The iron-responsive Fur regulon in Yersinia pestis.
ABSTRACT: The ferric uptake regulator (Fur) is a predominant bacterial regulator controlling the iron assimilation functions in response to iron availability. Our previous microarray analysis on Yersinia pestis defined the iron-Fur modulon. In the present work, we reannotated the iron assimilation genes in Y. pestis, and the resulting genes in complementation with those disclosed by microarray constituted a total of 34 genome loci (putative operons) that represent the potential iron-responsive targets of Fur. The subsequent real-time reverse transcription-PCR (RT-PCR) in conjunction with the primer extension analysis showed that 32 of them were regulated by Fur in response to iron starvation. A previously predicted Fur box sequence was then used to search against the promoter regions of the 34 operons; the homologue of the above box could be predicted in each promoter tested. The subsequent electrophoretic mobility shift assay (EMSA) demonstrated that a purified His(6) tag-fused Fur protein was able to bind in vitro to each of these promoter regions. Therefore, Fur is a global regulator, both an activator and a repressor, and directly controls not only almost all of the iron assimilation functions but also a variety of genes involved in various non-iron functions for governing a complex regulatory cascade in Y. pestis. In addition, real-time RT-PCR, primer extension, EMSA, and DNase I footprinting assay were used to elucidate the Fur regulation of the ybt locus encoding a virulence-required iron uptake system. By combining the published data on the YbtA regulation of ybt, we constructed a concise Fur/YbtA regulatory network with a map of the Fur-promoter DNA interactions within the ybt locus. The data presented here give us an overview of the iron-responsive Fur regulon in Y. pestis.
Project description:One requirement for the pathogenesis of Yersinia pestis, the causative agent of bubonic plague, is the yersiniabactin (Ybt) siderophore-dependent iron transport system that is encoded within a high-pathogenicity island (HPI) within the pgm locus of the Y. pestis chromosome. Nine gene products within the HPI have demonstrated functions in the nonribosomal peptide synthesis (NRPS)/polyketide (PK) synthesis or transport of Ybt. NRPS/PK synthetase or synthase enzymes are generally activated by phosphopantetheinylation. However, no products with similarities to known phosphopantetheinyl (P-pant) transferases were found within the pgm locus. We have identified a gene, ybtD, encoded outside the HPI and pgm locus, that is necessary for function of the Ybt system and has similarities to other P-pant transferases such as EntD of Escherichia coli. A deletion within ybtD yielded a strain (KIM6-2085+) defective in siderophore production. This strain was unable to grow on iron-deficient media at 37 degrees C but could be cross-fed by culture supernatants from Ybt-producing strains of Y. pestis. The promoter region of ybtD was fused to lacZ; beta-galactosidase expression from this reporter was not regulated by the iron status of the bacterial cells or by YbtA, a positive regulator of other genes of the ybt system. The ybtD mutant failed to express indicator Ybt proteins (high-molecular-weight protein 1 [HMWP1], HMWP2, and Psn), a pattern similar to those seen with several other ybt biosynthetic mutants. In contrast, cells containing a single amino acid substitution (S2908A) in the terminal thioesterase domain of HMWP2 failed to exhibit any ybt regulatory defects but did not elaborate extracellular Ybt under iron-deficient conditions.
Project description:The Yfe/Sit and Feo transport systems are important for the growth of a variety of bacteria. In Yersinia pestis, single mutations in either yfe or feo result in reduced growth under static (limited aeration), iron-chelated conditions, while a yfe feo double mutant has a more severe growth defect. These growth defects were not observed when bacteria were grown under aerobic conditions or in strains capable of producing the siderophore yersiniabactin (Ybt) and the putative ferrous transporter FetMP. Both fetP and a downstream locus (flp for fet linked phenotype) were required for growth of a yfe feo ybt mutant under static, iron-limiting conditions. An feoB mutation alone had no effect on the virulence of Y. pestis in either bubonic or pneumonic plague models. An feo yfe double mutant was still fully virulent in a pneumonic plague model but had an ?90-fold increase in the 50% lethal dose (LD(50)) relative to the Yfe(+) Feo(+) parent strain in a bubonic plague model. Thus, Yfe and Feo, in addition to Ybt, play an important role in the progression of bubonic plague. Finally, we examined the factors affecting the expression of the feo operon in Y. pestis. Under static growth conditions, the Y. pestis feo::lacZ fusion was repressed by iron in a Fur-dependent manner but not in cells grown aerobically. Mutations in feoC, fnr, arcA, oxyR, or rstAB had no significant effect on transcription of the Y. pestis feo promoter. Thus, the factor(s) that prevents repression by Fur under aerobic growth conditions remains to be identified.
Project description:The Gram-negative bacterium Yersinia pestis is the causative agent of the bubonic plague. Efficient iron acquisition systems are critical to the ability of Y. pestis to infect, spread and grow in mammalian hosts, because iron is sequestered and is considered part of the innate host immune defence against invading pathogens. We used a proteomic approach to determine expression changes of iron uptake systems and intracellular consequences of iron deficiency in the Y. pestis strain KIM6+ at two physiologically relevant temperatures (26 degrees C and 37 degrees C).Differential protein display was performed for three Y. pestis subcellular fractions. Five characterized Y. pestis iron/siderophore acquisition systems (Ybt, Yfe, Yfu, Yiu and Hmu) and a putative iron/chelate outer membrane receptor (Y0850) were increased in abundance in iron-starved cells. The iron-sulfur (Fe-S) cluster assembly system Suf, adapted to oxidative stress and iron starvation in E. coli, was also more abundant, suggesting functional activity of Suf in Y. pestis under iron-limiting conditions. Metabolic and reactive oxygen-deactivating enzymes dependent on Fe-S clusters or other iron cofactors were decreased in abundance in iron-depleted cells. This data was consistent with lower activities of aconitase and catalase in iron-starved vs. iron-rich cells. In contrast, pyruvate oxidase B which metabolizes pyruvate via electron transfer to ubiquinone-8 for direct utilization in the respiratory chain was strongly increased in abundance and activity in iron-depleted cells.Many protein abundance differences were indicative of the important regulatory role of the ferric uptake regulator Fur. Iron deficiency seems to result in a coordinated shift from iron-utilizing to iron-independent biochemical pathways in the cytoplasm of Y. pestis. With growth temperature as an additional variable in proteomic comparisons of the Y. pestis fractions (26 degrees C and 37 degrees C), there was little evidence for temperature-specific adaptation processes to iron starvation.
Project description:BACKGROUND: Environmental modulation of gene expression in Yersinia pestis is critical for its life style and pathogenesis. Using cDNA microarray technology, we have analyzed the global gene expression of this deadly pathogen when grown under different stress conditions in vitro. RESULTS: To provide us with a comprehensive view of environmental modulation of global gene expression in Y. pestis, we have analyzed the gene expression profiles of 25 different stress conditions. Almost all known virulence genes of Y. pestis were differentially regulated under multiple environmental perturbations. Clustering enabled us to functionally classify co-expressed genes, including some uncharacterized genes. Collections of operons were predicted from the microarray data, and some of these were confirmed by reverse-transcription polymerase chain reaction (RT-PCR). Several regulatory DNA motifs, probably recognized by the regulatory protein Fur, PurR, or Fnr, were predicted from the clustered genes, and a Fur binding site in the corresponding promoter regions was verified by electrophoretic mobility shift assay (EMSA). CONCLUSION: The comparative transcriptomics analysis we present here not only benefits our understanding of the molecular determinants of pathogenesis and cellular regulatory circuits in Y. pestis, it also serves as a basis for integrating increasing volumes of microarray data using existing methods.
Project description:BACKGROUND: Yersinia pestis synthesizes the attached biofilms in the flea proventriculus, which is important for the transmission of this pathogen by fleas. The hmsHFRS operons is responsible for the synthesis of exopolysaccharide (the major component of biofilm matrix), which is activated by the signaling molecule 3', 5'-cyclic diguanylic acid (c-di-GMP) synthesized by the only two diguanylate cyclases HmsT, and YPO0449 (located in a putative operonYPO0450-0448). METHODOLOGY/PRINCIPAL FINDINGS: The phenotypic assays indicated that the transcriptional regulator Fur inhibited the Y. pestis biofilm production in vitro and on nematode. Two distinct Fur box-like sequences were predicted within the promoter-proximal region of hmsT, suggesting that hmsT might be a direct Fur target. The subsequent primer extension, LacZ fusion, electrophoretic mobility shift, and DNase I footprinting assays disclosed that Fur specifically bound to the hmsT promoter-proximal region for repressing the hmsT transcription. In contrast, Fur had no regulatory effect on hmsHFRS and YPO0450-0448 at the transcriptional level. The detection of intracellular c-di-GMP levels revealed that Fur inhibited the c-di-GMP production. CONCLUSIONS/SIGNIFICANCE: Y. pestis Fur inhibits the c-di-GMP production through directly repressing the transcription of hmsT, and thus it acts as a repressor of biofilm formation. Since the relevant genetic contents for fur, hmsT, hmsHFRS, and YPO0450-0448 are extremely conserved between Y. pestis and typical Y. pseudotuberculosis, the above regulatory mechanisms can be applied to Y. pseudotuberculosis.
Project description:We have identified an approximately 22-kb region of the pgm locus of Yersinia pestis KIM6+ which encodes a number of iron-regulated proteins involved in the biosynthesis of the Y. pestis cognate siderophore, yersiniabactin (Ybt), and which is located immediately upstream of the pesticin/yersiniabactin receptor gene (psn). Sequence analysis and the construction of insertion and deletion mutants allowed us to determine the putative location of the irp1 gene and the positions of irp2, ybtT, and ybtE within the ybt operon. Mutations in the irp1, irp2, or ybtE gene yielded strains defective in siderophore production. Mutant strains were unable to grow on iron-deficient media at 37 degrees C but could be cross-fed by culture supernatants from yersiniabactin-producing strains of Y. pestis grown under iron-limiting conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of whole-cell extracts from Ybt+ and Ybt- strains grown in iron-deficient media revealed that expression of ybt-encoded proteins is not only iron regulated but also influenced by the presence of the siderophore itself. Finally, Y. pestis strains with mutations in either the psn or irp2 gene were avirulent in mice when inoculated subcutaneously.
Project description:BACKGROUND: The zinc uptake regulator Zur is a Zn2+-sensing metalloregulatory protein involved in the maintenance of bacterial zinc homeostasis. Up to now, regulation of zinc homeostasis by Zur is poorly understood in Y. pestis. RESULTS: We constructed a zur null mutant of Y. pestis biovar microtus strain 201. Microarray expression analysis disclosed a set of 154 Zur-dependent genes of Y. pestis upon exposure to zinc rich condition. Real-time reverse transcription (RT)-PCR was subsequently used to validate the microarray data. Based on the 154 Zur-dependent genes, predicted regulatory Zur motifs were used to screen for potential direct Zur targets including three putative operons znuA, znuCB and ykgM-RpmJ2. The LacZ reporter fusion analysis verified that Zur greatly repressed the promoter activity of the above three operons. The subsequent electrophoretic mobility shift assay (EMSA) demonstrated that a purified Zur protein was able to bind to the promoter regions of the above three operons. The DNase I footprinting was used to identify the Zur binding sites for the above three operons, verifying the Zur box sequence as predicted previously in gamma-Proteobacteria. The primer extension assay was further used to determine the transcription start sites for the above three operons and to localize the -10 and -35 elements. Zur binding sites overlapped the -10 sequence of its target promoters, which was consistent with the previous observation that Zur binding would block the entry of the RNA polymerase to repress the transcription of its target genes. CONCLUSION: Zur as a repressor directly controls the transcription of znuA, znuCB and ykgM-RpmJ2 in Y. pestis by employing a conserved mechanism of Zur-promoter DNA association as observed in gamma-Proteobacteria. Zur contributes to zinc homeostasis in Y. pestis likely through transcriptional repression of the high-affinity zinc uptake system ZnuACB and two alternative ribosomal proteins YkgM and RpmJ2.
Project description:We show that Yersinia enterocolitica strain Ye9 (bio-serotype 2/O:9) utilizes heme-containing molecules as an iron source. The Ye9 genome contains two multigenic clusters, hemPRSTUV-1 and hemPRST-2, encoding putative heme receptors HemR1 and HemR2, that share 62% amino acid identity. Expression of these proteins in an Escherichia coli mutant defective in heme biosynthesis allowed this strain to use hemin and hemoglobin as a source of porphyrin. The hemPRSTUV-1 and hemPRST-2 clusters are organized as operons, expressed from the phem-1 and weaker phem-2 promoters, respectively. Expression of both operons is negatively regulated by iron and the iron-responsive transcriptional repressor Fur. In addition, OmpR, the response regulator of two component system (TCSs) EnvZ/OmpR, represses transcription of both operons through interaction with binding sequences overlapping the -35 region of their promoters. Western blot analysis of the level of HemR1 in ompR, fur, and ompRfur mutants, showed an additive effect of these mutations, indicating that OmpR may regulate HemR expression independently of Fur. However, the effect of OmpR on the activity of the phem-1 promoter and on HemR1 production was observed in both iron-depleted and iron-replete conditions, i.e., when Fur represses the iron-regulated promoter. In addition, a hairpin RNA thermometer, composed of four uracil residues (FourU) that pair with the ribosome-binding site in the 5'-untranslated region (5'-UTR) of hemR1 was predicted by in silico analysis. However, thermoregulated expression of HemR1 could not be demonstrated. Taken together, these data suggest that Fur and OmpR control iron/heme acquisition via a complex mechanism based on negative regulation of hemR1 and hemR2 at the transcriptional level. This interplay could fine-tune the level of heme receptor proteins to allow Y. enterocolitica to fulfill its iron/heme requirements without over-accumulation, which might be important for pathogenic growth within human hosts.
Project description:Clostridium botulinum is a spore-forming bacterium that can produce a very powerful neurotoxin that causes botulism. In this study, we have investigated the Fur transcription regulators in Clostridium botulinum and Fur-regulated genes in Clostridium botulinum A ATCC 3502. We found that gene loss may be the main cause leading to the different numbers of Fur transcription regulators in different Clostridium botulinum strains. Meanwhile, 46 operons were found to be regulated by the Fur transcription regulator in Clostridium botulinum A ATCC 3502, involved in several functional classifications, including iron acquisition, iron utilization, iron transport, and transcription regulator. Under an iron-restricted medium, we experimentally found that a Fur transcription regulator (CBO1372) and two operons (DedA, CBO2610-CBO2614 and ABC transporter, CBO0845-CBO0847) are shown to be differentially expressed in Clostridium botulinum A ATCC 3502. This study has provided-us novel insights into the diversity of Fur transcription regulators in different Clostridium botulinum strains and diversity of Fur-targeted genes, as well as a better understanding of the dynamic changes in iron restriction occurring in response to this stress.
Project description:One prerequisite for the virulence of Yersinia pestis, causative agent of bubonic plague, is the yersiniabactin (Ybt) siderophore-dependent iron transport system that is encoded within a high-pathogenicity island (HPI) within the pgm locus of the Y. pestis chromosome. Several gene products within the HPI have demonstrated functions in the synthesis or transport of Ybt. Here we examine the roles of ybtU and ybtT. In-frame mutations in ybtT or ybtU yielded strains defective in siderophore production. Mutant strains were unable to grow on iron-deficient media at 37 degrees C but could be cross-fed by culture supernatants from a Ybt-producing strain of Y. pestis. The ybtU mutant failed to express four indicator Ybt proteins (HMWP1, HMWP2, YbtE, and Psn), a pattern similar to those for other ybt biosynthetic mutants. In contrast, strains carrying mutations in ybtT or ybtS (a previously identified gene required for Ybt biosynthesis) produced all four proteins at wild-type levels under iron-deprived conditions. To assess the effects of ybtT, -U, and -S mutations on transcription of ybt genes, reporter plasmids with ybtP or psn promoters controlling lacZ expression were introduced into these mutants. Normal iron-regulated beta-galactosidase activity was observed in the ybtT and ybtS mutants, whereas a significant loss of expression occurred in the DeltaybtU strain. These results show that ybtT and ybtU genes are involved in the biosynthesis of the Ybt siderophore and that a ybtU mutation but not ybtT or ybtS mutations affects transcription from the ybtP and psn promoters.