UMLS:C0348321 - FACTA Search

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Query: UMLS:C0348321 (Haemophilus)
15,372 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The crystal structure of HI0074 from Haemophilus influenzae, a protein of unknown function, has been determined at a resolution of 2.4 A. The molecules form an up-down, four-helix bundle, and associate into homodimers. The fold is most closely related to the substrate-binding domain of KNTase, yet the amino acid sequences of the two proteins exhibit no significant homology. Sequence analyses of completely and incompletely sequenced genomes reveal that the two adjacent genes, HI0074 and HI0073, and their close relatives comprise a new family of nucleotidyltransferases, with 15 members at the time of writing. The analyses also indicate that this is one of eight families of a large nucleotidyltransferase superfamily, whose members were identified based on the proximity of the nucleotide- and substrate-binding domains on the respective genomes. Both HI0073 and HI0074 were annotated "hypothetical" in the original genome sequencing publication. HI0073 was cloned, expressed, and purified, and was shown to form a complex with HI0074 by polyacrylamide gel electrophoresis under nondenaturing conditions, analytic size exclusion chromatography, and dynamic light scattering. Double- and single-stranded DNA binding assays showed no evidence of DNA binding to HI0074 or to HI0073/HI0074 complex despite the suggestive shape of the putative binding cleft formed by the HI0074 dimer.
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PMID:The HI0073/HI0074 protein pair from Haemophilus influenzae is a member of a new nucleotidyltransferase family: structure, sequence analyses, and solution studies. 1248 19

The gram-negative bacterium nontypeable Haemophilus influenzae (NTHI) is the predominant pathogen in chronic otitis media with effusion and, with Streptococcus pneumoniae and Moraxella catarrhalis, is a causative agent of acute otitis media. To identify potential virulence determinants, bacterial gene expression was monitored by differential fluorescence induction during early disease progression in one specific anatomical niche of a chinchilla model of NTHI-induced otitis media. Genomic DNA fragments from NTHI strain 86-028NP were cloned upstream of the promoterless gfpmut3 gene. NTHI strain 86-028NP served as the host for the promoter trap library. Pools of 2,000 transformants were inoculated into the left and right middle ear cavities of chinchillas. Middle ear effusions were recovered by epitympanic tap at 24 and 48 h, and clones containing promoter elements that were induced in vivo and producing green fluorescent protein were isolated by two-color fluorescence-activated cell sorting. Insert DNA was sequenced and compared to the complete genome sequence of H. influenzae strain Rd. In a screen of 16,000 clones, we have isolated 44 clones that contain unique gene fragments encoding biosynthetic enzymes, metabolic and regulatory proteins, and hypothetical proteins of unknown function. An additional eight clones contain gene fragments unique to our NTHI isolate. Using quantitative reverse transcription-PCR, we have confirmed that 26 clones demonstrated increased gene expression in vivo relative to expression in vitro. These data provide insight into the response of NTHI bacteria as they sense and respond to the middle ear microenvironment during early events of otitis media.
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PMID:Nontypeable Haemophilus influenzae gene expression induced in vivo in a chinchilla model of otitis media. 1276 Nov 30

The ferric uptake regulation (fur) gene was cloned and characterized from Actinobacillus pleuropneumoniae and it exhibited 97% amino acid sequence identity to the Haemophilus ducrey fur gene. The flanking regions of the fur gene included an upstream putative flavodoxin (fldA) gene and a downstream possible transmembrane protein gene of unknown function. A single promoter was identified by 5' rapid amplification of cDNA ends (RACE), but there were no sequences homologous to an Escherichia coli Fur box in the 5' upstream sequence. The A. pleuropneumoniae fur clone complemented an E. coli fur deletion mutant. Transcriptional analysis of the divergent promoters of the A. pleuropneumoniae toxin I operon (apxICABD)--and the Actinobacillus ferric uptake operon (afuABC) showed that Fur and calcium together positively regulated the transcription of apxICABD while Fur was a repressor for afuABC. Hemolytic activity was significantly induced by iron and calcium and Fur appeared to act as an activator under high calcium conditions and as a repressor under low calcium conditions. A possible regulator-binding site was suggested by the properties of a point mutation in 33 bp upstream of the apxIC gene. This point mutation affected ApxI and Afu expression in response to iron, calcium, or Fur. These results provide further proof that calcium and the A. pleuropneumoniae Fur protein play a role in the expression of ApxI and Afu.
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PMID:Cloning and characterization of the Actinobacillus pleuropneumoniae fur gene and its role in regulation of ApxI and AfuABC expression. 1450 29

Aminoacyl-tRNA synthetases are responsible for activating specific amino acids and transferring them onto cognate tRNA molecules. Due to the similarity in many amino acid side chains, certain synthetases misactivate non-cognate amino acids to an extent that would be detrimental to protein synthesis if left uncorrected. To ensure accurate translation of the genetic code, some synthetases therefore utilize editing mechanisms to hydrolyze non-cognate products. Previously class II Escherichia coli proline-tRNA synthetase (ProRS) was shown to exhibit pre- and post-transfer editing activity, hydrolyzing a misactivated alanine-adenylate (Ala-AMP) and a mischarged Ala-tRNAPro variant, respectively. Residues critical for the editing activity (Asp-350 and Lys-279) are found in a novel insertion domain (INS) positioned between motifs 2 and 3 of the class defining aminoacylation active site. In this work, we present further evidence that INS is responsible for editing in ProRS. We deleted the INS from wild-type E. coli ProRS to yield DeltaINS-ProRS. While DeltaINS-ProRS was still capable of misactivating alanine, the truncated construct was defective in hydrolyzing non-cognate Ala-AMP. When the INS domain was cloned and expressed as an independent protein, it was capable of deacylating a mischarged Ala-microhelixPro variant. Similar to full-length ProRS, post-transfer editing was abolished in a K279A mutant INS. We also show that YbaK, a protein of unknown function from Haemophilus influenzae with high sequence homology to the prokaryotic INS domain, was capable of deacylating Ala-tRNAPro and Ala-microhelixPro variants but not cognate Pro-tRNAPro. Thus, we demonstrate for the first time that an independently folded class II synthetase editing domain and a previously identified homolog can catalyze a hydrolytic editing reaction.
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PMID:An isolated class II aminoacyl-tRNA synthetase insertion domain is functional in amino acid editing. 1453 Feb 68

A strategy is described for the development of high-throughput screening assays against targets of unknown function that involves the use of nuclear magnetic resonance (NMR) spectroscopy. Using this approach, molecules that bind to the protein target are identified from an NMR-based screen of a library of substrates, cofactors, and other compounds that are known to bind to many proteins and enzymes. Once a ligand has been discovered, a fluorescent or radiolabeled analog of the ligand is synthesized that can be used in a high-throughput screen. The approach is illustrated in the development of a high-throughput screening assay against HI-0033, a conserved protein from Haemophilus influenzae whose function is currently unknown. Adenosine was found to bind to HI-0033 by NMR, and fluorescent analogs were rapidly identified that bound to HI-0033 in the submicromolar range. Using these fluorescent compounds, a fluorescence polarization assay was developed that is suitable for high-throughput screening and obtaining detailed structure-activity relationships for lead optimization.
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PMID:A strategy for high-throughput assay development using leads derived from nuclear magnetic resonance-based screening. 1459 58

In 1995, The Institute for Genomic Research completed the genomic sequence of a rough derivative of Haemophilus influenzae serotype d, strain KW20. This sequence, though extremely useful in understanding the basic biology of H. influenzae, has yet to provide significant insight into our understanding of disease caused by nontypeable H. influenzae (NTHI), because serotype d strains are not generally pathogens. In contrast, NTHI strains are frequently mucosal pathogens and are the primary pathogens of chronic otitis media as well as a significant cause of acute otitis media in children. Thus, it is of great importance to further understand their biology. We used a DNA-based microarray approach to identify genes present in a clinical isolate of NTHI that were absent from strain Rd. We also sequenced the genome of a second NTHI isolate from a child with chronic otitis media to threefold coverage and then used an array of bioinformatics tools to identify genes present in this NTHI strain but absent from strain Rd. These methods were complementary in approach and results. We identified, in both strains, homologues of H. influenzae lav, an autotransported protein of unknown function; tnaA, which encodes tryptophanase; as well as a homologue of Pasteurella multocida tsaA, which encodes an alkyl peroxidase that may play a role in protection against reactive oxygen species. We also identified a number of putative restriction-modification systems, bacteriophage genes and transposon-related genes. These data provide new insight that complements and extends our ongoing analysis of NTHI virulence determinants.
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PMID:Partial analysis of the genomes of two nontypeable Haemophilus influenzae otitis media isolates. 1510 13

In the study described here, we have taken steps to characterize the YjeE protein, an Escherichia coli protein of unknown function that is essential for bacterial viability. YjeE represents a protein family whose members are broadly conserved in bacteria, absent from eukaryotes and contain both Walker A and B motifs, characteristic of P-loop ATPases. We have revisited the dispensability of the yjeE gene in E. coli and describe efforts to probe the function of the YjeE protein with in vitro biochemistry. We have looked critically for ATPase activity in the recombinant E. coli protein and have made vigilant use of site-directed variants in the Walker A [K41A (Lys41-->Ala) and T42A] and putative Walker B (D80Q) motifs. We noted that any hydrolysis of ATP by the wild-type E. coli protein might be attributed to background ATPase, since it was not appreciably different from that of the variants. To overcome potential contaminants, we turned to crystalline pure YjeE protein from Haemophilus influenzae that was found to hydrolyse ATP at a slow rate (kcat=1 h(-1)). We have also shown high-affinity binding to YjeE by ADP using equilibrium dialysis (K(d)=32 microM) and by fluorescence resonance energy transfer from a conserved tryptophan in YjeE to a fluorescent derivative of ADP, 2'-/3'-O-(N-methylanthraniloyl)adenosine 5'-O-diphosphate (K(d)=8 microM). Walker motif variants were notably impaired for ADP binding and T42A and D80Q mutations in yjeE were incapable of complementing the yjeE deletion strain.
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PMID:Probing the active site of YjeE: a vital Escherichia coli protein of unknown function. 1532 1

Escherichia coli FtsK is a multifunctional protein that couples cell division and chromosome segregation. Its N-terminal transmembrane domain (FtsK(N)) is essential for septum formation, whereas its C-terminal domain (FtsK(C)) is required for chromosome dimer resolution by XerCD-dif site-specific recombination. FtsK(C) is an ATP-dependent DNA translocase. In vitro and in vivo data point to a dual role for this domain in chromosome dimer resolution (i) to directly activate recombination by XerCD-dif and (ii) to bring recombination sites together and/or to clear DNA from the closing septum. FtsK(N) and FtsK(C) are separated by a long linker region (FtsK(L)) of unknown function that is highly divergent between bacterial species. Here, we analysed the in vivo effects of deletions of FtsK(L) and/or of FtsK(C), of swaps of these domains with their Haemophilus influenzae counterparts and of a point mutation that inactivates the walker A motif of FtsK(C). Phenotypic characterization of the mutants indicated a role for FtsK(L) in cell division. More importantly, even though Xer recombination activation and DNA mobilization both rely on the ATPase activity of FtsK(C), mutants were found that can perform only one or the other of these two functions, which allowed their separation in vivo for the first time.
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PMID:FtsK activities in Xer recombination, DNA mobilization and cell division involve overlapping and separate domains of the protein. 1552 74

Natural competence for DNA uptake is common among bacteria but its evolutionary function is controversial. Resolving the dispute requires a detailed understanding of both how cells decide to take up DNA and how the DNA is processed during and after uptake. We have used whole-genome microarrays to follow changes in gene expression during competence development in wild-type Haemophilus influenzae cells, and to characterize dependence of competence-induced transcription on known regulatory factors. This analysis confirmed the existence of a postulated competence regulon, characterized by a promoter-associated 22 bp competence regulatory element (CRE) closely related to the cAMP receptor protein (CRP) binding consensus. This CRE regulon contains 25 genes in 13 transcription units, only about half of which have been previously associated with competence. The new CRE genes encode a periplasmic ATP-dependent DNA ligase, homologs of SSB, RadC and the Bacillus subtilis DNA uptake protein ComEA, and eight genes of unknown function. Competence-induced transcription of genes in the CRE regulon is strongly dependent on cAMP, consistent with the known role of catabolite regulation in competence. Electrophoretic mobility-shift assays confirmed that CRE sequences are a new class of CRP-binding site. The essential competence gene sxy is induced early in competence development and is required for competence-induced transcription of CRE-regulon genes but not other CRP-regulated genes, suggesting that Sxy may act as an accessory factor directing CRP to CRE sites. Natural selection has united these 25 genes under a common regulatory mechanism. Elucidating this mechanism, and the functions of the genes, will provide a valuable window into the evolutionary function of natural competence.
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PMID:A novel CRP-dependent regulon controls expression of competence genes in Haemophilus influenzae. 1576 66

HI1506 is a 128-residue hypothetical protein of unknown function from Haemophilus influenzae. It was originally annotated as a shorter 85-residue protein, but a more detailed sequence analysis conducted in our laboratory revealed that the full-length protein has an additional 43 residues on the C terminus, corresponding with a region initially ascribed to HI1507. As part of a larger effort to understand the functions of hypothetical proteins from Gram-negative bacteria, and H. influenzae in particular, we report here the three-dimensional solution NMR structure for the corrected full-length HI1506 protein. The structure consists of two well-defined domains, an alpha/beta 50-residue N-domain and a 3-alpha 32-residue C-domain, separated by an unstructured 30-residue linker. Both domains have positively charged surface patches and weak structural homology with folds that are associated with RNA binding, suggesting a possible functional role in binding distal nucleic acid sites.
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PMID:Solution structure of HI1506, a novel two-domain protein from Haemophilus influenzae. 1740 Sep 15

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