Hemin uptake system of Yersinia enterocolitica: similarities with other TonB-dependent systems in gram-negative bacteria.

The hemin receptor HemR of Yersinia enterocolitica was identified as a 78 kDa iron regulated outer membrane protein. Cells devoid of the HemR receptor as well as cells mutated in the tonB gene were unable to take up hemin as an iron source. The hemin uptake operon from Y. enterocolitica was cloned in Escherichia coli K12 and was shown to encode four proteins: HemP (6.5 kDa), HemR (78 kDa), HemS (42 kDa) and HemT (27 kDa). When expressed in E.coli hemA aroB, a plasmid carrying genes for HemP and HemR allowed growth on hemin as a porphyrin source. Presence of genes for HemP, HemR and HemS was necessary to allow E.coli hemA aroB cells to use hemin as an iron source. The nucleotide sequence of the hemR gene and its promoter region was determined and the amino acid sequence of the HemR receptor deduced. HemR has a signal peptide of 28 amino acids and a typical TonB box at its amino-terminus. Upstream of the first gene in the operon (hemP), a well conserved Fur box was found which is in accordance with the iron-regulated expression of HemR.     

Haem iron-transport system in enterohaemorrhagic Escherichia coli O157:H7

In this study, we identified the iron-transport systems of Escherichia coli O157:H7 strain EDL933. This strain synthesized and transported enterobactin and had a ferric citrate transport system but lacked the ability to produce or use aerobactin. It used haem and haemoglobin, but not transferrin or lactoferrin, as iron sources. We cloned the gene encoding an iron-regulated haem-transport protein and showed that this E. coli haem-utilization gene ( chuA ) encoded a 69 kDa outer membrane protein that was synthesized in response to iron limitation. Expression of this protein in a laboratory strain of E. coli was sufficient for utilization of haem or haemoglobin as iron sources. Mutation of the chromosomal chuA and tonB genes in E. coli O157:H7 demonstrated that the utilization of haemin and haemoglobin was ChuA- and TonB-dependent. Nucleotide sequence analysis of chuA revealed features characteristic of TonB-dependentFur-regulated, outer membrane iron-transport proteins. It was highly homologous to the shuA gene of Shigella dysenteriae and less closely related to hemR of Yersinia enterocolitica and hmuR of Yersinia pestis . A conserved Fur box was identified upstream of the chuA gene, and regulation by Fur was confirmed.     

Cloning of the Serratia marcescens hasF gene encoding the Has ABC exporter outer membrane component: a TolC analogue

The Serratia marcescens haemophore HasA is secreted by an ABC exporter comprising three envelope proteins. The ABC protein (ATP-binding cassette) HasD and the MFP protein (membrane fusion protein) HasE but not the outer membrane component have been isolated previously. In Escherichia coli , TolC, the outer membrane component of the haemolysin transporter, can form a hybrid exporter with HasD and HasE. This hybrid secretes HasA and the very similar metalloproteases from S. marcescens and Erwinia chrysanthemi . By analogy, the genuine exporter was predicted to secrete metalloproteases. The hasF gene was thus cloned from S. marcescens into an E. coli tolC mutant carrying hasD and hasE genes, by screening for a proteolytic phenotype on skimmed-milk plates. hasF encodes a protein sharing 74% identity with the E. coli TolC protein. Anti-TolC antibodies cross-reacted with a protein with an apparent molecular weight of 53 kDa in E. coli expressing hasF and in S. marcescens . hasF is unlinked to the has cluster and, unlike the has operon, is not iron regulated. hasF complements some of the tolC phenotypes, including drug- and detergent sensitivities and haemolysin secretion but not colicin E1 uptake. This suggests that the various functions of TolC could correspond to distinct domains on the protein.     

Cloning and expression in Escherichia coli of a recA homologue from Mycobacterium tuberculosis.

A 3.8 kb PstI fragment of Mycobacterium tuberculosis was cloned in a recA-deleted Escherichia coli by selecting transformants with increased EMS resistance. The cloned fragment restored homologous recombination in Hfr crosses and conferred resistance to long wave (302 nm) but not short wave (254 nm) UV light. E. coli containing the 3.8 kb PstI fragment produced a 38-40 kDa protein which cross-reacted with antibodies raised against the E. coli RecA protein. The cloned DNA thus probably encodes a RecA homologue.     

Genetics and regulation of heme iron transport in Shigella dysenteriae and detection of an analogous system in Escherichia coli O157:H7.

Shigella species can use heme as the sole source of iron. In this work, the heme utilization locus of Shigella dysenteriae was cloned and characterized. A cosmid bank of S. dysenteriae serotype 1 DNA was constructed in an Escherichia coli siderophore synthesis mutant incapable of heme transport. A recombinant clone, pSHU12, carrying the heme utilization system of S. dysenteriae was isolated by screening on iron-poor medium supplemented with hemin. Transposon insertional mutagenesis and subcloning identified the region of DNA in pSHU12 responsible for the phenotype of heme utilization. Minicell analysis indicated that a 70-kDa protein encoded by this region was sufficient to allow heme utilization in E. coli. Synthesis of this protein, designated Shu (Shigella heme uptake), was induced by iron limitation. The 70-kDa protein is located in the outer membrane and binds heme, suggesting it is the S. dysenteriae heme receptor. Heme iron uptake was found to be TonB dependent in E. coli. Transformation of an E. coli hemA mutant with the heme utilization subclone, pSHU262, showed that heme could serve as a source of porphyrin as well as iron, indicating that the entire heme molecule is transported into the bacterial cell. DNA sequences homologous to shu were detected in strains of S. dysenteriae serotype 1 and E. coli O157:H7.     

Cloning and characterization of the Vibrio cholerae genes encoding the utilization of iron from haemin and haemoglobin

Vibrio cholerae can utilize haemin or haemoglobin as its sole source of iron. Four haem utilization mutants of a classical strain of V. cholerae were isolated. These mutations were complemented with pHUT1, a cosmid clone isolated from a library of wild-type CA401 DNA. Two independent Tn5 insertions into the cloned sequence disrupted function in all of the complemented mutants. Escherichia coli 1017 transformed with pHUT1 failed to utilize haemin as an iron source; a second plasmid containing a different cloned fragment of V. cholerae DNA (pHUT3) was required in addition to pHUT1 to reconstitute the system in E. coli. Minicell analysis and SDS-PAGE of protein fractions indicate that pHUT10 (a subclone of p>HUT1) encodes a 26 kDa inner membrane protein, and pHUT3 encodes a 77 kDa outer membrane protein. Loss of either protein by Tn5 mutagenesis abolishes haem utilization in E. coli. An E. coli hemA mutant that cannot synthesize porphyrins was transformed with the recombinant plasmids to determine whether the plasmids encoded the ability to transport the porphyrin as well as the iron. The transformants grew aerobically in media containing haemin, whereas the parental strain was unable to grow under these conditions. This indicates that V. cholerae haem-iron utilization genes allow transport of the entire haem moiety into the cell.     

Absence of DNA sequence homology with genes of the Escherichia coli hemB locus in Shiga-toxin producing E. coli (STEC) O157 strains

By molecular cloning of chromosomal DNA of a human faecal Escherichia coli O6:non-motile strain, we identified a 1350-bp DNA segment which is commonly present in laboratory and wild-type E. coli strains but had no homology to DNA of Shiga-toxin producing E. coli O157, O145 and enteropathogens E. coli O55 strains. The nucleotide sequence of the 1350-bp segment cloned on plasmid pEO67 was determined (GenBank accession number AF087670) and a 97.2% sequence homology was found to a region of the E. coli hemB locus with an unknown gene function. The introduction of pEO67 into an STEC O157:H- strain had a stimulating effect on the growth of the recipient strain which was most expressed when bacteria were grown in iron depleted M9 medium with hemin added as the exogenous iron source. This growth effect was not observed with E. coli K-12 carrying pEO67. We suggest that the cloned gene is involved in iron uptake of E. coli and that the alteration in this part of the hemB locus is clonally inherited in genetically closely related STEC O157 and O55 strains.     

The glutamate uptake regulatory protein (Grp) of Zymomonas mobilis and its relation to the global regulator Lrp of Escherichia coli.

After being expressed in Escherichia coli JC5412, which is defective in glutamate transport, a Zymomonas mobilis gene which enabled this strain to grow on glutamate was cloned. This gene encodes a protein with 33% amino acid identity to the leucine-responsive regulatory protein (Lrp) of E. coli. Although overall glutamate uptake in E. coli was increased, the protein encoded by the cloned fragment repressed the secondary H+/glutamate transport system GltP by interaction with the promoter region of the gltP gene. It also repressed the secondary, H(+)-coupled glutamate uptake system of Z. mobilis, indicating that at least one role of this protein in Z. mobilis is to regulate glutamate transport. Consequently, it was designated Grp (for glutamate uptake regulatory protein). When expressed in E. coli, Grp repressed the secondary H+/glutamate transport system GltP by binding to the regulatory regions of the gltP gene. An lrp mutation in E. coli was complemented in trans with respect to the positive expression regulation of ilvIH (coding for acetohydroxy acid synthase III) by a plasmid which carries the grp gene. The expression of grp is autoregulated, and in Z. mobilis, it depends on growth conditions. The putative presence of a homolog of Grp in E. coli is discussed.     

Protein II influences ferrichrome-iron transport in Escherichia coli K12.

Ferrichrome-promoted iron uptake in Escherichia coli K12 is strictly dependent upon the tonA gene product, a 'minor' outer membrane protein. By selection for mutants of E. coli resistant to phages which require 'major' outer membrane proteins as receptors, strains with pronounced protein deficiencies were constructed. Such strains were tested for anomalous behaviour of ferrichrome transport. No significant differences in iron uptake were detected in E. coli K12 strains with markedly reduced amounts of protein I. However, a reduction in the initial velocity (up to 40%) was observed in E. coli deficient in outer membrane protein II. This difference was only evident when cells were grown under iron-starvation conditions; it was abolished when cells were grown in rich medium. Kinetic parameters for ferrichrome transport were determined for maximum velocity but for Km; double reciprocal plots showed a biphasic nature, probably attributable to a limited number of outer membrane binding sites and to the multi-component nature of the ferrichrome-iron transport system.     

Characterization of the ferrous iron uptake system of Escherichia coli.

Escherichia coli has an iron(II) transport system (feo) which may make an important contribution to the iron supply of the cell under anaerobic conditions. Cloning and sequencing of the iron(II) transport genes revealed an open reading frame (feoA) possibly coding for a small protein with 75 amino acids and a membrane protein with 773 amino acids (feoB). The upstream region of feoAB contained a binding site for the regulatory protein Fur, which acts with iron(II) as a corepressor in all known iron transport systems of E. coli. In addition, a Fnr binding site was identified in the promoter region. The FeoB protein had an apparent molecular mass of 70 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was localized in the cytoplasmic membrane. The sequence revealed regions of homology to ATPases, which indicates that ferrous iron uptake may be ATP driven. FeoA or FeoB mutants could be complemented by clones with the feoA or feoB gene, respectively.     

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.     

人FGF-21基因的克隆、表达及其调节脂肪细胞糖代谢的活性

成纤维细胞生长因子FGF-21是FGF家族的一个新成员, 最近的研究发现其具有调节血糖的作用, 有望成为治疗2型糖尿病的基因药物。文章应用RT-PCR技术, 从成人肝脏中克隆人源的FGF-21成熟蛋白基因, 并将其克隆到T载体上, 经测序鉴定后, 将其亚克隆到原核表达载体pSUMO上, 转入大肠杆菌Rosetta(DE3)中。鉴定阳性克隆后, 用IPTG诱导FGF-21表达, 并用Ni-NTA柱进行亲和层析纯化。以3T3-L1脂肪细胞的葡萄糖吸收实验来鉴定FGF-21表达产物促进糖吸收的活性。结果表明, FGF-21成熟蛋白基因大小为546 bp, 测序结果与GenBank数据库中的序列一致。SDS-PAGE与Western blotting结果表明: 人源FGF-21成熟蛋白大小19.4 kDa, 经3T3-L1脂肪细胞的葡萄糖吸收实验验证其具有促进葡萄糖吸收的生物活性, 并且GLUT1是FGF-21发挥生物学作用的终末执行单位。     

High-affinity iron uptake systems present in Erwinia carotovora subsp. carotovora include the hydroxamate siderophore aerobactin.

The phytopathogenic bacterium Erwinia carotovora subsp. carotovora W3C105 produced the hydroxamate siderophore aerobactin under iron-limiting conditions. A survey of 22 diverse strains of E. carotovora revealed that strain W3C105 alone produced aerobactin. The ferric-aerobactin receptor of strain W3C105 was an 80-kDa protein, identified by immunoblots of Sarkosyl-soluble proteins obtained from E. carotovora cells grown in iron-depleted medium and probed with antiserum raised against the 74-kDa ferric-aerobactin receptor encoded by the pColV-K30 plasmid of Escherichia coli. Genes determining aerobactin biosynthesis and uptake were localized to an 11.3-kb EcoRI-HindIII chromosomal fragment of strain W3C105. A 10-kb subclone of the fragment conferred on E. coli DH5 alpha both aerobactin biosynthesis and uptake, determined by cloacin DF13 sensitivity, the presence of the 80-kDa receptor protein, and iron-independent growth of E. coli clones. The aerobactin biosynthesis genes of E. carotovora W3C105 hybridized to those of the pColV-K30 plasmid of E. coli, but the restriction patterns of the aerobactin regions of E. coli and E. carotovora differed. Although the aerobactin region of enteric bacteria is commonly flanked by IS1-like sequences, IS1 sequences were not detected in the genomic DNA or the cloned aerobactin region of E. carotovora. E. coli DH5 alpha cells harboring cloned aerobactin biosynthesis genes from E. carotovora W3C105 produced greater quantities of aerobactin and the 80-kDa ferric-aerobactin receptor when grown in iron-limited than in iron-replete medium. Strain W3C105 grew on an iron-limited medium, whereas derivatives that lacked a functional aerobactin iron acquisition system did not grow on the medium. These results provide evidence for the occurrence and heterogeneity of aerobactin as a high-affinity iron uptake system of both clinical and phytopathogenic species of the Enterobacteriaceae. Although future studies may reveal a role for aerobactin in the virulence or ecology of strain W3C105, a functional aerobactin iron acquisition system is not necessary for the pathogenicity of E. carotovora.     

Molecular cloning of a possible excretion protein of Vibrio cholerae

Abstract The gene for a Vibrio cholerae protein of about kDa (kilodalton) has been cloned and its location within the 1.9-kb cloned DNA fragment determined by transposon insertion and deletion analyses. The proteins encoded within the various plasmids have been analyzed in Escherichia coli K-12 minicells. The 25-kDa protein when expressed in E. coli K-12 allows the release of the periplasmic deoxyribonuclease. It is a minor protein suggesting that the release of DNase is not an artefact due to membrane damage. It is possible that this protein functions as part of an excretion system.
Results with transposon Tn 1725 insertions suggest that it contains a termination site in one orientation and a promoter in the other.     

Characterization of HasB, a Serratia marcescens TonB-like protein specifically involved in the haemophore-dependent haem acquisition system

In Gram-negative bacteria, the TonB-ExbB-ExbD inner membrane multiprotein complex is required for active transport of diverse molecules through the outer membrane. We present evidence that Serratia marcescens, like several other Gram-negative bacteria, has two TonB proteins: the previously characterized TonBSM, and also HasB, a newly identified component of the has operon that encodes a haemophore-dependent haem acquisition system. This system involves a soluble extracellular protein (the HasA haemophore) that acquires free or haemoprotein-bound haem and presents it to a specific outer membrane haemophore receptor (HasR). TonBSM and HasB are significantly similar and can replace each other for haem acquisition. However, TonBSM, but not HasB, mediates iron acquisition from iron sources other than haem and haemoproteins, showing that HasB and TonBSM only display partial redundancy. The reconstitution in Escherichia coli of the S. marcescens Has system demonstrated that haem uptake is dependent on the E. coli ExbB, ExbD and TonB proteins and that HasB is non-functional in E. coli. Nevertheless, a mutation in the HasB transmembrane anchor domain allows it to replace TonBEC for haem acquisition. As the change affects a domain involved in specific TonBEC-ExbBEC interactions, HasB may be unable to interact with ExbBEC, and the HasB mutation may allow this interaction. In E. coli, the HasB mutant protein was functional for haem uptake but could not complement the other TonBEC-dependent functions, such as iron siderophore acquisition, and phage DNA and colicin uptake. Our findings support the emerging hypothesis that TonB homologues are widespread in bacteria, where they may have specific functions in receptor-ligand uptake systems.     

The hmu locus of Yersinia pestis is essential for utilization of free haemin and haem-protein complexes as iron sources

Yersinia pestis strains utilize haem and several haem-protein complexes as sole sources of iron. In this study, the haemin uptake locus (hmu) of Y. pestis KIM6+ was selected from a genomic library by trans-duction into an Escherichia coli siderophore synthesis (entC) mutant. Recombinant plasmids containing a common 16 kb BamHI insert were isolated that allowed E. coli entC to use haemin as an iron source. An 8.6 kb region of this insert was found to be essential for haemin utilization and encoded at least five proteins with molecular masses of 79/77, 44, 37, 35, and 30/27.5 kDa. A 10.9 kb Clal fragment containing the hmu locus showed varying degrees of homology to genomic DNA from Yersinia pseudotuberculosis, Yersinia enter-ocolitica, and other genera of Enterobacteriaceae. An E. coli hemA aroB strain harbouring cloned hmu genes used haemin as both an iron and porphyrin source but only on iron-poor medium, suggesting that haemin uptake is tightly iron regulated. Additionally, haemoglobin and myoglobin were used as iron sources by an E. coli entC (pHMU2.2) strain. Deletion of the hmu locus from Y. pestis KIM6+ chromosome generated a mutant that grew poorly on iron-depleted medium containing free haemin as well as mammalian haem-protein complexes including haemoglobin, haemoglobin-haptoglobin, myoglobin, haem-haemopexin, and haem-albumin unless it was complemented with cloned hmu genes.     

The metal permease ZupT from Escherichia coli is a transporter with a broad substrate spectrum

The Escherichia coli zupT (formerly ygiE) gene encodes a cytoplasmic membrane protein (ZupT) related to members of the eukaryotic ZIP family of divalent metal ion transporters. Previously, ZupT was shown to be responsible for uptake of zinc. In this study, we show that ZupT is a divalent metal cation transporter of broad substrate specificity. An E. coli strain with a disruption in all known iron uptake systems could grow in the presence of chelators only if zupT was expressed. Heterologous expression of Arabidopsis thaliana ZIP1 could also alleviate iron deficiency in this E. coli strain, as could expression of indigenous mntH or feoABC. Transport studies with intact cells showed that ZupT facilitates uptake of 55Fe2+ similarly to uptake of MntH or Feo. Other divalent cations were also taken up by ZupT, as shown using 57Co2+. Expression of zupT rendered E. coli cells hypersensitive to Co2+ and sensitive to Mn2+. ZupT did not appear to be metal regulated: expression of a Phi(zupT-lacZ) operon fusion indicated that zupT is expressed constitutively at a low level.