Biomedical subjects
Publications and source records attributed to Volkmar Braun.
The crystal structures of two transport proteins associated with a fragment of the TonB protein have been reported for the first time in two recent papers. TonB is implicated in transferring energy from the cytoplasmic membrane to the outer membrane of Gram-negative bacteria. The very similar structures of the protein complexes define the mode of interactions of TonB with active transport proteins.
Studies of the ferric citrate transport genes in Escherichia coli K-12 have revealed a novel type of transcriptional regulation. The inducer, ferric citrate, binds to an outer membrane protein and must not be transported into the cells to initiate transcription of the ferric citrate transport genes. Rather, a signaling cascade from the cell surface across the outer membrane, the periplasm, and the cytoplasmic membrane into the cytoplasm transmits information on the presence of the inducer in the culture medium into the cytoplasm, where gene transcription occurs. The outer membrane protein FecA serves as a signal receiver and as a signal transmitter across the outer membrane. The FecR protein serves as a signal receiver in the periplasm and as a signal transmitter across the cytoplasmic membrane into the cytoplasm, where the FecI sigma factor is activated to bind RNA polymerase and specifically initiate transcription of the fecABCDE transport genes by binding to the promoter upstream of the fecA gene. Transcription of the fecI fecR regulatory genes is repressed by Fe2+ bound to the Fur repressor protein. Under iron-limiting conditions, Fur is not loaded with Fe2+, the fecI and fecR genes are transcribed, and the FecI and FecR proteins are synthesized and respond to the presence of ferric citrate in the medium when ferric citrate binds to the FecA protein. Regulation of the fec genes represents the paradigm of a growing number of gene regulation systems involving transmembrane signaling across three cellular compartments.
The antibiotic albomycin is highly effective against Streptococcus pneumoniae, with an MIC of 10 ng/ml. The reason for the high efficacy was studied by measuring the uptake of albomycin into S. pneumoniae. Albomycin was transported via the system that transports the ferric hydroxamates ferrichrome and ferrioxamine B. These two ferric hydroxamates antagonized the growth inhibition by albomycin and salmycin. Cross-inhibition of the structurally different ferric hydroxamates to both antibiotics can be explained by the similar iron coordination centers of the four compounds. [(55)Fe(3+)]ferrichrome and [(55)Fe(3+)]ferrioxamine B were taken up by the same transport system into S. pneumoniae. Mutants in the adjacent fhuD, fhuB, and fhuG genes were transport inactive and resistant to the antibiotics. Albomycin, ferrichrome, ferrioxamine B, and salmycin bound to the isolated FhuD protein and prevented degradation by proteinase K. The fhu locus consisting of the fhuD, fhuB, fhuG, and fhuC genes determines a predicted ABC transporter composed of the FhuD binding lipoprotein, the FhuB and FhuG transport proteins, and the FhuC ATPase. It is concluded that active transport of albomycin mediates the high antibiotic efficacy in S. pneumoniae.
Ferric citrate transport in Escherichia coli involves proteins encoded by the fec genes, including the transport and signaling protein FecA and the signal transducing protein FecR. Randomly isolated FecA point mutants showed a reduced interaction with FecR and a reduced transcription initiation of the ferric citrate transport genes. The mutations were localized on one side of the FecA signaling domain, which might form the interface to FecR. Some of the mutants showed strongly reduced iron transport rates, which suggests that the signaling domain affects the structure of the FecA transporter domain.
A new Escherichia coli phage, named Rtp, was isolated and shown to be closely related to phage T1. Electron microscopy revealed that phage Rtp has a morphologically unique tail tip consisting of four leaf-like structures arranged in a rosette, whereas phage T1 has thinner, flexible leaves that thicken toward the ends. In contrast to T1, Rtp did not require FhuA and TonB for infection. The 46.2-kb genome of phage Rtp encodes 75 open reading frames, 47 of which are homologous to phage T1 genes. Like phage T1, phage Rtp encodes a large number of small genes at the genome termini that exhibit no sequence similarity to known genes. Six predicted genes larger than 300 nucleotides in the highly homologous region of Rtp are not found in T1. Two predicted HNH endonucleases are encoded at positions different from those in phage T1. The sequence similarity of rtp37, -38, -39, -41, -42, and -43 to equally arranged genes of lambdoid phages suggests a common tail assembly initiation complex. Protein Rtp43 is homologous to the lambda J protein, which determines lambda host specificity. Since the two proteins differ most in the C-proximal area, where the binding site to the LamB receptor resides in the J protein, we propose that Rtp43 contributes to Rtp host specificity. Lipoproteins similar to the predicted lipoprotein Rtp45 are found in a number of phages (encoded by cor genes) in which they prevent superinfection by inactivating the receptors. We propose that, similar to the proposed function of the phage T5 lipoprotein, Rtp45 prevents inactivation of Rtp by adsorption to its receptor during cells lysis. Rtp52 is a putative transcriptional regulator, for which 10 conserved inverted repeats were identified upstream of genes in the Rtp genome. In contrast, the much larger E. coli genome has only one such repeat sequence.
In Escherichia coli K-12, transcription of the ferric citrate transport genes fecABCDE is initiated by binding of diferric dicitrate to the outer membrane protein FecA which elicits a signaling cascade from the cell surface to the cytoplasm. The FecI sigma factor is only active in the presence of FecR, which transfers the signal across the cytoplasmic membrane. In other bacteria, fecIRA homologues control iron transport gene transcription by siderophores other than citrate. However, in most cases, the FecI homologues are active in the absence of the FecR homologues, which might function as anti-sigma factors. Since not all E. coli strains contain a fec system, we determined the occurrence of fec genes in selected Enterobacteriaceae and the dependence of FecI activity on FecR. Incomplete FecIRA systems were chromosomally encoded in Enterobacter aerogenes strains and plasmid-encoded in K. pneumoniae. E. coli B, Photorhabdus luminescens and one of three Klebsiella pneumoniae strains had a functional FecIRA regulatory system as in E. coli K-12. The cytoplasmic N-terminal FecR fragments caused constitutive FecI activity in the absence of ferric citrate. The PCR-generated mutant FecI(D40G) was inactive and FecI(S15P) was partially active. FecR of E. coli K-12 activated FecI of all tested strains except FecI encoded on the virulence plasmid pLVPK of K. pneumoniae, which differed from E. coli K-12 FecI by having mutations in region 4, which is important for interaction with FecR. The C-terminally truncated FecR homologue of pLVPK was inactive. pLVPK-encoded FecA contains a 38-residue sequence in front of the signal sequence that did not prevent processing and proper integration of FecA into the outer membrane of E. coli and lacks the signaling sequence required for transcription initiation of the fec transport genes, making it induction-incompetent but transport-competent. The evidence indicates that fecIRABCDE genes are acquired by horizontal DNA transfer and can undergo debilitating mutations.
Studies of the ferric citrate transport genes in Escherichia coli K-12 have revealed a novel type of transcriptional regulation. The inducer, ferric citrate, binds to an outer membrane protein and must not be transported into the cells to initiate transcription of the ferric citrate transport genes. Rather, a signaling cascade from the cell surface across the outer membrane, the periplasm, and the cytoplasmic membrane into the cytoplasm transmits information on the presence of the inducer in the culture medium into the cytoplasm, where gene transcription occurs. The outer membrane protein FecA serves as a signal receiver and as a signal transmitter across the outer membrane. The FecR protein serves as a signal receiver in the periplasm and as a signal transmitter across the cytoplasmic membrane into the cytoplasm, where the FecI sigma factor is activated to bind RNA polymerase and specifically initiate transcription of the fecABCDE transport genes by binding to the promoter upstream of the fecA gene. Transcription of the fecI fecR regulatory genes is repressed by Fe(2+) bound to the Fur repressor protein. Under iron-limiting conditions, Fur is not loaded with Fe(2+), the fecI and fecR genes are transcribed, and the FecI and FecR proteins are synthesized and respond to the presence of ferric citrate in the medium when ferric citrate binds to the FecA protein. Regulation of the fec genes represents the paradigm of a growing number of gene regulation systems involving transmembrane signaling across three cellular compartments.
Analysis of the genome sequence of Caulobacter crescentus predicts 67 TonB-dependent outer membrane proteins. To demonstrate that among them are proteins that transport nutrients other than chelated Fe(3+) and vitamin B(12)-the substrates hitherto known to be transported by TonB-dependent transporters-the outer membrane protein profile of cells grown on different substrates was determined by two-dimensional electrophoresis. Maltose induced the synthesis of a hitherto unknown 99.5-kDa protein, designated here as MalA, encoded by the cc2287 genomic locus. MalA mediated growth on maltodextrins and transported [(14)C]maltodextrins from [(14)C]maltose to [(14)C]maltopentaose. [(14)C]maltose transport showed biphasic kinetics, with a fast initial rate and a slower second rate. The initial transport had a K(d) of 0.2 microM, while the second transport had a K(d) of 5 microM. It is proposed that the fast rate reflects binding to MalA and the second rate reflects transport into the cells. Energy depletion of cells by 100 microM carbonyl cyanide 3-chlorophenylhydrazone abolished maltose binding and transport. Deletion of the malA gene diminished maltose transport to 1% of the wild-type malA strain and impaired transport of the larger maltodextrins. The malA mutant was unable to grow on maltodextrins larger than maltotetraose. Deletion of two C. crescentus genes homologous to the exbB exbD genes of Escherichia coli abolished [(14)C]maltodextrin binding and transport and growth on maltodextrins larger than maltotetraose. These mutants also showed impaired growth on Fe(3+)-rhodotorulate as the sole iron source, which provided evidence of energy-coupled transport. Unexpectedly, a deletion mutant of a tonB homolog transported maltose at the wild-type rate and grew on all maltodextrins tested. Since Fe(3+)-rhodotorulate served as an iron source for the tonB mutant, an additional gene encoding a protein with a TonB function is postulated. Permeation of maltose and maltotriose through the outer membrane of the C. crescentus malA mutant was slower than permeation through the outer membrane of an E. coli lamB mutant, which suggests a low porin activity in C. crescentus. The pores of the C. crescentus porins are slightly larger than those of E. coli K-12, since maltotetraose supported growth of the C. crescentus malA mutant but failed to support growth of the E. coli lamB mutant. The data are consistent with the proposal that binding of maltodextrins to MalA requires energy and MalA actively transports maltodextrins with K(d) values 1,000-fold smaller than those for the LamB porin and 100-fold larger than those for the vitamin B(12) and ferric siderophore outer membrane transporters. MalA is the first example of an outer membrane protein for which an ExbB/ExbD-dependent transport of a nutrient other than iron and vitamin B(12) has been demonstrated.
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The ferric citrate transport system of Escherichia coli is the first example of a transcription initiation mechanism that starts at the cell surface. The inducer, ferric citrate, binds to an outer membrane transport protein, and without further transport elicits a signal that is transmitted across the outer membrane, the periplasm, and the cytoplasmic membrane into the cytoplasm. Signal transfer across the three subcellular compartments is mediated by the outer membrane transport protein that interacts in the periplasm with a cytoplasmic transmembrane protein. The latter is required for activation of a sigma factor which belongs to the extracytoplasmic function sigma factor family. A similar kind of transcription regulation has been demonstrated in Pseudomonas putida, P. aeruginosa, Serratia marcescens, Klebsiella pneumoniae, Aerobacter aerogenes, Bordetella pertussis, B. bronchseptica, B. avium, and Ralstonia solanacearum. The genomes of P. putida, P. aeruginosa, Nitrosomonas europaea, Bacteroides thetaiotaomicron and Caulobacter crescentus predict the existence of many more such transcriptional regulatory devices.
Infection of Escherichia coli K-12 by phages T1 and phi 80 requires the FhuA outer membrane protein and the TonB protein. Mutations in the N-terminal globular domain close to the predicted channel in the beta-barrel of FhuA were created. The FhuA Delta 107-111 N104K K110D L111P mutant and the FhuA(L(109)DPNGLK(110)) insertion mutant were sensitive to phage T1, but nearly resistant to phage phi 80. FhuA Delta 107-111 N104K K110D L111P mediated phage T1 infection in a tonB mutant without formation of TonB-independent phage T1 host-range mutants. The FhuA mutants showed no altered sensitivity to phage T5. Although the phages share overlapping binding sites in FhuA, the structural alterations elicited by the mutations resulted in very different phage sensitivities. In the FhuA deletion mutant, the TonB requirement for phage T1 infection was partially bypassed.
Bacteria have developed a series of iron-scavenging and transport systems. The expression of many of the iron utilization genes is tightly regulated by the Fe2+ loaded Fur repressor protein. In this study, the Fur titration assay (FURTA) was used to screen for DNA fragments from a genomic DNA library of Photobacterium damselae ssp. piscicida containing potential Fe2+ Fur binding sites or iron binding-proteins which withdraw iron from Fur. One of the clones encoded a tonB gene and adjacent a functionally related exbB gene. An additional and complete tonB exbB exbD gene cluster was identified and sequenced. A gene homologous to the ferritin gene was found whose FURTA-positive phenotype may be explained by its iron-binding ability. Genes encoding a putative complete iron-regulated outer membrane transport protein and a pseudogene of a transport protein were found. The FURTA assay also revealed iron regulation of the AraC type transcriptional regulation.
For Serratia marcescens, secreted hemolysin/cytotoxin is not only secreted but also activated by an outer membrane protein. Excluding posttranslational processing by mass spectrometry, the conformation of active and inactive ShlA derivatives strongly differed in electrophoretic mobilities, gel permeation chromatography, sensitivity to trypsin, circular dichroism, and intrinsic fluorescence. We concluded that ShlB interacts with ShlA during secretion and imposes a conformational change in ShlA to form the active hemolysin.
Replacement of glutamate 176, the only charged amino acid in the third transmembrane helix of ExbB, with alanine (E176A) abolished ExbB activity in all determined ExbB-dependent functions of Escherichia coli. Combination of the mutations T148A in the second transmembrane helix and T181A in the third transmembrane helix, proposed to form part of a proton pathway through ExbB, also resulted in inactive ExbB. E176 and T148 are strictly conserved in ExbB and TolQ proteins, and T181 is almost strictly conserved in ExbB, TolQ, and MotA.
Precise deletions of cell surface-exposed loops of FhuA resulted in mutants of Escherichia coli with distinct phenotypes. Deletion of loop 3 or 11 inactivated ferrichrome transport activity. Deletion of loop 8 inactivated receptor activity for colicin M and the phages T1, T5, and phi80. The loop 7 deletion mutant was colicin M resistant but fully phage sensitive. The loop 4 deletion mutant was resistant to the TonB-dependent phages T1 and phi80 but fully sensitive to the TonB-independent phage T5. The phenotypes of the deletion mutants revealed important sites for the multiple FhuA transport and receptor activities. The ligand binding sites are nonidentical and are distributed among the entire exposed surface. Presumably, FhuA evolved as a ferrichrome transporter and was subsequently used as a receptor by the phages and colicin M, which selected the same as well as distinct loops as receptor sites.
The FecA outer membrane protein of Escherichia coli functions as a transporter of ferric citrate and as a signal receiver and signal transmitter for transcription initiation of the fec transport genes. Three FecA regions for which functional roles have been predicted from the crystal structures were mutagenized: (i) loops 7 and 8, which move upon binding of ferric citrate and close the entrance to the ferric citrate binding site; (ii) the dinuclear ferric citrate binding site; and (iii) the interface between the globular domain and the beta-barrel. Deletion of loops 7 and 8 abolished FecA transport and induction activities. Deletion of loops 3 and 11 also inactivated FecA, whereas deletion of loops 9 and 10 largely retained FecA activities. The replacement of arginine residue R365 or R380 and glutamine Q570, which are predicted to serve as binding sites for the negatively charged dinuclear ferric citrate, with alanine resulted in inactive FecA, whereas the binding site mutant R438A retained approximately 50% of the FecA induction and transport activities. Residues R150, E541, and E587, conserved among energy-coupled outer membrane transporters, are predicted to form salt bridges between the globular domain and the beta-barrel and to contribute to the fixation of the globular domain inside the beta-barrel. Mutations E541A and E541R affected FecA induction and transport activity slightly, whereas mutations E587A and E587R more strongly reduced FecA activity. The double mutations R150A E541R and R150A E587R nearly abolished FecA activity. Apparently, the salt bridges are less important than the individual functions these residues seem to have for FecA activity. Comparison of the properties of the FecA, FhuA, FepA, and BtuB transporters indicates that although they have very similar crystal structures, the details of their functional mechanisms differ.
Ferric iron is transported into Escherichia coli by a number of chelating compounds. Iron transport through the outer membrane by citrate, ferrichrome, enterobactin, aerobactin, yersiniabactin, and heme is catalyzed by highly specific proteins and across the cytoplasmic membrane by ABC transport systems with lower specificity. Transport across the outer membrane requires energy, which is provided by the proton motive force of the cytoplasmic membrane and transmitted to the outer membrane via the TonB-ExbB-ExbD proteins. Binding of substrates induces large long-range structural changes in the transport proteins, but does not open the channel. It is thought that the channel is opened by energy input from the cytoplasmic membrane. Although a basic understanding of how the transport proteins might function has been obtained from the crystal structures of three outer membrane proteins of E. coli and from many genetic and biochemical experiments, numerous fundamental questions still remain open. Transcription of the transport protein genes is regulated by the Fur protein, which when loaded with ferrous iron functions as a repressor. Fur also positively regulates genes of iron-containing proteins by repressing synthesis of an anti-sense RNA. Regulation of ferric citrate transport genes via a transmembrane device has become the paradigm of the regulation of a variety of systems, including the hypersensitivity response of plants to bacterial infections.
Induction of the ferric citrate transport genes of Escherichia coli K-12 involves a signalling cascade that starts at the cell surface and proceeds to the cytoplasm. Three specific proteins are involved: FecA in the outer membrane, FecR in the cytoplasmic membrane, and FecI in the cytoplasm. The binding of dinuclear ferric citrate to FecA causes substantial structural changes in FecA, triggering the signal cascade. The amino-proximal end of FecA interacts with the carboxy-proximal end of FecR in the periplasm. FecR then transmits the signal across the cytoplasmic membrane into the cytoplasm and activates the FecI sigma factor, which binds to the RNA polymerase core enzyme and directs the RNA polymerase to the promoter upstream of the fecABCDE transport genes to initiate transcription. Transcription of the fecIR regulatory genes and the fec transport genes is repressed by the Fur protein loaded with Fe(2+). Therefore, transcription of the fec transport genes is subjected to double control: cells first detect iron deficiency and respond by synthesis of the regulatory proteins FecI and FecR, which initiate transcription of the fec transport genes, provided ferric citrate is available. FecI belongs to the extracytoplasmic function sigma factors, which are widespread among bacteria. With the recent sequencing of complete microbial genomes, it has become apparent that the FecIRA cascade is now a paradigm for the regulatory control of FecI family sigmas in Gram-negative bacteria.