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Query: UNIPROT:O75191 (H. influenzae)
 4,961 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have developed a new micro-broth-dilution assay for determining the antimicrobial susceptibility of Haemophilus influenzae. This assay is based on the ability of viable H. influenzae to reduce nitrates to nitrites. Bacterial viability is detected by a positive nitrite reaction rather than visible turbidity. The nitrate reduction assay was compared with a standard microassay using 51 isolates of H. influenzae and six beta-lactam antibiotics. Although there was good agreement between the two methods, the nitrate reduction assay was more sensitive in detecting viable bacteria, and so established a more accurate estimate of the minimal inhibitory concentration. The nitrate reduction assay offered the additional advantage that it could be used to determine the minimal bactericidal concentration without having to subculture the broth. Ampicillin, penicillin, and cefamandole were equally effective in vitro against susceptible strains (minimal inhibitory concentrations, 0.125 to 0.5 mug/ml), whereas all three antibiotics were ineffective against two beta-lactamase-producing strains. Using the nitrate reduction assay, resistance to cefamandole was detectable with inoculum sizes ranging from 10(4) to 10(6) colony-forming units per ml, while the turbidity assay detected resistance only with the largest inoculum.
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PMID:Nitrate reduction: new method for testing the antibiotic susceptibility of Haemophilus influenzae. 30 65

The ability of Haemophilus influenzae, H. parainfluenzae and H. paraphrophilus to utilize iron complexes, iron-proteins and exogenous microbial siderophores was evaluated. In a plate bioassay, all three species used not only ferric nitrate but also the iron chelates ferric citrate, ferric nitrilotriacetate and ferric 2,3-dihydroxybenzoate. Each Haemophilus species examined also used haemin, haemoglobin and haem-albumin as iron sources although only H. influenzae could acquire iron from transferrin or from haemoglobin complexed with haptoglobin. None of the haemophili obtained iron from ferritin or lactoferrin or from the microbial siderophores aerobactin or desferrioxamine B. However, the phenolate siderophore enterobactin supplied iron to both H. parainfluenzae and H. paraphrophilus, and DNA isolated from both organisms hybridized with a DNA probe prepared from the Escherichia coli ferric enterobactin receptor gene fepA. In addition, a monospecific polyclonal antiserum raised against the E. coli 81 kDa ferric enterobactin receptor (FepA) recognized an iron-repressible outer membrane protein (OMP) in H. parainfluenzae of between 80 and 82 kDa (depending on the strain). This anti-FepA serum did not cross-react with any of the OMPs of H. paraphrophilus or H. influenzae. The OMPs of each Haemophilus species were also probed with antisera raised against the 74 kDa Cir or 74 kDa IutA (aerobactin receptor) proteins of E. coli. Apart from one H. parainfluenzae strain (NCTC 10665), in which an OMP of about 80 kDa cross-reacted with the anti-IutA sera, no cross-reactivity was observed between Cir, IutA and the OMPs of H. influenzae, H. parainfluenzae or H. paraphrophilus.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Utilization of enterobactin and other exogenous iron sources by Haemophilus influenzae, H. parainfluenzae and H. paraphrophilus. 215 Apr 14

During growth in high concentrations of iron nitrate, H. influenzae produces compounds reactive in biochemical assays for hydroxamates. Mixing experiments established that nitrate was responsible for inducing these compounds. Analysis by 1H and 13C NMR and high resolution mass spectrometry identified the active species as 2,2-bis(3'-indolyl)indoxyl. Bacterial production of the latter compound has been previously observed only in Pseudomonas aureofaciens. A mutant defective in the production of 2,2-bis(3'-indolyl)indoxyl was constructed by marker insertion. The formation of indole and 2,2-bis (3'-indolyl)indoxyl was quantitated by reverse-phase high pressure liquid chromatography during growth in high concentrations of nitrate. The mutant produced high concentrations of indole, but only minimal amounts of 2,2-bis(3'-indolyl)indoxyl, and also proved to be defective in nitrate reduction. These data suggest that indole may function as an electron donor for nitrate reductase in H. influenzae.
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PMID:Production and oxidation of indole by Haemophilus influenzae. 781 18

The absolute requirement for elemental iron and the porphyrin nucleus for growth of Haemophilus influenzae led us to investigate the role of iron and hemin in regulation of expression of the H. influenzae transferrin receptor. H. influenzae type b strain H1689 was grown in brain heart infusion broth supplemented with beta-NAD and either 10 or 0.1 microgram of hemin ml-1. Transferrin-binding ability was determined with a dot blot assay using human transferrin-horseradish peroxidase conjugate. Cells grown in media with 0.1 microgram of hemin ml-1 bound transferrin, but organisms grown in media with 10 micrograms ml-1 did not. In hemin-restricted media, transferrin binding occurred despite addition of up to 10 mM ferric nitrate, ferric citrate, or ferric PPi, whereas addition of 10 micrograms of hemoglobin ml-1 repressed expression. The breadth of species distribution of this mode of regulation was determined with strains previously characterized by multilocus enzyme electrophoresis. When grown in hemin-restricted media, 24 of 28 type b strains and 52 of 57 serologically nontypeable strains exhibited transferrin binding, although none did so in hemin- and iron-sufficient media. Strain H1689 and serologically nontypeable strain HI1423 grown in heat-inactivated pooled normal human serum, human cerebrospinal fluid, or human breast milk exhibited transferrin binding. Growth in these fluids with 10 micrograms of added hemin ml-1 abolished transferrin binding, whereas addition of 10 mM ferric nitrate did not. These data suggest that the transferrin receptor of H. influenzae is regulated by levels of hemin but not elemental iron alone and that this property is widely distributed among several major cloned families in the species.
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PMID:Expression of the Haemophilus influenzae transferrin receptor is repressible by hemin but not elemental iron alone. 840 90

Identifying the complete transcriptional regulatory network for an organism is a major challenge. For each regulatory protein, we want to know all the genes it regulates, that is, its regulon. Examples of known binding sites can be used to estimate the binding specificity of the protein and to predict other binding sites. However, binding site predictions can be unreliable because determining the true specificity of the protein is difficult because of the considerable variability of binding sites. Because regulatory systems tend to be conserved through evolution, we can use comparisons between species to increase the reliability of binding site predictions. In this article, an approach is presented to evaluate the computational predictions of regulatory sites. We combine the prediction of transcription units having orthologous genes with the prediction of transcription factor binding sites based on probabilistic models. We augment the sets of genes in Escherichia coli that are expected to be regulated by two transcription factors, the cAMP receptor protein and the fumarate and nitrate reduction regulatory protein, through a comparison with the Haemophilus influenzae genome. At the same time, we learned more about the regulatory networks of H. influenzae, a species with much less experimental knowledge than E. coli. By studying orthologous genes subject to regulation by the same transcription factor, we also gained understanding of the evolution of the entire regulatory systems.
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PMID:A comparative genomics approach to prediction of new members of regulons. 1128 72

The napB gene of the pathogenic bacterium Haemophilus influenzae encodes a dihaem cytochrome c, the small subunit of a heterodimeric periplasmic nitrate reductase similar to those found in other bacteria. In order to obtain sufficient protein for biophysical studies, we aimed to overproduce the recombinant dihaem protein in Escherichia coli. Initial expression experiments indicated that the NapB signal peptide was not cleaved by the leader peptidase of the host organism. Apocytochrome was formed under aerobic, semi-aerobic and anaerobic growth conditions in either Luria--Bertani or minimal salts medium. The highest amounts of apo-NapB were produced in the latter medium, and the bulk was inserted into the cytoplasmic membrane. The two haem groups were covalently attached to the pre-apocytochrome only under anaerobic growth conditions, and with 2.5 mM nitrite or at least 10 mM nitrate supplemented to the minimal salts growth medium. In order to obtain holocytochrome, the gene sequence encoding mature NapB was cloned in-frame with the E. coli ompA (outer membrane protein A) signal sequence. Under anaerobic conditions, NapB was secreted into the periplasmic space, with the OmpA signal peptide being correctly processed and with both haem c groups attached covalently. Unless expressed in the DegP-protease-deficient strain HM125, some of the recombinant NapB polypeptides were N-terminally truncated as a result of proteolytic activity. Under aerobic growth conditions, co-expression with the E. coli ccm (cytochrome c maturation) genes resulted in a higher yield of holocytochrome c. The pure recombinant NapB protein showed absorption maxima at 419, 522 and 550 nm in the reduced form. The midpoint reduction potentials of the two haem groups were determined to be -25 mV and -175 mV. These results support our hypothesis that the Nap system fulfils a nitrate-scavenging role in H. influenzae.
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PMID:Overproduction, purification and novel redox properties of the dihaem cytochrome c, NapB, from Haemophilus influenzae. 1138 94

Periplasmic nitrate reductase (napFDAGHBC operon product) functions in anaerobic respiration. Transcription initiation from the Escherichia coli napF operon control region is activated by the Fnr protein in response to anaerobiosis and by the NarQ-NarP two-component regulatory system in response to nitrate or nitrite. The binding sites for the Fnr and phospho-NarP proteins are centered at positions -64.5 and -44.5, respectively, with respect to the major transcription initiation point. The E. coli napF operon is a rare example of a class I Fnr-activated transcriptional control region, in which the Fnr protein binding site is located upstream of position -60. To broaden our understanding of napF operon transcriptional control, we studied the Haemophilus influenzae Rd napF operon control region, expressed as a napF-lacZ operon fusion in the surrogate host E. coli. Mutational analysis demonstrated that expression required binding sites for the Fnr and phospho-NarP proteins centered at positions -81.5 and -42.5, respectively. Transcription from the E. coli napF operon control region is activated by phospho-NarP but antagonized by the orthologous protein, phospho-NarL. By contrast, expression from the H. influenzae napF-lacZ operon fusion in E. coli was stimulated equally well by nitrate in both narP and narL null mutants, indicating that phospho-NarL and -NarP are equally effective regulators of this promoter. Overall, the H. influenzae napF operon control region provides a relatively simple model for studying synergistic transcription by the Fnr and phospho-NarP proteins acting from class I and class II locations, respectively.
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PMID:Fnr-, NarP- and NarL-dependent regulation of transcription initiation from the Haemophilus influenzae Rd napF (periplasmic nitrate reductase) promoter in Escherichia coli K-12. 1619 62

The metabolic activity of H. influenzae can be followed quantitatively by measurement of the nitrite produced in a medium containing 0.2 per cent potassium or sodium nitrate. When X-factor, or hemin, and other specific substances required for the optimum growth of H. influenzae, are present in excess, the nitrite produced by this organism is quantitatively related to the concentration of V-factor, or total coenzyme. This quantitative relationship has been demonstrated for five strains of H. influenzae. It has been shown that various media, which in the past have been used for the determination of coenzyme by growth of H. influenzae, have in many instances been deficient in X-factor and that this substance rather than coenzyme has been the specific factor limiting growth. When 0.5 per cent blood is added to a basal proteose-peptone medium the specific requirements for optimum growth and metabolic activity of H. influenzae, other than coenzyme, are met, and a large number of specific biocatalysts and nutritive substances added to this medium are without effect in stimulating further growth. The foregoing studies have formed the basis for a quantitative method for the determination of total coenzyme in blood and tissue. This method is being described elsewhere.
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PMID:STUDIES ON THE NUTRITION OF HEMOPHILUS INFLUENZAE : I. THE RELATIONSHIP BETWEEN THE UTILIZATION OF COENZYME AND HEMIN AND THE REDUCTION OF NITRATE. 1987 Dec 32

1. Iron protoporphyrin IX was required for the growth of H. influenzae. It could be replaced by protoporphyrin IX. When grown on protoporphyrin evidence was obtained for the presence of Fe porphyrin in the organism. It was concluded that the organism could insert iron into the protoporphyrin ring. 2. In the smooth strains, other porphyrins containing no iron such as deutero-, hemato-, meso-, and coproporphyrins could not replace protoporphyrin for growth. Since protoporphyrin has two vinyl groups which other porphyrins lack, it was concluded that the two vinyl groups were essential for growth. 3. When porphyrins lacking vinyl groups were converted chemically into iron porphyrins and then supplied to the organisms it was found that these iron porphyrins supported growth. It was concluded that the "smooth" organisms were able to insert iron only into the porphyrin containing the vinyl groups; i.e., protoporphyrin. One function of the vinyl groups then was to permit iron to be inserted biologically into the porphyrin ring. 4. An anomalous behavior in the rough Turner strain was observed and discussed. This organism was able to insert iron into mesoporphyrin at low concentrations but was inhibited by this compound at higher concentrations. In all other reactions with the porphyrins this rough strain behaved in the same was as did the smooth strains. 5. All strains which were grown on iron porphyrins lacking vinyl groups could not reduce nitrate to nitrite. When grown on protoporphyrin or Fe protoporphyrin reduction of nitrate occurred. It was concluded that the nitrate-reducing mechanism required the presence of the vinyl groups either for its formation or function. 6. The porphyrins lacking iron and lacking vinyl groups inhibited the growth of H. influenzae on Fe protoporphyrin. The inhibition between a porphyrin and Fe protoporphyrin was a competitive one. It was suggested that the porphyrin inhibited the growth-promoting properties of Fe protoporphyrin by attaching on to a particular apoprotein, thus preventing the formation of a heme catalyst. Likewise, competition between two growth-promoting Fe porphyrins for apoenzymes could be shown to occur. 7. Protoporphyrin and Fe protoporphyrin supported growth. When their propionic acid side chains were esterified they no longer supported growth. It was suggested that the esterified carboxyl groups could not attach to the specific apoproteins to form the heme enzymes and so could not act to support growth. For the same reason the inhibitory action of porphyrins lacking vinyl groups could be prevented by esterifying their propionic acid groups.
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PMID:THE PORPHYRIN REQUIREMENTS OF HAEMOPHILUS INFLUENZAE AND SOME FUNCTIONS OF THE VINYL AND PROPIONIC ACID SIDE CHAINS OF HEME. 1987 73

Haemophilus influenzae is a host adapted human pathogen known to contribute to a variety of acute and chronic diseases of the upper and lower respiratory tract as well as the middle ear. At the sites of infection as well as during growth as a commensal the environmental conditions encountered by H. influenzae will vary significantly, especially in terms of oxygen availability, however, the mechanisms by which the bacteria can adapt their metabolism to cope with such changes have not been studied in detail. Using targeted metabolomics the spectrum of metabolites produced during growth of H. influenzae on glucose in RPMI-based medium was found to change from acetate as the main product during aerobic growth to formate as the major product during anaerobic growth. This change in end-product is likely caused by a switch in the major route of pyruvate degradation. Neither lactate nor succinate or fumarate were major products of H. influenzae growth under any condition studied. Gene expression studies and enzyme activity data revealed that despite an identical genetic makeup and very similar metabolite production profiles, H. influenzae strain Rd appeared to favor glucose degradation via the pentose phosphate pathway, while strain 2019, a clinical isolate, showed higher expression of enzymes involved in glycolysis. Components of the respiratory chain were most highly expressed during microaerophilic and anaerobic growth in both strains, but again clear differences existed in the expression of genes associated e.g., with NADH oxidation, nitrate and nitrite reduction in the two strains studied. Together our results indicate that H. influenzae uses a specialized type of metabolism that could be termed "respiration assisted fermentation" where the respiratory chain likely serves to alleviate redox imbalances caused by incomplete glucose oxidation, and at the same time provides a means of converting a variety of compounds including nitrite and nitrate that arise as part of the host defence mechanisms.
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PMID:Metabolic versatility in Haemophilus influenzae: a metabolomic and genomic analysis. 2462 22


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