacking the inflammasome constituents ASC or NLRP-3. IL-1β processing was hyperactive in these mice, due to a new, non-canonical mechanism involving the matrix metalloproteinase 7- (MMP-7). ASC and NLRP-3 served as transcriptional repressors of MMP7 and as a result, Mmp7 was markedly overexpressed in the bladder epithelium of Asc-/- and Nlrp3-/- mice. The resulting IL-1β hyper-activation loop included a large number of IL-1β-dependent pro-inflammatory genes and the IL-1 receptor antagonist Anakinra inhibited their expression and rescued susceptible Asc-/- mice from bladder pathology. An MMP inhibitor had a similar therapeutic effect. Finally, elevated levels of IL-1β and MMP-7 were detected in patients with acute cystitis, suggesting a potential role as biomarkers and immunotherapeutic targets. The results reproduce important aspects of human acute cystitis in the murine model and provide a comprehensive molecular framework for the pathogenesis and immunotherapy of acute cystitis, one of the most common infections in man. Trial Registration: The clinical studies were approved by the Human Ethics Committee at Lund University (approval numbers LU106-02, LU236-99 and Clinical Trial Registration RTP-A2003, International Committee of Medical Journal Editors, www.clinicaltrials.gov)