Purification and characterization of a chimeric enzyme from Haemophilus influenzae Rd that exhibits glutathione-dependent peroxidase activity

While belonging to the same family of antioxidant enzymes, members of the peroxiredoxins do not necessarily employ one and the same method for their reduction. Most representatives become reduced with the aid of thioredoxin, whereas some members use AhpF, tryparedoxin, or cyclophilin A. Recent research on a new peroxiredoxin isoform (type C) from Populus trichocarpa has shown that these particular types may also use glutaredoxin instead of thioredoxin. This finding is supported by the occurrence of chimeric proteins composed of a peroxiredoxin and glutaredoxin region. A gene encoding such a fusion protein is enclosed in the Haemophilus influenzae Rd genome. We expressed the H. influenzae protein, denoted here as PGdx, in Escherichia coli and purified the recombinant enzyme. In vitro assays demonstrate that PGdx, in the presence of dithiothreitol or glutathione, is able to protect supercoiled DNA against the metal ion-catalyzed oxidation-system. Enzymatic assays did, indeed, characterize PGdx as a peroxidase, requiring the glutathione redox cycle for the reduction of hydrogen peroxide (k(cat)/K(m) 5.01 x 10(6) s(-1) m(-1)) as well as the small organic hydroperoxide tert-butylhydroperoxide (k(cat)/K(m) 5.67 x 10(4) s(-1) m(-1)). Enzymatic activity as function of the glutathione concentration deviated from normal Michaelis-Menten kinetics, giving a sigmoidal pattern with an apparent Hill coefficient of 2.9. Besides the formation of a disulfide-linked PGdx dimer, it was also shown by mass spectrometric analysis that cysteine 49, which is equivalent to the active site cysteine of the peroxiredoxins, undergoes glutathionylation during purification under nonreducing conditions. Based on these results, we propose a model for the catalytic mechanism.     

The genome of Haemophilus influenzae Rd has a unique NotI site

Previous analysis of physical maps of Haemophilus influenzae, which is circular and 1.9 Mb in length [Lee and Smith, J. Bacteriol. 170 (1988) 4402-4405; Kauc et al., J. Bacteriol. 171 (1989) 2474-2479], did not detect any NotI (GCGGCCGC) restriction sites. A transposon, Tn916, was constructed to contain a NotI linker cloned into its NciI site and introduced into the H. influenzae chromosome. NotI digestion of chromosomes containing a Tn916-associated NotI site followed by separation of fragments by field-inversion gel electrophoresis revealed the presence of two fragments obtained by two NotI cuts, one in Tn916 and the other, a unique, 'natural' NotI site in the original chromosomal DNA. The examination of other Haemophilus strains demonstrated the presence of one or more NotI sites in all of those tested.     

Structural characterization of lipid A from nontypeable and type f Haemophilus influenzae: variability of fatty acid substitution

Lipid A isolated by mild acid hydrolysis from lipopolysaccharides of 22 nontypeable and 2 type f Haemophilus influenzae strains was investigated using electrospray ionization coupled to quadrupole ion trap mass spectrometry. The lengths, positions, and number of acyl chains in the lipid A molecule were determined using multiple-step tandem mass spectrometry (MSn). All of the analyzed strains showed a major lipid A molecule comprising beta-2-amino-2-deoxy-D-glucopyranose-(1-->6)-alpha-2-amino-2-deoxy-D-glucopyranose phosphorylated at the C4' and C1 positions. The C2/C2' and C3/C3' positions were substituted by amide-linked and ester-linked 3-hydroxytetradecanoic acid chains, respectively. The fatty acid chains on C3' and C2' were further esterified by tetradecanoic acid chains. In all strains, minor amounts of lipid A molecules with different acylation patterns were identified. Thus, structures comprising the hexaacylated lipid A with the C2 or C3 position being substituted by 3-hydroxydecanoic acid, and hexaacylated lipid A with the C3 and C3' positions being substituted by 3-hydroxydodecanoic or dodecanoyloxytetradecanoic acid, respectively, were found. In addition, lipid A with an acetyl group attached to the 3-hydroxytetradecanoic acid groups attached to the C2 or C3 position was detected in two nontypeable H. influenzae strains.     

Alterations in erythrocyte membrane lipid and fatty acid composition in Chediak-Higashi syndrome

Chediak-Higashi syndrome (CHS) is an autosomal recessive disease characterized by the presence of abnormally large cytoplasmic organelles in all body granule producing cells. The molecular mechanism for this disease is still unknown. Functional disorders in membrane-related processes have been reported. Erythrocyte membranes from four CHS patients and 15 relatives including obligatory heterozygous were studied to examine potential alterations in the lipid and fatty acid profile of erythrocyte membranes associated with this syndrome. Plasma concentrations of cholesterol, triglycerides, phospholipids, and apolipoproteins AI and B100, and the lipid components of very low-, intermediate-, low- and high-density lipoproteins were also determined. CHS erythrocyte membranes were found to be enriched with lipids in relation to protein and to show: (1) an increase in cholesterol and choline-containing phospholipids (sphingomyelin and phosphatidylcholine) that predominate in the outer monolayer, which is higher than the increase in phosphatidylserine and phosphatidylethanolamine, that are chiefly limited to the inner monolayer in normal red blood cells; (2) a relative palmitic acid and saturated fatty acid increase and arachidonic acid and unsaturated fatty acid decrease, this resulting in a lower unsaturation index than controls. Changes in CHS erythrocyte membrane lipids seem to be unrelated to serum lipid disorders as plasma lipid and apolipoprotein concentrations were apparently in the normal range, with the exception of a modest hypertriglyceridemia in patients and relatives and a decreased concentration of HDL cholesterol in patients. These findings indicate that CHS erythrocyte membranes contain an abnormal lipid matrix with which membrane proteins are defectively associated. The anomalous CHS membrane composition can be explained on the postulated effects of the CHS1/Lyst gene.     

Deoxyribonucleic acid-binding properties and membrane protein composition of a competence-deficient mutant of Haemophilus influenzae.

A mutant of Haemophilus influenzae was isolated which was completely unable to take up double-stranded homologous deoxyribonucleic acid (DNA) at normal physiological conditions but which took up DNA equally as well as the wild type at low pH (pH 4.4). The properties of the mutant provide evidence for the existence of two different mechanisms for DNA entry in the H. influenzae transformation system. With the aid of the mutant the optimal conditions for entry of DNA by these two mechanisms were determined, and the dependence of entry and the specific transforming activity of the entered DNA on competence was examined. The mechanism of entry of DNA at neutral pH, which is not functioning in the mutant, effected entry of homologous DNA only, whereas the mechanism involved in entry of DNA at low pH also effected entry of heterologous DNA. This suggests that the mutant is lacking a protein which recognizes the specific base sequence(s) required for entry. Comparison of the protein composition of the membranes of mutant cells subjected to a growth regimen provoking competence in wild-type cells with that of competent wild-type cells revealed that the mutant is impaired in the synthesis of a protein with a molecular weight of 22,500.     

Regulation of membrane fatty acid composition by temperature in mutants of Arabidopsis with alterations in membrane lipid composition

Background  

A wide range of cellular responses occur when plants are exposed to elevated temperature, including adjustments in the unsaturation level of membrane fatty acids. Although membrane bound desaturase enzymes mediate these adjustments, it is unknown how they are regulated to achieve these specific membrane compositions. Furthermore, the precise roles that different membrane fatty acid compositions play in photosynthesis are only beginning to be understood. To explore the regulation of the membrane composition and photosynthetic function in response to temperature, we examined the effect of temperature in a collection of mutants with altered membrane lipid fatty acid composition.     

Assessment of the metabolic capabilities of Haemophilus influenzae Rd through a genome-scale pathway analysis

The annotated full DNA sequence is becoming available for a growing number of organisms. This information along with additional biochemical and strain-specific data can be used to define metabolic genotypes and reconstruct cellular metabolic networks. The first free-living organism for which the entire genomic sequence was established was Haemophilus influenzae. Its metabolic network is reconstructed herein and contains 461 reactions operating on 367 intracellular and 84 extracellular metabolites. With the metabolic reaction network established, it becomes necessary to determine its underlying pathway structure as defined by the set of extreme pathways. The H. influenzae metabolic network was subdivided into six subsystems and the extreme pathways determined for each subsystem based on stoichiometric, thermodynamic, and systems-specific constraints. Positive linear combinations of these pathways can be taken to determine the extreme pathways for the complete system. Since these pathways span the capabilities of the full system, they could be used to address a number of important physiological questions. First, they were used to reconcile and curate the sequence annotation by identifying reactions whose function was not supported in any of the extreme pathways. Second, they were used to predict gene products that should be co-regulated and perhaps co-expressed. Third, they were used to determine the composition of the minimal substrate requirements needed to support the production of 51 required metabolic products such as amino acids, nucleotides, phospholipids, etc. Fourth, sets of critical gene deletions from core metabolism were determined in the presence of the minimal substrate conditions and in more complete conditions reflecting the environmental niche of H. influenzae in the human host. In the former case, 11 genes were determined to be critical while six remained critical under the latter conditions. This study represents an important milestone in theoretical biology, namely the establishment of the first extreme pathway structure of a whole genome.     

Inhibition of transformation and transfection in Haemophilus influenzae Rd9 by lysogeny.

Haemophilus influenzae Rd9 lysogenic for temperate bacteriophage N3 was found to be virtually nontransformable and nontransfectable. This inhibition of transformation and transfection was due partly to the decreased capacity of competent lysogenic cells for irreversible binding of deoxyribonucleic acid (DNA) and partly to some events taking place after adsorption of the DNA. The unadsorbed DNA was not degraded by the competent lysogenic cells.     

Cloning and characterization of the Haemophilus influenzae Rd rec-1+ gene.

The Haemophilus influenzae Rd rec-1+ gene was cloned from a partial chromosomal digest into a plasmid vector as a 20-kilobase-pair (kbp) BstEII fragment and then subcloned. The smallest subclone with rec-1+ activity carried a 3.1-kbp EcoRI fragment. The identity of the rec-I gene in these clones was confirmed by transforming an Rd strain carrying a leaky rec-1 mutation (recA4) to resistance to methyl methanesulfonate (MMS) by using whole or digested plasmids. It was demonstrated that the Rec+ phenotype of the MMSr transformants was linked to the strA, novAB, and mmsA loci, as expected if the recA4 allele had been replaced by rec-1+. In growing cultures (rec-1 or rec+), all rec-1+-carrying plasmids induced near-maximal levels of transformability when their hosts reached stationary phase; these levels are 100 to 1,000 times higher than the values seen with strains not carrying a Rec plasmid. Transfer of the 3.1-kbp subclone was greatly reduced compared with transfer of similarly sized vector plasmids, and the resulting transformants grew slowly; this suggests an explanation of my failure to directly clone this fragment from chromosomal DNA digests. Transfer of a rec-1+ plasmid to a very poorly genetically transformable H. influenzae Rb strain resulted in greatly increased transformability. Transfer of such plasmids to a noncompetent H. influenzae Rc strain did not render this strain competent. It is suggested that transformability of Rd and Rb strains is limited by rec-1 expression but that the noncompetence of Rc has some other basis.     

Temperature-dependent changes in phospholipid and fatty acid composition and membrane lipid fluidity of Yersinia enterocolitica

Temperature-dependent compositional changes of phospholipids and their fatty acids were analysed in Yersinia enterocolitica grown at 5°, 25° and 37°C. The relative amounts of the four phospholipids, phosphatidylethanolamine (75–78%), phosphatidylglycerol (10–11%), cardiolipin (<7%) and lysophosphatidylethanolamine (<5%), were essentially the same at all growth temperatures. The degree of fatty acid unsaturation of the four phospholipids increased with decrease in growth temperature, mainly due to an increase of C_16:1 and C_18:1 and a corresponding decrease of C_16;0, C_18:0 and cyclo C_17:0. An electron spin resonance spectroscopic study of the membrane lipids showed that membrane lipid fluidity was enhanced by decreasing the growth temperatures. The changes in fatty acid composition of phospholipids in response to varied temperatures were consistent with the temperature-dependent changes in the membrane lipid fluidity of Y. enterocolitica , and were similar to those reported for other bacteria.     

Gene HI1472 of Haemophilus influenzae Rd is a novel gene involved in DNA repair

A chimeric plasmid, pJPuvr4, consists of a 16.7 kbp Haemophilus influenzae Rd chromosomal DNA insert at the EcoRI site of vector pJ1-8. This plasmid complements the UV and gamma ray sensitivity of the mutant strain MBH4. This plasmid carries the wild type allele of gene uvr4 which was localised to a 3.8 kbp DraI fragment, with an internal EcoRI site. Partial sequencing of the gene and its alignment with the published genome sequence of H. influenzae Rd revealed uvr4 to be HI1472. HI1472 is a putatively identified open reading frame (ORF), which has been assigned no function so far. The partial sequence did show nt database match with 3D exon of N cadherin gene of homosepians and moaA gene of H. influenzae. Cadherins are involved in cell adhesion, cell to cell contact and morphogenesis in homosepians and moaA gene codes for molybdenum biosynthesis subunitA. This report implicates HI1472 of Haemophilus influenzae Rd in DNA repair. Nucleotide sequence obtained for the gene uvr4 was compared with the published sequence of gene HI1472. A wild type strain variation was observed at the 592nd nucleotide position corresponding to a change from aspartic acid to threonine.     

Overexpression and biochemical characterization of beta-1,3-N-acetylgalactosaminyltransferase LgtD from Haemophilus influenzae strain Rd

The lipopolysaccharide of capsule deficient Haemophilus influenzae strain Rd contains an N-acetylgalactosamine residue attached to the terminal globotriose moiety in the Hex5 glycoform. Genome analysis identified an open reading frame HI1578, referred to as lgtD, whose amino acid sequence shows significant level of similarity to a number of bacterial glycosyltransferases involved in lipopolysaccharide biosynthesis. To investigate its function, overexpression and biochemical characterization were performed. Most of the protein was obtained in a highly soluble and active form. By using standard glycosyltransferase assay and HPLC, we show that LgtD is an N-acetylgalactosaminyltransferase with high donor substrate specificity and globotriose is a highly preferred acceptor substrate for the enzyme. The K_m for UDP-GalNAc and globotriose are 58 μM and 8.6 mM, respectively. The amino acid sequence of the enzyme shows the conserved features of family II glycosyltransferases. This is the first N-acetylgalactosaminyltransferase identified from H. influenzae, which shows potential application in large-scale synthesis of globo-series oligosaccharides.     

The fatty acid composition of Haemophilus and Pasteurella multocida cells as a phylogenetic marker

When grown on meat-peptone agar with heated blood, different Haemophilus species (H. influenzae, H. parahaemolyticus, H. parasuis, H. pleuropneumoniae), including different H. influenzae serovars (a, b, c, d, e, f), and Pasteurella multocida have identical fatty acid composition, characterized by the prevalence of fatty acids with 16 carbon atoms, constituting about 70% and more of the total number of fatty acids, and a low level of fatty acids with 18 carbon atoms. P. multocida strains cultivated on meat-peptone agar with unheated blood have a greatly increased content of fatty acids with 18 carbon atoms, while the content of fatty acids with 16 carbon atoms is much lower. The identity of fatty acid composition under similar cultivation conditions, together with their similarity in other phenetic signs, is indicative of close phylogenic relationship between bacteria belonging to the genus Haemophilus and P. multocida.     

Molecular cloning and characterization of the HmrM multidrug efflux pump from Haemophilus influenzae Rd

We cloned a gene responsible for norfloxacin resistance from the chromosomal DNA of Haemophilus influenzae Rd, and designated the gene as hmrM. HmrM showed sequence similarity with NorM of Vibrio parahaemolyticus and YdhE of Escherichia coli and others that belong to the MATE family multidrug efflux pumps. The recombinant plasmid carrying the hmrM gene conferred elevated resistance not only to norfloxacin but also to acriflavine, 4 ', 6-diamidino-2-phenylindole, doxorubicin, ethidium bromide, tetraphenylphosphonium chloride, Hoechst 33342, daunomycin, berberine, and sodium deoxycholate in Escherichia coli KAM32, a drug-hypersensitive strain. We observed an Na+-dependent efflux of ethidium and an ethidium-induced efflux of Na+ in E. coli KAM32 cells harboring the plasmid carrying the hmrM gene. These results indicate that HmrM is an Na+/drug antiporter-type multidrug efflux pump. A difference in substrate preference was observed between HmrM, NorM, and YdhE.     

Pore characteristics of nontypeable Haemophilus influenzae outer membrane protein P5 in planar lipid bilayers

The structure of outer membrane protein P5 of NTHi, a homolog of Escherichia coli OmpA, was investigated by observing its pore characteristics in planar lipid bilayers. Recombinant NTHi P5 was overexpressed in E. coli and purified using ionic detergent, LDS-P5, or nonionic detergent, OG-P5. LDS-P5 and OG-P5 could not be distinguished by their migration on SDS-PAGE gels; however, when incorporated into planar bilayers of DPhPC between symmetric aqueous solutions of 1 M KCl at 22 degrees C, LDS-P5 formed narrow pores (58 +/- 6 pS) with low open probability, whereas OG-P5 formed large pores (1.1 +/- 0.1 nS) with high open probability (0.99). LDS-P5 narrow pores were gradually and irreversibly transformed into large pores, indistinguishable from those formed by OG-P5, at temperatures >or=40 degrees C; the process took 4-6 h at 40 degrees C or 35-45 min at 42 degrees C. Large pores were stable to changes in temperatures; however, large pores were rapidly converted to narrow pores when exposed to LDS at room temperatures, indicating acute sensitivity of this conformer to ionic detergent. These studies suggest that narrow pores are partially denatured forms and support the premise that the native conformation of NTHi P5 is that of a large monomeric pore.     

Transposon mutagenesis, characterization, and cloning of transformation genes of Haemophilus influenzae Rd.

A plasmid library of PstI fragments of Haemophilus influenzae Rd genomic DNA was mutagenized in Escherichia coli with mini-Tn10kan. The mutagenized PstI fragments were introduced by transformation into the H. influenzae chromosome, and kanamycin-resistant transformants were screened for the transformation-deficient phenotype by a cyclic AMP-DNA plate method. Fifty-four mutant strains containing 24 unique insertions that mapped to 10 different PstI fragments were isolated. Strains carrying unique insertions were tested individually for DNA uptake, transformation efficiency, UV sensitivity, and growth rate. The transformation frequencies of these mutants were decreased by factors of 10(-2) to 10(-6). Five of the mutants had normal competence-induced DNA uptake, and the rest were variably deficient in competence development. Three strains were moderately UV sensitive. All strains but one had doubling times within 50% of that of the wild type. Mutated genes were cloned into an H. influenzae-E. coli shuttle vector, and wild-type loci were recovered by in vivo recombinational exchange. Hybridization of these clones to SmaI genomic fragments separated in pulsed-field gels showed that these insertions were not clustered in a particular region of the chromosome.