Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0348321 (Haemophilus)
 15,372 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ATP-dependent DNase from Hemophilus influenzae digests double-stranded linear DNA molecules exonucleolytically while hydrolyzing large amounts of ATP to ADP. Various cross-linked linear duplex DNA molecules are partially resistant to the exonuclease action. Vaccinia DNA, containing natural terminal cross-links (probably in the form of terminal single-stranded loops), is much more slowly degraded than comparable "open-ended" DNA molecules, and ATP is consumed at a proportionately lower rate. It is postulated that the vaccinia DNA molecules undergo slow terminal cleavage by the single strand specific endonuclease activity of the enzyme, and are then rapidly degraded by the double strand exonuclease activity. Phage T7 DNA, containing an average of 100 4',5'8-trimethylpsoralen cross-links/molecule at random internal sites, is digested only to the extent of 2 to 3%. However, ATP hydrolysis continues at a linear rate long after DNA digestion has ceased. A stable enzyme-DNA complex is formed as demonstrated by co-sedimentation of DNA and ATPase activity in sucrose gradients. The hypothesis is advanced that the enzyme digests exonucleolytically to the first cross-link at each end of the DNA molecules where further movement is prevented. The enzyme then remains bound at the cross-links and functions continuously as an ATPase.
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PMID:Action of ATP-dependent DNase from Hemophilus influenzae on cross-linked DNA molecules. 13 99

A nucleotide pyrophosphatase isolated from Haemophilus influenzae was purified to electrophoretic homogeneity and characterized with respect to molecular weight, substrate specificity, pH profile, thermal stability, functional group involvement, and effectiveness of selective inhibition. The enzyme catalyzes the hydrolysis of NAD to NMN and AMP and appears located appropriately to facilitate the internalization of NAD needed to satisfy the V-factor growth requirement of the organism. In the processing of NAD and structurally related substrates, the enzyme exhibited negative cooperativity. Structural alterations in the purine moiety of these dinucleotide substrates had pronounced effects on the negative cooperativity of the enzyme. AMP, ADP, and several related nucleotides were observed to be effective substrate-competitive inhibitors of the enzyme. Several of the dinucleotides serving as substrates for the nucleotide pyrophosphatase were evaluated with respect to substituting for NAD in supporting growth of the organism. AMP and ADP inhibited growth of the organism when NAD served as V-factor, and this inhibition correlated well with the inhibitory effects of these nucleotides on the purified nucleotide pyrophosphatase.
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PMID:Characterization of Haemophilus influenzae nucleotide pyrophosphatase. An enzyme of critical importance for growth of the organism. 300 42

The lipooligosaccharide (LOS) of nontypeable Haemophilus influenzae (NTHi) is an important factor in pathogenesis and virulence. In an attempt to elucidate the genes involved in LOS biosynthesis, we have cloned the rfaE gene from NTHi 2019 by complementing a Salmonella typhimurium rfaE mutant strain with an NTHi 2019 plasmid library. The rfaE mutant synthesizes lipopolysaccharide (LPS) lacking heptose, and the rfaE gene is postulated to be involved in ADP-heptose synthesis. Retransformation with the plasmid containing 4 kb of NTHi DNA isolated from a reconstituted mutant into rfaE mutants gave wild-type LPS phenotypes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis confirmed the conversion of the rfaE mutant LPS to a wild-type LPS phenotype. Sequence analysis of a 2.4-kb BglII fragment revealed two open reading frames. One open reading frame encodes the RfaE protein with a molecular weight of 37.6 kDa, which was confirmed by in vitro transcription and translation, and the other encodes a polypeptide highly homologous to the Escherichia coli HtrB protein. These two genes are transcribed from the same promoter region into opposite directions. Primer extension analysis of the rfaE gene revealed a single transcription start site at 37 bp upstream of the predicted translation start site. The upstream promoter region contained a sequence (TA AAAT) homologous to the -10 region of the bacterial sigma 70-dependent promoters at an appropriate distance (7 bp), but not sequence resembling the consensus sequence of the -35 region was found. These studies demonstrate the ability to use complementation of defined LPS defects in members of the family Enterobacteriaceae to identify LOS synthesis genes in NTHi.
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PMID:Molecular cloning and characterization of the nontypeable Haemophilus influenzae 2019 rfaE gene required for lipopolysaccharide biosynthesis. 786 52

The genomic organization of the chromosomal cps region that is responsible for capsular polysaccharide synthesis in Klebsiella pneumoniae Chedid (O1:K2) was investigated. Deletion analyses and Southern hybridization studies suggested that the central region of the cloned 29-kb BamHI fragment is indispensable for K2 capsular polysaccharide synthesis. The 24,329-bp nucleotide sequence of the Klebsiella cps region was determined and deposited in the EMBL and GenBank databases through DDBJ and assigned accession number D21242. Nineteen possible open reading frames (ORFs) were identified in the sequenced area. Among them, 13 ORFs are very close to each other. Six of the 19 ORFs show considerable nucleotide sequence similarities to Salmonella typhimurium cpsG, cpsB, rfbP, and orf2.8, Escherichia coli gnd, and Haemophilus influenzae bexD, respectively. Moreover, the deduced amino acid sequence of the ORF10 product demonstrated a highly hydrophobic profile and showed putative membrane topology similarity to Rickettsia prowazekii ATP/ADP translocase. Nucleotide sequence similar to the sigma 54-dependent promoter, as well as the usual -35 and -10 sequences, were identified just upstream of ORF3, which is the first ORF in the polycistronic structure. Furthermore, a sequence (GGGCGGTAGCGT) found just downstream of the sigma 54-dependent promoter-like sequence was generally conserved among gene clusters implicated in cell surface polysaccharide synthesis, such as Salmonella rfb and viaB and E. coli kpsMT and rfaQPG. A possible transcriptional terminator with a hairpin loop structure found just downstream of ORF15 that is a homolog of E. coli gnd. K2 capsular polsaccharide biosynthesis in E. coli K-12 depends on cpsB (mannose-1-phosphate guanyltransferase gene), and Klebsiella cpsB, found in the downstream region of the polycistronic structure, was able to complement cpsB of E. coli. Results of transposon insertion and promoter-cloning analyses were consistent with the results of nucleotide sequence analysis.
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PMID:Genomic organization of the Klebsiella pneumoniae cps region responsible for serotype K2 capsular polysaccharide synthesis in the virulent strain Chedid. 789 2

We have determined that gene HI#1181 of Haemophilus influenzae is a homolog of Escherichia coli gmhA (previously designated lpcA) (J. S. Brooke and M. A. Valvano, J. Biol. Chem. 271:3608-3614, 1996), which encodes a phosphoheptose isomerase catalyzing the first step of the biosynthesis of ADP-L-glycero-D-manno heptose. Mutations in this gene are associated with a heptoseless core lipopolysaccharide which determines an increased outer membrane permeability to hydrophobic compounds. The cloned H. influenzae gmhA restored the synthesis of a complete core in the gmhA-deleted E. coli strain chi711. Amino acid sequence comparisons of the GmhA proteins of E. coli and H. influenzae with other proteins in the databases revealed the existence of a novel family of phosphosugar a1do-keto isomerases.
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PMID:Molecular cloning of the Haemophilus influenzae gmhA (lpcA) gene encoding a phosphoheptose isomerase required for lipooligosaccharide biosynthesis. 865 17

Haemophilus influenzae is an important human pathogen. The lipooligosaccharide (LOS) of H. influenzae has been implicated as a virulence determinant. To better understand the assembly of LOS in nontypeable H. influenzae (NtHi), we have cloned and characterized the rfaD and rfaF genes of NtHi 2019, which encode the ADP-L-glycero-D-manno-heptose-6-epimerase and heptosyltransferase II enzymes, respectively. This cloning was accomplished by the complementation of Salmonella typhimurium lipopolysaccharide (LPS) biosynthesis gene mutants. These deep rough mutants are novobiocin susceptible until complemented with the appropriate gene. In this manner, we are able to use novobiocin resistance to select for specific NtHi LOS inner core biosynthesis genes. Such a screening system yielded a plasmid with a 4.8-kb insert. This plasmid was able to complement both rfaD and rfaF mutants of S. typhimurium. The LPS of these complemented strains appeared identical to the wild-type Salmonella LPS. The genes encoding the rfaD and rfaF genes from NtHi 2019 were sequenced and found to be similar to the analogous genes from S. typhimurium and Escherichia coli. The rfaD gene encodes a polypeptide of 35 kDa and the rfaF encodes a protein of 39 kDa, as demonstrated by in vitro transcription-translation studies. Isogenic mutants which demonstrated truncated LOS consistent with inner core biosynthesis mutants were constructed in the NtHi strain 2019. Primer extension analysis demonstrated the presence of a strong promoter upstream of rfaD but suggested only a very weak promoter upstream of rfaF. Complementation studies, however, suggest that the rfaF gene does have an independent promoter. Mass spectrometric analysis shows that the LOS molecules expressed by H. influenzae rfaD and rfaF mutant strains have identical molecular masses. Additional studies verified that in the rfaD mutant strain, D-glycero-D-manno-heptose is added to the LOS molecule in place of the usual L-glycero-D-manno-heptose. Finally, the genetic organizations of the inner core biosynthesis genes of S. typhimurium, E. coli, and several strains of H. influenzae were examined, and substantial differences were uncovered.
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PMID:Identification of the ADP-L-glycero-D-manno-heptose-6-epimerase (rfaD) and heptosyltransferase II (rfaF) biosynthesis genes from nontypeable Haemophilus influenzae 2019. 911 77

The periplasmic nucleotide pyrophosphatase from Haemophilus parasuis was purified 750-fold to electrophoretic homogeneity through salt fractionation and ion-exchange and affinity chromatography. The purified enzyme was monomeric with an apparent M(r) of 70,000 and catalyzed the hydrolysis of the pyrophosphate bond of NAD to yield NMN and AMP as products. The enzyme exhibited negative cooperativity in the hydrolysis of a number of pyridine dinucleotides and structurally-related pyrophosphate compounds as indicated by biphasic double-reciprocal plots and Hill coefficients of 0.5. The kinetic parameters, K(m) and Vm, determined titrimetrically and analyzed through computer programs, were used to compare the relative effectiveness of dinucleotides containing nitrogen bases other than nicotinamide or adenine to that of NAD. Effective substrate-competitive inhibition of the pyrophosphatase was observed with purine and pyrimidine nucleoside diphosphates in the low micromolar concentration range. Although less effective, N1-alkylnicotinamide chlorides also inhibited competitively with respect to the substrate, NAD. In addition to being an effective inhibitor of the purified enzyme, adenosine diphosphate also inhibited growth of H. parasuis at a low micromolar concentration. This inhibition of growth correlates well with inhibition of the periplasmic pyrophosphatase which is supported by the fact that adenosine diphosphate does not effectively inhibit growth when the pyrophosphatase is by-passed by growth on nicotinamide mononucleotide. These observations are all consistent with the periplasmic nucleotide pyrophosphatase being essential for the growth of the organism on NAD and therefore, a very important enzyme with respect to the pathogenesis of the organism. 3-Aminopyridine mononucleotide, which also inhibited growth of H. parasuis at a low micromolar concentration, did not effectively inhibit the purified pyrophosphatase and a different target enzyme needs to be considered to explain growth inhibition by this derivative.
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PMID:Characterization of H. parasuis periplasmic nucleotide pyrophosphatase as a potential target enzyme for inhibition of growth. 945 36

The intermediate steps in the biosynthesis of the ADP-L-glycero-D-manno-heptose precursor of inner core lipopolysaccharide (LPS) are not yet elucidated. We isolated a mini-Tn10 insertion that confers a heptoseless LPS phenotype in the chromosome of Escherichia coli K-12. The mutation was in a gene homologous to the previously reported rfaE gene from Haemophilus influenzae. The E. coli rfaE gene was cloned into an expression vector, and an in vitro transcription-translation experiment revealed a polypeptide of approximately 55 kDa in mass. Comparisons of the predicted amino acid sequence with other proteins in the database showed the presence of two clearly separate domains. Domain I (amino acids 1 to 318) shared structural features with members of the ribokinase family, while Domain II (amino acids 344 to 477) had conserved features of the cytidylyltransferase superfamily that includes the aut gene product of Ralstonia eutrophus. Each domain was expressed individually, demonstrating that only Domain I could complement the rfaE::Tn10 mutation in E. coli, as well as the rfaE543 mutation of Salmonella enterica SL1102. DNA sequencing of the rfaE543 gene revealed that Domain I had one amino acid substitution and a 12-bp in-frame deletion resulting in the loss of four amino acids, while Domain II remained intact. We also demonstrated that the aut::Tn5 mutation in R. eutrophus is associated with heptoseless LPS, and this phenotype was restored following the introduction of a plasmid expressing the E. coli Domain II. Thus, both domains of rfaE are functionally different and genetically separable confirming that the encoded protein is bifunctional. We propose that Domain I is involved in the synthesis of D-glycero-D-manno-heptose 1-phosphate, whereas Domain II catalyzes the ADP transfer to form ADP-D-glycero-D-manno-heptose.
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PMID:The rfaE gene from Escherichia coli encodes a bifunctional protein involved in biosynthesis of the lipopolysaccharide core precursor ADP-L-glycero-D-manno-heptose. 1062 97

A hypothetical protein encoded by the gene YjeE of Haemophilus influenzae was selected as part of a structural genomics project for X-ray analysis to assist with the functional assignment. The protein is considered essential to bacteria because the gene is present in virtually all bacterial genomes but not in those of archaea or eukaryotes. The amino acid sequence shows no homology to other proteins except for the presence of the Walker A motif G-X-X-X-X-G-K-T that indicates the possibility of a nucleotide-binding protein. The YjeE protein was cloned, expressed, and the crystal structure determined by the MAD method at 1.7-A resolution. The protein has a nucleotide-binding fold with a four-stranded parallel beta-sheet flanked by antiparallel beta-strands on each side. The topology of the beta-sheet is unique among P-loop proteins and has features of different families of enzymes. Crystallization of YjeE in the presence of ATP and Mg2+ resulted in the structure with ADP bound in the P-loop. The ATPase activity of YjeE was confirmed by kinetic measurements. The distribution of conserved residues suggests that the protein may work as a "molecular switch" triggered by ATP hydrolysis. The phylogenetic pattern of YjeE suggests its involvement in cell wall biosynthesis.
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PMID:Crystal structure of the YjeE protein from Haemophilus influenzae: a putative Atpase involved in cell wall synthesis. 1211 91

The rfaE (WaaE) gene of Salmonella typhimurium is known to be located at 76min on the genetic map outside of the rfa gene cluster encoding core oligosaccharide biosynthesis of lipopolysaccharide(LPS). The rfaE mutant synthesizes heptose-deficient LPS; its LPS consists of only lipid A and 3-deoxy-D-manno-octulosonic acid (KDO), and the rfaE gene is believed to be involved in the formation of ADP-L-glycero-D-manno-heptose. Mutants, which make incomplete LPS, are known as rough mutants. Salmonella typhimurium deep-rough mutants affected in the heptose region of the inner core often show reduced growth rate, sensitivity to high temperature and hypersensitivity to hydrophobic antibiotics. We have cloned the rfaE gene of S. typhimurium. The chromosomal region carrying this gene was isolated by screening a genomic library of S. typhimurium using the complementation of S. typhimurium rfaE mutant. The 2.6-Kb insert in the plasmid pHEPs appears to carry a functional rfaE gene. SL1102 (rfaE543) makes heptose-deficient LPS and has a deep rough phenotype, but pHEPs complement the rfaE543 mutation to give the smooth phenotype. The sensitivity of SL1102 to bacteriophages (P22.c2, Felix-O, Br60) which use LPS as their receptor for adsorption is changed to that of wild-type strain. The permeability barrier of SL1102 to hydrophobic antibiotics (novobiocin) is restored to that of wild-type. LPS produced by SL1102 (rfaE543) carrying pHEPs makes LPS indistinguishable from that of smooth strains. The rfaE gene encoded a polypeptide of 477 amino acid residues highly homologous to the S. enterica rfaE protein (98% identity), E. coli (93% identity), Yersenia pestis (85% identity), Haemophilus influenzae (70% identity) and Helicobacter pyroli (41% identity) with a molecular weight 53 kDa.
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PMID:Molecular cloning and functional expression of the rfaE gene required for lipopolysaccharide biosynthesis in Salmonella typhimurium. 1244 67


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