Identification and analysis of a gene encoding a Fur-like protein of Staphylococcus epidermidis

Abstract A gene ( fur ) for a Fur-like protein was identified on a 1.1 kb chromosomal DNA fragment of Staphylococcus epidermidis BN 280; the fur gene is followed by an open reading frame coding for the N-terminus of a putative Superoxide dismutase. Within the − 35 promoter region of both genes, a sequence motif was detected with low similarity to Fur-binding regulatory DNA segments, the so-called Fur boxes. Fur titration in Escherichia coli strain H1717 demonstrated that the E. coli Fur protein binds to the Fur box of the promoter region of the S. epidermidis fur gene. The S. epidermidis Fur protein was expressed in E. coli as indicated by the formation of inactive dimers with the chimeric repressor CI(N)-Fur(C) (Stojiljkovic I. and Hantke. K. (1995) Mol. Gen. Genet. 247, 199–205), but was not able to complement the Fur mutation in E. coli H1681.     

Expression and secretion of outer surface protein (OSP-A) of Borrelia burgdorferi from Escherichia coli

Abstract Outer surface protein A (OspA) is encoded by the ospA gene from Borrelia burgdorferi . This protein induces immunity against infection in mice. The cloning and expression of OspA in Escherichia coli have been previously described, but the secretion of OspA into culture media in E. coli has not yet been reported. In this report we demonstrate that a chimeric OspA protein was secreted into culture media by E. coli when it also harbors the hemolysin secretion genes hlyBD . The OspA fusion protein was also overexpressed from a T7 promoter and purified by immobilized metal ion chromatography. This was possible because the fusion protein contains ix histidyl residues in its N-terminus.     

Identification of a Functional fur Gene in Bradyrhizobium japonicum

The recent identification of the iron response regulator (Irr) in Bradyrhizobium japonicum raised the question of whether the global regulator Fur is present in that organism. A fur gene homolog was isolated by the functional complementation of an Escherichia coli fur mutant. The B. japonicum Fur bound to a Fur box DNA element in vitro, and a fur mutant grown in iron-replete medium was derepressed for iron uptake activity. Thus, B. japonicum expresses at least two regulators of iron metabolism.     

Identification and cloning of a fur regulatory gene in Yersinia pestis.

Yersinia pestis is one of many microorganisms responding to environmental iron concentrations by regulating the synthesis of proteins and an iron transport system(s). In a number of bacteria, expression of iron uptake systems and other virulence determinants is controlled by the Fur regulatory protein. DNA hybridization analysis revealed that both pigmented and nonpigmented cells of Y. pestis possess a DNA locus homologous to the Escherichia coli fur gene. Introduction of a Fur-regulated beta-galactosidase reporter gene into Y. pestis KIM resulted in iron-responsive beta-galactosidase activity, indicating that Y. pestis KIM expresses a functional Fur regulatory protein. A cloned 1.9-kb ClaI fragment of Y. pestis chromosomal DNA hybridized specifically to the fur gene of E. coli. The coding region of the E. coli fur gene hybridized to a 1.1-kb region at one end of the cloned Y. pestis fragment. The failure of this clone to complement an E. coli fur mutant suggests that the 1.9-kb clone does not contain a functional promoter. Subcloning of this fragment into an inducible expression vector restored Fur regulation in an E. coli fur mutant. In addition, a larger 4.8-kb Y. pestis clone containing the putative promoter region complemented the Fur- phenotype. These results suggest that Y. pestis possesses a functional Fur regulatory protein capable of interacting with the E. coli Fur system. In Y. pestis Fur may regulate the expression of iron transport systems and other virulence factors in response to iron limitation in the environment. Possible candidates for Fur regulation in Y. pestis include genes involved in ferric iron transport as well as hemin, heme/hemopexin, heme/albumin, ferritin, hemoglobin, and hemoglobin/haptoglobin utilization.     

The gonococcal fur regulon: identification of additional genes involved in major catabolic,recombination, and secretory pathways

In this study, we have characterized the in vitro binding of Neisseria gonorrhoeae Fur to several well-defined iron transport genes, as well as to additional genes involved in major catabolic, secretory, and recombination pathways of gonococci. The gonococcal Fur protein was recombinantly expressed in Escherichia coli HBMV119. Fur was isolated from inclusion bodies and partially purified by ion-exchange chromatography. Gonococcal Fur was found to bind to the promoter/operator region of a gene encoding the previously identified Fur-regulated periplasmic binding protein (FbpA) in a metal ion-dependent fashion, demonstrating that purified Fur is functional. In silico analysis of the partially completed gonococcal genome (FA1090) identified Fur boxes in the promoters of several genes, including tonB, fur, recN, secY, sodB, hemO, hmbR, fumC, a hypothetical gene (Fe-S homolog), and the opa family of genes. By using purified gonococcal Fur, we demonstrate binding to the operator regions of tonB, fur, recN, secY, sodB, hemO, hmbR, fumC, the Fe-S homolog gene, and the opa gene family as determined by an electrophoretic mobility shift assay. While gonococcal Fur was demonstrated to bind to the promoter regions of all 11 opa genes (opaA through -K), we did not detect binding of purified E. coli Fur with 8 of the 11 opa members, indicating that target DNA sequence specificities between these two closely related proteins exist. Furthermore, we observed differences in the relative strengths of binding of gonococcal Fur for these different genes, which most likely reflect a difference in affinity between gonococcal Fur and its DNA targets. This is the first report that definitively demonstrates the binding of gonococcal Fur to its own promoter/operator region, as well as to the opa family of genes that encode surface proteins. Our results demonstrate that the gonococcal Fur protein binds to the regulatory regions of a broad array of genes and indicates that the gonococcal Fur regulon is larger than originally proposed.     

The Lyme disease agent Borrelia burgdorferi requires BB0690, a Dps homologue, to persist within ticks

Borrelia burgdorferi survives in an enzootic cycle, and Dps proteins protect DNA against damage during starvation or oxidative stress. The role of a Dps homologue encoded by Borrelia in spirochaete survival was assessed. Dps-deficient spirochaetes were infectious in mice via needle-inoculation at the dose of 10(5) spirochaetes. Larval ticks successfully acquired Dps-deficient spirochaetes via a blood meal on mice. However, after extended periods within unfed nymphs, the Dps-deficient spirochaetes failed to be transmitted to a new host when nymphs fed. Our data suggest that Dps functions to protect the spirochaetes during dormancy in unfed ticks, and in its absence, the spirochaetes become susceptible during tick feeding. dps is differentially expressed in vivo- low in mice and high in ticks - but constitutively expressed in vitro, showing little change during growth or in response to oxidative stress. Borrelia Dps forms a dodecameric complex capable of sequestering iron. The Dps-deficient spirochaetes showed no defect in starvation and oxidative stress assays, perhaps due to the lack of iron in spirochaetes grown in vitro. Dps is critical for spirochaete persistence within ticks, and strategies to interfere with Dps could potentially reduce Borrelia populations in nature and thereby influence the incidence of Lyme disease.     

The extended promoters for two outer membrane lipoprotein genes of Borrelia spp. uniquely include a T-rich region

OspA and B proteins of Borrelia burgdorferi and Vmp proteins of Borrelia hermsii are abundant outer membrane lipoproteins, whose expression varies with the environment. The genes for these proteins have the '-35' and '-10' elements of a sigma70-type promoter. Deletions of the promoters for these genes were analysed with a chloramphenicol acetyltransferase (CAT) reporter gene and plasmid constructs that were stably maintained in Escherichia coli or transiently transfected into B. burgdorferi. Reporter expression was measured as susceptibility of transformed E. coli cells to chloramphenicol and the CAT activity of E. coli and B. burgdorferi lysates in vitro. Presence of the '-10' element was essential for full activity in both B. burgdorferi and E. coli. Upstream of the '-35' elements of the ospAB and vmp promoters were tracts with Ts in 16 of 20 positions for B. burgdorferi and 18 of 20 positions for B. hermsii. Deletion of the T-rich region from the ospAB or vmp promoter caused a greater reduction of CAT activity in B. burgdorferi than in E. coli. The findings indicate that ospAB and vmp promoters are extended promoters with two parts: (i) a core region containing typical '-35' and '-10' elements and (ii) a unique T-rich region.     

Characterization of the Vibrio anguillarum fur gene: role in regulation of expression of the FatA outer membrane protein and catechols.

The chromosomally encoded Vibrio anguillarum fur gene was characterized. The amino acid sequence of the Fur protein showed a very high degree of homology with those of V. cholerae and V. vulnificus. The degree of homology was lower, although still high, with the Escherichia coli and Yersinia pestis Fur amino acid sequences, while the lowest degree of homology was found with the Pseudomonas aeruginosa Fur protein. The C-terminal portion of Fur is the least conserved region among these Fur proteins. Within this portion, two regions spanning amino acids 105 to 121 and 132 to the end are the least conserved. A certain degree of variation is also present in the N termini spanning amino acids 28 to 46. Regulation of expression of the V. anguillarum fur gene by iron was not detected by immunoblot analysis. Mutations in the cloned fur gene were generated either by site-directed mutagenesis (the Lys-77 was changed to a Gly to generate the derivative FurG77) or by insertion of a DNA fragment harboring the aph gene in the same position. FurG77 was impaired in its ability to regulate a reporter gene with the Fur box in its promoter, while the insertion mutant was completely inactive. V. anguillarum fur mutants were obtained by isolating manganese-resistant derivatives. In one of these mutants, which encoded a Fur protein with an apparent lower molecular weight, the regulation of the production of catechols and synthesis of the outer membrane protein FatA were partially lost. In the case of another mutant, no protein was detected by anti-Fur serum. This derivative showed a total lack of regulation of biosynthesis of catechols and FatA protein by iron.