Identification and Characterization of pvuA, a Gene Encoding the Ferric Vibrioferrin Receptor Protein in Vibrio parahaemolyticus

We previously reported that Vibrio parahaemolyticus expresses two outer membrane proteins of 78 and 83 kDa concomitant with production of siderophore vibrioferrin in response to iron starvation stress and that these proteins are the ferric vibrioferrin receptor and heme receptor, respectively (S. Yamamoto, T. Akiyama, N. Okujo, S. Matsuura, and S. Shinoda, Microbiol. Immunol. 39:759-766, 1995; S. Yamamoto, Y. Hara, K. Tomochika, and S. Shinoda, FEMS Microbiol. Lett. 128:195-200, 1995). In this study, the Fur titration assay (FURTA) system was applied to isolate DNA fragments containing a potential Fur box from a genomic DNA library of V. parahaemolyticus WP1. Sequencing a 3.2-kb DNA insert in one FURTA-positive clone revealed that an amino acid sequence deduced from a partial gene, which was preceded by a full-length gene (psuA) encoding a receptor for a siderophore of unknown origin, was consistent with the N-terminal amino acid sequence of the 78-kDa ferric vibrioferrin receptor. Then, the full-length gene (pvuA) encoding the ferric vibrioferrin receptor was cloned and characterized. The deduced protein encoded by pvuA displayed the highest similarity (31% identity; 48% similarity) to RumA, a ferric rhizoferrin receptor of Morganella morganii. Primer extension and Northern blot analyses indicated that psuA and pvuA constitute an operon which is transcribed from a Fur-repressed promoter upstream of psuA. The product of the pvuA gene and its function were confirmed by generating a pvuA-disrupted mutant, coupled with genetic complementation studies. A mutant with disruption in the upstream psuA gene also displayed a phenotype impaired in the utilization of ferric vibrioferrin.     

Selectivity of Ferric Enterobactin Binding and Cooperativity of Transport in Gram-Negative Bacteria

The ligand-gated outer membrane porin FepA serves Escherichia coli as the receptor for the siderophore ferric enterobactin. We characterized the ability of seven analogs of enterobactin to supply iron via FepA by quantitatively measuring the binding and transport of their ⁵⁹Fe complexes. The experiments refuted the idea that chirality of the iron complex affects its recognition by FepA and demonstrated the necessity of an unsubstituted catecholate coordination center for binding to the outer membrane protein. Among the compounds we tested, only ferric enantioenterobactin, the synthetic, left-handed isomer of natural enterobactin, and ferric TRENCAM, which substitutes a tertiary amine for the macrocyclic lactone ring of ferric enterobactin but maintains an unsubstituted catecholate iron complex, were recognized by FepA (K_d ≈ 20 nM). Ferric complexes of other analogs (TRENCAM-3,2-HOPO; TREN-Me-3,2-HOPO; MeMEEtTAM; MeME-Me-3,2-HOPO; K₃MECAMS; agrobactin A) with alterations to the chelating groups and different net charge on the iron center neither adsorbed to nor transported through FepA. We also compared the binding and uptake of ferric enterobactin by homologs of FepA from Bordetella bronchisepticus, Pseudomonas aeruginosa, and Salmonella typhimurium in the native organisms and as plasmid-mediated clones expressed in E. coli. All the transport proteins bound ferric enterobactin with high affinity (K_d ≤ 100 nM) and transported it at comparable rates (≥50 pmol/min/10⁹ cells) in their own particular membrane environments. However, the FepA and IroN proteins of S. typhimurium failed to efficiently function in E. coli. For E. coli, S. typhimurium, and P. aeruginosa, the rate of ferric enterobactin uptake was a sigmoidal function of its concentration, indicating a cooperative transport reaction involving multiple interacting binding sites on FepA.     

Direct Measurements of the Outer Membrane Stage of Ferric Enterobactin Transport: POSTUPTAKE BINDING*

When Gram-negative bacteria acquire iron, the metal crosses both the outer membrane (OM) and the inner membrane, but existing radioisotopic uptake assays only measure its passage through the latter bilayer, as the accumulation of the radionuclide in the cytoplasm. We devised a methodology that exclusively observes OM transport and used it to study the uptake of ferric enterobactin (FeEnt) by Escherichia coli FepA. This technique, called postuptake binding, revealed previously unknown aspects of TonB-dependent transport reactions. The experiments showed, for the first time, that despite the discrepancy in cell envelope concentrations of FepA and TonB (∼35:1), all FepA proteins were active and equivalent in FeEnt uptake, with a maximum turnover number of ∼5/min. FepA-mediated transport of FeEnt progressed through three distinct phases with successively decreasing rates, and from its temperature dependence, the activation energy of the OM stage was 33–35 kcal/mol. The accumulation of FeEnt in the periplasm required the binding protein and inner membrane permease components of its overall transport system; postuptake binding assays on strains devoid of FepB, FepD, or FepG did not show uptake of FeEnt through the OM. However, fluorescence labeling data implied that FepA was active in the ΔfepB strain, suggesting that FeEnt entered the periplasm but then leaked out. Further experiments confirmed this futile cycle; cells without FepB transported FeEnt across the OM, but it immediately escaped through TolC.     

Identification and Characterization of a Seven Transmembrane Hormone Receptor Using Differential Display

Targeted mutagenesis analysis has shown that theCmybproto-oncogene, which encodes a sequence-specific DNA binding protein, is required for normal murine fetal liver erythropoiesis and myelopoiesis. To identify novel genes involved in hematopoiesis, differential display analysis was conducted using total liver RNA isolated from 14.5-day postcoitusCmybwildtype, heterozygous, and homozygous mutant littermates. Using 4 oligo(dT) 3′ primers and 5 arbitrary decamers as 5′ primers, 22 differentially expressed genes have been identified. Eight putatively novel genes were identified from 12 cDNAs that were sequenced. One gene, initially designated DD7A5-7, is primarily expressed in cells of the myeloid lineage. The full-length DD7A5-7 cDNA is 3239 nucleotides, encoding a putative protein of 931 amino acids. The protein is a member of a family of hormone receptors containing 7 transmembrane segments. The receptor also contains 7 epidermal growth factor-like (Egf-like) motifs at the amino terminal of the predicted protein. The gene is alternatively spliced, resulting in the deletion of one or more copies of theEgf-like motif. DD7A5-7 maps to mouse Chromosome 17 and is the putative homologue ofEMR1,a recently describedEgf-like module containing mucin-like hormone receptor with 7 transmembrane segments in humans. Our results indicate that theCmybmutant fetuses represent a unique resource for identifying genes involved in hematopoiesis.     

Identification and Characterization of a New Bocavirus Species in Gorillas

A novel parvovirus, provisionally named Gorilla Bocavirus species 1 (GBoV1), was identified in four stool samples from Western gorillas (Gorilla gorilla) with acute enteritis. The complete genomic sequence of the new parvovirus revealed three open reading frames (ORFs) with an organization similar to that of known bocaviruses. Phylogenetic analysis using complete capsid and non structural (NS) gene sequence suggested that the new parvovirus is most closely related to human bocaviruses (HBoV). However, the NS ORF is more similar in length to the NS ORF found in canine minute virus and bovine parvovirus than in HBoV. Comparative genetic analysis using GBoV and HBoV genomes enabled characterization of unique splice donor and acceptor sites that appear to be highly conserved among all four HBoV species, and provided evidence for expression of two different NS proteins in all primate bocaviruses. GBoV is the first non-human primate bocavirus identified and provides new insights into the genetic diversity and evolution of this highly prevalent and recently discovered group of parvoviruses.     

Identification and Characterization of a New Lipoprotein, NlpI, in Escherichia coli K-12

We report here the identification of a new lipoprotein, NlpI, in Escherichia coli K-12. The NlpI structural gene (nlpI) is located between the genes pnp (polynucleotide phosphorylase) and deaD (RNA helicase) at 71 min on the E. coli chromosome. The nlpI gene encodes a putative polypeptide of approximately 34 kDa, and multiple lines of evidence clearly demonstrate that NlpI is indeed a lipoprotein. An nlpI::cm mutation rendered growth of the cells osmotically sensitive, and incubation of the insertion mutant at an elevated temperature resulted in the formation of filaments. The altered phenotype of the mutant was a direct consequence of the mutation in nlpI, since it was complemented by the wild-type nlpI gene alone. Overexpression of the unaltered nlpI gene in wild-type cells resulted in the loss of the rod morphology and the formation of single prolate ellipsoids and pairs of prolate ellipsoids joined by partial constrictions. NlpI may be important for an as-yet-undefined step in the overall process of cell division.     

Characterization of erythromycin resistance in Campylobacter coli and Campylobacter jejuni isolated from pig offal in New Zealand

AIMS: To determine the level and mechanism(s) of antimicrobial resistance in Campylobacter isolates obtained from human and environmental sources from South Canterbury, New Zealand. METHODS AND RESULTS: A total of 251 Campylobacter isolates were tested for susceptibility to ciprofloxacin, erythromycin, nalidixic acid and tetracycline using disc diffusion assays. Five pig offal isolates were observed to be highly erythromycin resistant, with minimal inhibitory concentrations determined to be >/=256 microg ml(-1). Nucleotide sequencing of the 23S ribosomal DNA (rDNA) in these resistant isolates identified an A --> G change at Escherichia coli position 2059 that has been previously implicated in erythromycin resistance in Campylobacter coli. Macrorestriction profiling using pulsed-field gel electrophoresis showed these isolates were nonclonal. CONCLUSIONS: The majority of Campylobacter isolates from South Canterbury remain sensitive to the most clinically relevant antimicrobial agents. Our results support other reports showing that specific variations in the 23S rDNA contribute to erythromycin resistance. SIGNIFICANCE AND IMPACTS OF THE STUDY: This study defines the baseline frequency of antimicrobial resistance associated with Campylobacter isolates from South Canterbury, and discusses the likely molecular mechanisms conferring erythromycin resistance in this organism. Resistance to erythromycin in these isolates is not linked to a dominant Campylobacter clone and has likely arisen independently in different genetic lines exposed to selective antimicrobial pressure.     

Identification and Characterization of Endothelin Receptor Subtype B in Rat Retina

Abstract: The presence of immunoreactive (IR) endothelin (ET)-1 and ET-1 receptors in rat retina has been studied by radioimmunoassay and receptor assay, respectively. The specific binding of ¹²⁵I-ET-1 to rat retinal particulate preparations was saturable. Apparent equilibrium conditions were established within 120–140 min. Scatchard analysis of binding data indicated a single class of high-affinity binding sites with a K _D of 35 ± 11 p M and a B_max of 168 ± 60 fmol/mg of protein. ¹²⁵I-ET-1 binding to retinal particulate preparations was not inhibited by 1 μ M concentrations of somatostatin, atrial natriuretic factor, brain natriuretic peptide, thyroid-stimulating hormone, growth hormone, or insulin. The three endothelin isoforms, ET-1,-2, and-3, had similar affinity for the receptor. Cross-linking of ¹²⁵I-ET-1 to retinal particulate preparations with disuccinimidyl suberate resulted in the labeling of two bands with apparent molecular masses of 52 and 34 kDa. We have established a highly sensitive and specific radioimmunoassay for ET-1. The concentration of IR-ET-1 in rat retina was 35 ± 10 fmol/g wet weight. The demonstration of specific high-affinity ETB receptors and the presence of IR-ET-1 suggest that the peptide may act as a neurotransmitter or neuro-modulator in the retina.     

Identification and Characterization of a Novel Lysophosphatidic Acid Receptor, p2y5/LPA6

p2y5 is an orphan G protein-coupled receptor that is closely related to the fourth lysophosphatidic acid (LPA) receptor, LPA₄. Here we report that p2y5 is a novel LPA receptor coupling to the G₁₃-Rho signaling pathway. “LPA receptor-null” RH7777 and B103 cells exogenously expressing p2y5 showed [³H]LPA binding, LPA-induced [³⁵S]guanosine 5′-3-O-(thio)triphosphate binding, Rho-dependent alternation of cellular morphology, and G_s/13 chimeric protein-mediated cAMP accumulation. LPA-induced contraction of human umbilical vein endothelial cells was suppressed by small interfering RNA knockdown of endogenously expressed p2y5. We also found that 2-acyl-LPA had higher activity to p2y5 than 1-acyl-LPA. A recent study has suggested that p2y5 is an LPA receptor essential for human hair growth. We confirmed that p2y5 is a functional LPA receptor and propose to designate this receptor LPA₆.     

Overproduction in Escherichia coli and Characterization of a Soybean Ferric Leghemoglobin Reductase

We previously cloned and sequenced a cDNA encoding soybean ferric leghemoglobin reductase (FLbR), an enzyme postulated to play an important role in maintaining leghemoglobin in a functional ferrous state in nitrogen-fixing root nodules. This cDNA was sub-cloned into an expression plasmid, pTrcHis C, and overexpressed in Escherichia coli. The recombinant FLbR protein, which was purified by two steps of column chromatography, was catalytically active and fully functional. The recombinant FLbR cross-reacted with antisera raised against native FLbR purified from soybean root nodules. The recombinant FLbR, the native FLbR purified from soybean (Glycine max L.) root nodules, and dihydrolipoamide dehydrogenases from pig heart and yeast had similar but not identical ultraviolet-visible absorption and fluorescence spectra, cofactor binding, and kinetic properties. FLbR shared common structural features in the active site and prosthetic group binding sites with other pyridine nucleotide-disulfide oxidoreductases such as dihydrolipoamide dehydrogenases, but displayed different microenvironments for the prosthetic groups.     

Characterization of CetA and CetB, a bipartite energy taxis system in Campylobacter jejuni

The energy taxis receptor Aer, in Escherichia coli , senses changes in the redox state of the electron transport system via an flavin adenine dinucleotide cofactor bound to a PAS domain. The PAS domain (a sensory domain named after three proteins P er, A RNT and S im, where it was first identified) is thought to interact directly with the Aer HAMP domain to transmit this signal to the highly conserved domain (HCD) found in chemotaxis receptors. An apparent energy taxis system in Campylobacter jejuni is composed of two proteins, CetA and CetB, that have the domains of Aer divided between them. CetB has a PAS domain, while CetA has a predicted transmembrane region, HAMP domain and the HCD. In this study, we examined the expression of cetA and cetB and the biochemical properties of the proteins they encode. cetA and cetB are co-transcribed independently of the flagellar regulon. CetA has two transmembrane helices in a helical hairpin while CetB is a peripheral membrane protein tightly associated with the membrane. CetB levels are CetA dependent. Additionally, we demonstrated that both CetA and CetB participate in complexes, including a likely CetB dimer and a complex that may include both CetA and CetB. This study provides a foundation for further characterization of signal transduction mechanisms within CetA/CetB.     

Cloning and Characterization of a Campylobacter jejuni Iron-Uptake Operon

We report that C. jejuni modifies its outer membrane protein (OMP) repertoire when cultivated under iron-limiting conditions such as during incubation with epithelial cells. To identify genes encoding de novo expressed OMPs, a C. jejuni cosmid library was screened with antisera raised against proteins expressed in the presence of epithelial cells. A single clone was identified encoding an 80-kDa antigen. Sequence analysis of subclones identified an operon of three open reading frames (ORFs) encoding proteins that are homologous to the E. coli ferrichrome uptake system encoded by the fhu locus. Under low-iron conditions, C. jejuni expressed the 80-kDa OMP, indicating that its expression is regulated by the presence of iron. Southern blot analysis indicated that six of eleven isolates of C. jejuni harbor a fhuA homolog which, like all other DNA in this region sequenced thus far, is strikingly GC-rich (65%) compared with the C. jejuni genome (35% G+C). Received: 19 June 2000 / Accepted: 30 August 2000     

Identification and molecular characterisation of CmeB, a Campylobacter jejuni multidrug efflux pump

A multidrug efflux pump gene (cmeB) was identified from the published Campylobacter jejuni genome sequence. Secondary structural analysis showed that the gene encoded a protein belonging to the resistance nodulation cell division (RND) family of efflux transporters. The gene was inactivated by insertional mutagenesis. Compared with the wild-type strain (NCTC 11168), the resultant knockout strain (NCTC 11168-cmeB::kan(r)) displayed increased susceptibility to a range of antibiotics including beta-lactams, fluoroquinolones, macrolides, chloramphenicol, tetracycline, ethidium bromide, the dye acridine orange and the detergent sodium dodecyl sulfate. Accumulation of ciprofloxacin was increased in the knockout mutant, but carbonyl cyanide m-chlorophenyl hydrazone, a proton motive force inhibitor, had less effect upon ciprofloxacin accumulation in the knockout mutant compared with NCTC 11168. These data show that the identified gene encodes an RND-type multi-substrate efflux transporter, which contributes to intrinsic resistance to a range of structurally unrelated compounds in C. jejuni. This efflux pump has been named CmeB (for Campylobacter multidrug efflux).     

Insertion Mutagenesis of the Ferric Pyoverdine Receptor FpvA of Pseudomonas aeruginosa: Identification of Permissive Sites and a Region Important for Ligand Binding

Insertion of an 18-amino-acid-encoding sequence within the fpvA gene identified permissive sites at residues Y350, A402, R451, R521, and R558, consistent with these residues occurring in extramembranous loop regions of the protein. Insertions at R451, R521, and R558 did not adversely affect receptor function, although insertions at Y350 and A402 compromised ferric pyoverdine binding and uptake. The latter region likely contributes to or interacts with the ligand-binding site.     

Detecting the Dominant T and B Epitopes of Klebsiella pneumoniae Ferric Enterobactin Protein (FepA) and Introducing a Single Epitopic Peptide as Vaccine Candidate

International Journal of Peptide Research and Therapeutics - Klebsiella pneumoniae causes various human infections. Ferric enterobactin protein (FepA) is a conserved protein of K. pneumoniae with...