TonB-dependent maltose transport by Caulobacter crescentus.

We have shown previously that Caulobacter crescentus grows on maltodextrins which are actively transported across the outer membrane by the MalA protein. Evidence for energy-coupled transport was obtained by deletion of the exbB exbD genes which abolished transport. However, removal of the TonB protein, which together with the ExbB ExbD proteins is predicted to form an energy-coupling device between the cytoplasmic membrane and the outer membrane, left transport unaffected. Here we identify an additional tonB gene encoded by the cc2334a ORF, which when deleted abolished maltose transport. MalA contains a TonB box that reads EEVVIT and is predicted to interact with TonB. Replacement of valine number 15 in the TonB box by proline abolished maltose transport. Maltose was transported across the cytoplasmic membrane by the MalY protein (CC2283). Maltose transport was induced by maltose and repressed by the MalI protein (CC2284). In addition to MalA, MalY and MalI, the mal locus encodes two predicted cytoplasmic alpha-amylases (CC2285 and CC2286) and a periplasmic glucoamylase (CC2282). The TonB dependence together with the previously described ExbB ExbD dependence demonstrates energy-coupled maltose transport across the outer membrane. MalY is involved in maltose transport across the cytoplasmic membrane by a presumably ion-coupled mechanism.

The colicin G, H and X determinants encode microcins M and H47, which might utilize the catecholate siderophore receptors FepA, Cir, Fiu and IroN.

The colicin G producer Escherichia coli CA46, the colicin H producer E. coli CA58 and E. coli Nissle 1917 (DSM 6601) were shown to produce microcin H47 and the newly described microcin M. Both microcins were exported like colicin V by an RND-type export system, including TolC. The gene cluster encoding microcins H47 and M in strains CA46 and CA58 is nearly identical to that in strain DSM 6601, except that two additional genes are included. A Fur box identified in front of the microcin-encoding genes explained the observed iron regulation of microcin production. The catecholate siderophore receptors Fiu, Cir and FepA from E. coli and IroN, Cir and FepA from Salmonella were identified as receptors for microcins M, H47 and E492. IroN takes up the glucose-containing catecholate siderophore salmochelin, whose synthesis is encoded in the iro gene cluster found in Salmonella and certain, often uropathogenic, E. coli strains. A gene in this iro cluster, iroB, which encodes a putative glycosyltransferase, was also found in the microcin H47/M and microcin E492 gene clusters. These microcins could aid the producing strain in competing against enterobacteria that utilize catecholate siderophores.

Dual repression by Fe(2+)-Fur and Mn(2+)-MntR of the mntH gene, encoding an NRAMP-like Mn(2+) transporter in Escherichia coli.

The uptake of Mn(2+), a cofactor for several enzymes in Escherichia coli, is mediated by MntH, a proton-dependent metal transporter, which also recognizes Fe(2+) with lower affinity. MntH belongs to the NRAMP family of eukaryotic Fe(2+) and Mn(2+) transporters. In E. coli strains with chromosomal mntH-lacZ fusions, mntH was partially repressed by both Mn(2+) and Fe(2+). Inactivation of fur resulted in the loss of Fe(2+)-dependent repression of mntH transcription, demonstrating that Fe(2+) repression depends on the global iron regulator Fur. However, these fur mutants still showed Mn(2+)-dependent repression of mntH. The Mn(2+)-responsive transcriptional regulator of mntH was identified as the gene product of o155 (renamed MntR). mntR mutants were impaired in Mn(2+) but not Fe(2+) repression of mntH transcription. Binding of purified MntR to the mntH operator was manganese dependent. The binding region was localized by DNase I footprinting analysis and covers a nearly perfect palindrome. The Fur binding site, localized within 22 nucleotides of the mntH operator by in vivo operator titration assays, resembles the Fur-box consensus sequence.

The zinc-responsive regulator Zur and its control of the znu gene cluster encoding the ZnuABC zinc uptake system in Escherichia coli.

The synthesis of the Escherichia coli zinc transporter, encoded by the znuACB gene cluster, is regulated in response to the intracellular zinc concentration by the zur gene product. Inactivation of the zur gene demonstrated that Zur acts as a repressor when binding Zn(2+). Eight chromosomal mutant zur alleles were sequenced to correlate the loss of Zur function with individual mutations. Wild-type Zur and ZurDelta46-91 formed homo- and heterodimers. Dimerization was independent of metal ions since it also occurred in the presence of metal chelators. Using an in vivo titration assay, the znu operator was narrowed down to a 31-base pair region overlapping the translational start site of znuA. This location was confirmed by footprinting assays. Zur directly binds to a single region comprising a nearly perfect palindrome. Zinc chelators completely inhibited and Zn(2+) in low concentrations enhanced DNA binding of Zur. No evidence for autoregulation of Zur was found. Zur binds at least 2 zinc ions/dimer specifically. Although most of the mutant Zur proteins bound to the znu operator in vitro, no protection was observed in in vivo footprinting experiments. Analysis of the mutant Zur proteins suggested an amino-terminal DNA contact domain around residue 65 and a dimerization and Zn(2+)-binding domain toward the carboxyl-terminal end.

Subunit vaccination of mice against new world cutaneous leishmaniasis: comparison of three proteins expressed in amastigotes and six adjuvants.

A mixture of well-defined recombinant antigens together with an adjuvant that preferentially stimulates specific gamma interferon (IFN-gamma)-secreting helper type 1 CD4(+) T cells (Th1 cells) presents a rational option for a vaccine against leishmaniasis. The potential of this approach was investigated in murine infections with Leishmania mexicana, which are characterized by the absence of a parasite-specific Th1 response and uncontrolled parasite proliferation. A mixture of three antigens (glycoprotein 63, cysteine proteinases, and a membrane-bound acid phosphatase), which are all expressed in amastigotes, the mammalian stage of the parasite, were used for the immunization of C57BL/6 mice in combination with six adjuvants (interleukin 12 [IL-12], Detox, 4'-monophosphoryl lipid A, QS-21, Mycobacterium bovis BCG, and Corynebacterium parvum). All six vaccine formulations containing the mixture of recombinant antigens were protective against challenge infections with promastigotes, the insect stage of the parasite, in that mice controlled and healed infections but developed transient and, in certain cases, accentuated disease. The most effective adjuvants were IL-12 followed by Detox. Further studies using these two adjuvants showed that a similar protective effect was observed with a mixture of the corresponding native proteins, and mice which had controlled the infection showed a preponderance of IFN-gamma-secreting CD4(+) T cells in the lymph nodes draining the lesion. Using the recombinant proteins individually, it is shown that the relatively abundant cysteine proteinases and glycoprotein 63, but not the acid phosphatase, are able to elicit a protective response. The results are discussed in comparison to previous studies with subunit vaccines and with respect to cell biological aspects of antigen presentation in Leishmania-infected macrophages.

SufS is a NifS-like protein, and SufD is necessary for stability of the [2Fe-2S] FhuF protein in Escherichia coli.

Escherichia coli fhuF mutants, a sufS::MudI mutant, and a sufD::MudI mutant were found to have the same phenotype: the inability to use ferrioxamine B as an iron source in a plate assay. In addition, the sufS and sufD genes were shown to be regulated by the iron-dependent Fur repressor. Sequence analysis revealed that the sufS open reading frame corresponds to orf f406. The protein SufS belongs to the family of NifS-like proteins, which supply sulfur for [Fe-S] centers. The protein FhuF contains a [2Fe-2S] center. A mutation in the upstream sufD gene (orf f423) caused the same phenotype. The T7 expression system and a His tag allow the isolation in good yield of the FhuF protein from a wild-type strain. In contrast, overproduction of the protein in a DeltasufD strain failed. Radioactive labeling of N-His-FhuF with [35S]methionine showed that the protein was unstable in the DeltasufD mutant.

The ZnuABC high-affinity zinc uptake system and its regulator Zur in Escherichia coli.

In Escherichia coli, lacZ operon fusions were isolated that were derepressed under iron repletion and repressed under iron depletion. Two fusions were localized in genes that formed an operon whose gene products had characteristics of a binding protein-dependent transport system. The growth defect of these mutants on TY medium containing 5mM EGTA was compensated for by the addition of Zn2+. In the presence of 0.5mM EGTA, only the parental strain was able to take up 65Zn2+. This high-affinity transport was energized by ATP. The genes were named znuACB (for zinc uptake; former name yebLMI) and localized at 42 min on the genetic map of E. coli. At high Zn2+ concentrations, the znu mutants took up more 65Zn2+ than the parental strain. The high-affinity 65Zn2+ uptake was repressed by growth in the presence of 10 microM Zn2+. A znuA-lacZ operon fusion was repressed by 5 microM Zn2+ and showed a more than 20-fold increase in beta-galactosidase activity when Zn2+ was bound to 1.5 microM TPEN [tetrakis-(2-pyridylmethyl) ethylenediamine]. To identify the Zn2+-dependent regulator, constitutive mutants were isolated and tested for complementation by a gene bank of E. coli. A complementing gene, yjbK of the E. coli genome, was identified and named zur (for zinc uptake regulation). The Zur protein showed 27% sequence identity with the iron regulator Fur. High-affinity 65Zn2+ transport of the constitutive zur mutant was 10-fold higher than that of the uninduced parental strain. An in vivo titration assay suggested that Zur binds to the bidirectional promoter region of znuA and znuCB.