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 acquisition of iron is an essential component in the pathogenesis of Yersinia pestis, the agent of bubonic and pneumonic plague. A cosmid library derived from the genomic DNA ofY. pestis KIM6+ was used for transduction of anEscherichia coli mutant (SAB11) defective in the biosynthesis of the siderophore enterobactin. Recombinant plasmids which had a common 13-kb BamHI fragment were isolated from SAB11 transductants in which growth but not enterobactin synthesis was restored on media containing the iron chelator EDDA [ethylenediamine-di(o-hydroxyphenyl acetic acid)]. Subcloning and transposon mutagenesis revealed a 5.6-kb region, designated yfe, essential for SAB11 growth stimulation. In vitro transcription-translation analysis identified polypeptides of 18, 29.5, 32, and 33 kDa encoded by the yfe locus. Sequence analysis shows this locus to be comprised of five genes in two separate operons which have potential Fur-binding sequences in both promoters. A putative polycistronic operon, yfeABCD, is Fur regulated and responds to iron and manganese. A functional Fur protein is required for the observed manganese repression of this operon. This operon encodes polypeptides which have strong similarity to the ATP-binding cassette (ABC) family of transporters and include a periplasmic binding protein (YfeA), an ATP-binding protein (YfeB), and two integral membrane proteins (YfeC and -D), which likely function in the acquisition of inorganic iron and possibly other ions. The ∼21-kDa protein encoded by the separately transcribedyfeE gene may be located in the cell envelope, since ayfeE::TnphoA fusion is PhoA+. Mutations in this gene abrogate growth of SAB11 on iron-chelated media.

A Yersinia pestis fur mutation was constructed by insertionally disrupting the fur open reading frame.Analysis of a Fur-regulated 0-galactosidase reporter gene revealed a loss of iron regulation as a result of the fur mutation. trans complementation with the cloned Y. pestisfur gene restored iron regulation. The expression of most iron-regulated proteins was also deregulated by this mutation; however, a number of iron-repressible and two iron-inducible polypeptides retained normal regulation. Mutations infur or hmsH, a gene encoding an 86-kDa surface protein required for hemin storage, increased the sensitivity of Y. pestis cells to the bacteriocin pesticin. Interestingly, the Y. pestis fur mutant lost temperature control of hemin storage; however, expression of the HmsH polypeptide was not deregulated. When grown with excess iron, a Y. pestisfur mutant possessing the 102-kb pigmentation locus exhibited severe growth inhibition and a dramatic increase in the number of spontaneous nonpigmented chromosomal deletion mutants present at late log phase. These results suggest that the Fur protein of Y. pestis is an important global regulator and that a separate Fur-independent iron regulatory system may exist.Iron is an essential element that must often be acquired from iron-deficient environments. The regulation of diverse genes belonging to global iron limitation stimulons has been described previously for a number of bacteria, including Escherichia coli (6,10,37,47,51), Vibrio species (20, 25, 69), yersiniae (12,13,65,68), Neisseria gonorrhoeae (4), Neisseria meningitidis (73), Pseudomonas aeruginosa (60), shigellae (52), Salmonella typhimurium (18), Corynebacterium diphtheriae (5, 62), and Serratia marcescens (58). Many of these iron-regulated factors are involved in virulence, especially acquisition of iron from the host (9, 27, 51), while the roles of other genes in virulence or iron acquisition are less clearly defined. The coordinate regulation of these iron-regulated genes in a number of bacterial species is controlled by the Fur (ferric uptake regulation) system (4,10,11,18,20,25,33,58,60,67,68).The Fur protein is a negative transcriptional regulator that binds DNA when complexed with ferrous iron or other divalent metal ions. Fur-regulated genes possess operator regions with Fur binding sequences (Fur boxes). These -21-bp regions contain inverted repeats which are bound by the Fur-Fe2" complex, thereby preventing transcription. Insufficient cytoplasmic ferrous iron results in apo-Fur and relieves this repression (6, 47).In Yersinia pestis, iron starvation at 370C induces over 30 iron-repressible proteins (44) and an energy-dependent, highaffinity inorganic-iron uptake system which is inhibited by relatively weak iron chelators (56