Biomedical subjects

V Braun

Publications and source records attributed to V Braun.

At least 127 records · Page 7Linked to original sources

PubMed1994

Energy-coupled colicin transport through the outer membrane of Escherichia coli K-12: mutated TonB proteins alter receptor activities and colicin uptake.

The current model of TonB-dependent colicin transport through the outer membrane of Escherichia coli proposes initial binding to receptor proteins, vectorial release from the receptors and uptake into the periplasm from where the colicins, according to their action, insert into the cytoplasmic membrane or enter the cytoplasm. The uptake is energy-dependent and the TonB protein interacts with the receptors as well as with the colicins. In this paper we have studied the uptake of colicins B and Ia, both pore-forming colicins, into various tonB point mutants. Colicin Ia resistance of the tonB mutant (G186D, R204H) was consistent with a defective Cir receptor-TonB interaction while colicin Ia resistance of E. coli expressing TonB of Serratia marcescens, or TonB of E. coli carrying a C-terminal fragment of the S. marcescens TonB, seemed to be caused by an impaired colicin Ia-TonB interaction. In contrast, E. coli tonB (G174R, V178I) was sensitive to colicin Ia and resistant to colicin B unless TonB, ExbB and ExbD were overproduced which resulted in colicin B sensitivity. The differential effects of tonB mutations indicate differences in the interaction of TonB with receptors and colicins.

Bacterial Proteins↗

PubMed1994

Energy-dependent receptor activities of Escherichia coli K-12: mutated TonB proteins alter FhuA receptor activities to phages T5, T1, phi 80 and to colicin M.

The activity of the FhuA receptor in the outer membrane of Escherichia coli is dependent on the TonB, ExbB and ExbD proteins which are anchored to the cytoplasmic membrane. Only infection by phage T5 occurs independently of TonB, ExbB and ExbD. In this paper we describe mutated FhuA proteins which displayed either an increased or decreased FhuA activity to phage T5 when combined with mutated TonB proteins. These results suggest conformational changes in FhuA by TonB which are recognized by phage T5. Similar results were obtained with colicin M and the phages T1 and phi 80. It is proposed that the FhuA mutant proteins assume conformations which are either improved or impaired by the TonB derivatives. For the direct interaction of FhuA with TonB regions which are located outside the TonB box of FhuA and the region around residue 160 of TonB are important.

Bacterial Outer Membrane Proteins↗

PubMed1994

Colicins: structures, modes of action, transfer through membranes, and evolution.

This article intends to inform a broader audience on a fascinating class of protein toxins (bacteriocins) which usually kill only cells of the same species. Those who gained a deeper interest in bacteriocins can find a comprehensive description of the field in a recent book based on a conference (James et al. 1992), and in more specialized review articles dealing with certain aspects (Pugsley 1984a, b), or certain colicins (De Graaf and Oudega 1986; Harkness and Olschläger 1991; Lazdunski et al. 1988). The older literature has been reviewed by Brandis and Smarda (1971), Reeves (1972), Hardy (1975) and Konisky (1982).

Bacterial Outer Membrane Proteins↗

PubMed1994

Inactivation of FhuA at the cell surface of Escherichia coli K-12 by a phage T5 lipoprotein at the periplasmic face of the outer membrane.

Inactivation of phage T5 by lysed cells after phage multiplication is prevented by a phage-encoded lipoprotein (Llp) that inactivates the FhuA outer membrane receptor protein (K. Decker, V. Krauel, A. Meesmann, and K. Heller, Mol. Microbiol. 12:321-332, 1994). Using FhuA derivatives carrying insertions of 4 and 16 amino acid residues and point mutations, we determined whether FhuA inactivation is caused by binding of Llp to FhuA and which regions of FhuA are important for inactivation by Llp. Cells expressing Llp were resistant not only to phage T5 but to all FhuA ligands tested, such as phage phi 80, colicin M, and albomycin, and they were strongly reduced in the uptake of ferrichrome. Most of the FhuA derivatives which were not affected by Llp were, according to a previously published FhuA transmembrane topology model, located in periplasmic turns and in the TonB box close to the periplasm. Since the ligands bind to the cell surface, interaction of FhuA with Llp in the periplasm may induce a FhuA conformation which impairs binding of the ligands. This conclusion was supported by the increase rather than decrease of colicin M sensitivity of two mutants in the presence of Llp. The only Llp-resistant FhuA derivatives with mutations at the cell surface contained insertions of 16 residues in the loop that determines the permeability of the FhuA channel and serves as the principal binding site for all FhuA ligands. This region may be inactivated by steric hindrance in that a portion of Llp penetrates into the channel. Outer membranes prepared with 0.25% Triton X-100 from cells expressing Llp contained inactivated FhuA, suggesting Llp to be an outer membrane protein whose interaction with FhuA was not abolished by Triton X-100. Llp solubilized in 1.1% octylglucoside prevented T5 inactivation by FhuA dissolved in octylglucoside.

Amino Acid Sequence↗

PubMed1994

Selective peripheral denervation for the treatment of spasmodic torticollis.

The results of selective peripheral denervation in 50 patients with spasmodic torticollis are presented. Of our patients, 76% reported a significant improvement or disappearance of their dystonia. The mean follow-up is 25 months. There were no major side effects. We recommend the procedure to patients who primarily have responded to botulinum toxin therapy and had become secondary nonresponders or to those refusing further injections while still responding. The results are much less promising in patients who are primary nonresponders to botulinum toxin. Some remarkable histological findings are presented. The posterior branches of the cervical roots frequently showed signs of severe compression neuropathy. In three cases, a functional motor nerve regeneration was proved. Among all surgical options, selective peripheral denervation provides the best result and has the fewest side effects.

Adolescent↗

PubMed1993

Topology of the ExbB protein in the cytoplasmic membrane of Escherichia coli.

The ExbB protein together with the ExbD and TonB proteins is involved in energy-coupled transport across the outer membrane of Escherichia coli. To understand this unusual process it is required to determine the subcellular location of ExbB and its transmembrane arrangement. Using ExbB-beta-lactamase fusion proteins as reporters for a periplasmic versus a cytoplasmic location of the fusion sites, and accessibility of ExbB in spheroplasts and cell lysates to aminopeptidase K, trypsin, and proteinase K, we arrived at a model of ExbB topology in the cytoplasmic membrane. Starting with the N terminus in the periplasm ExbB contains three transmembrane segments (residues 16-39, 128-155, 162-194) a small periplasmic loop and two large portions in the cytoplasm. Two of the 18 fusion proteins studied, ExbB34-beta-lactamase and ExbB41-beta-lactamase, conferred a high ampicillin resistance. Protease experiments revealed a high respectively low percentage of the molecules in a reverse transmembrane orientation. Both proteins were lacking positive charges at the inner side of the cytoplasmic membrane which determine the orientation of transmembrane segments.

Bacterial Proteins↗

PubMed1993

Conversion of the FhuA transport protein into a diffusion channel through the outer membrane of Escherichia coli.

The FhuA receptor protein is involved in energy-coupled transport of Fe3+ via ferrichrome through the outer membrane of Escherichia coli. Since no energy source is known in the outer membrane it is assumed that energy is provided through the action of the TonB, ExbB and ExbD proteins, which are anchored to the cytoplasmic membrane. By deleting 34 amino acid residues of a putative cell surface exposed loop, FhuA was converted from a ligand specific transport protein into a TonB independent and nonspecific diffusion channel. The FhuA deletion derivative FhuA delta 322-355 formed stable channels in black lipid membranes, in contrast to wild-type FhuA which did not increase membrane conductance. The single-channel conductance of the FhuA mutant channels was at least three times larger than that of the general diffusion porins of E. coli outer membrane. It is proposed that the basic structure of FhuA in the outer membrane is a channel formed by beta-barrels. Since the loop extending from residue 316 to 356 is part of the active site of FhuA, it probably controls the permeability of the channel. The transport-active conformation of FhuA is mediated by a TonB-induced conformational change in response to the energized cytoplasmic membrane. The ferrichrome transport rate into cells expressing FhuA delta 322-355 increased linearly with increasing substrate concentration (from 0.5 to 20 microM), in contrast to FhuA wild-type cells, which displayed saturation at 5 microM. This implies that in wild-type cells ferrichrome transport through the outer membrane is the rate-limiting step and that TonB, ExbB and ExbD are only required for outer membrane transport.

Amino Acid Sequence↗

PubMed1993

Domains of colicin M involved in uptake and activity.

Colicin M inhibits murein biosynthesis by interfering with bactoprenyl phosphate carrier regeneration. It belongs to the group B colicins the uptake of which through the outer membrane depends on the TonB, ExbB and ExbD proteins. These colicins contain a sequence, called the TonB box, which has been implicated in transport via TonB. Point mutations were introduced by PCR into the TonB box of the structural gene for colicin M, cma, resulting in derivatives that no longer killed cells. Mutations in the tonB gene suppressed, in an allele-specific manner, some of the cma mutations, suggesting that interaction of colicin M with TonB may be required for colicin M uptake. Among the hydroxylamine-generated colicin M-inactive cma mutants was one which carried cysteine in place of arginine at position 115. This colicin derivative still bound to the FhuA receptor and killed cells when translocated across the outer membrane by osmotic shock treatment. It apparently represents a new type of transport-deficient colicin M. Additional hydroxylamine-generated inactive derivatives of colicin M carried mutations centered on residues 193-197 and 223-252. Since these did not kill osmotically shocked cells the mutations must be located in a region which is important for colicin M activity. It is concluded that the TonB box at the N-terminal end of colicin M must be involved in colicin uptake via TonB across the outer membrane and that the C-terminal portion of the molecule is likely to contain the activity domain.

Alleles↗

PubMed1993

The TonB protein of Yersinia enterocolitica and its interactions with TonB-box proteins.

The tonB gene is required for energy-dependent transport processes across the outer membrane of gram-negative bacteria. Using the antibiotics albomycin and ferrimycin, a tonB mutant of Yersinia enterocolitica was isolated. Comparison of the tonB mutant with the parent strain revealed that in Y. enterocolitica the uptake of ferrioxamine, ferrichrome, pesticin and heme is TonB-dependent. The tonB gene from Y. enterocolitica was sequenced and found to be similar to those of other Enterobacteria. The Y. enterocolitica tonB gene complemented a Y. enterocolitica tonB mutant. In contrast, some TonB functions of an Escherichia coli tonB mutant were not restored by the tonB gene of Y. enterocolitica. The observed differences in the ability to complement E. coli TonB functions correlated with the degree to which the TonB boxes of the receptors and colicins differed from the TonB box consensus sequence. Furthermore, the N-terminal membrane anchor of the TonB proteins and the TolA protein are likely to form an alpha-helix with an identical sequence motif (SHLS) located at one face of the alpha-helix, suggesting this region to be involved in the functional cross-talk between the TonB-ExbBD- and TolABQR-dependent transport systems across the outer membrane.

Amino Acid Sequence↗

PubMed1993

Enterobacterial hemolysins: activation, secretion and pore formation.

Two types of enterobacterial hemolysins have been studied in detail: the Escherichia coli alpha-hemolysin and the Serratia marcescens hemolysin. Although they have similar properties, they differ entirely in the number and structure of the proteins that determine their hemolytic activities, in the mechanism and the subcellular location of activation and in their secretion mechanisms.

Bacterial Proteins↗

PubMed1993

Activation and secretion of Serratia hemolysin.

The hemolysin of Serratia marcescens (ShlA) is secreted into the culture medium and forms small pores of a defined size in erythrocytes and in black lipid membranes. The protein is synthesized as an inactive precursor of 1608 residues which is translocated across the cytoplasmic membrane by the Sec-export system. In the absence of the outer membrane protein ShlB, the ShlA protein (designated ShlA*) stays in the periplasm and displays about 0.1% of the activity of the secreted form. Secretion of ShlA with the help of ShlB is accompanied by its conversion to the hemolytic form. A ShlA derivative consisting of the N-terminal 238 residues of ShlA is secreted by ShlB, showing that the secretion signal resides in the amino terminal part of ShlA. ShlA* can be activated in vitro by a cell lysate containing ShlB, the activated ShlA remains hemolytic upon removal of ShlB. The assumed covalent modification of ShlA* by ShlB occurs in the N-terminus of ShlA since an amino terminal fragment (M(r) 28,000) secreted by ShlB, and a trypsin fragment of ShlA (M(r) 15,000) are both able to convert ShlA* to a hemolytic protein. In contrast to the permanent modification of ShlA* by ShlB, ShlA activity achieved by complementation with the ShlA fragments is abolished upon removal of the fragments. Apparently, the N-terminal portion of ShlA contains the information for secretion through the outer membrane and for insertion into the erythrocyte membrane. This information is lacking in ShlA* formed in the absence of ShlB but contained in the ShlA fragments formed in the presence of ShlB. The latter bind to ShlA* and direct ShlA* into the erythrocyte membrane. The fragments themselves are too short to build pores. The HpmA hemolysin of Proteus mirabilis shows extensive homology to ShlA. In vitro activation of HpmA* by ShlB and complementation by the 28 kDa ShlA fragment indicates a common activation mechanism.

Amino Acid Sequence↗

PubMed1993

Survey on newly characterized iron uptake systems of Yersinia enterocolitica.

Iron assimilation systems have been shown to be important for virulence in Yersinia enterocolitica. In principle, iron transport in Y. enterocolitica is similarly organized as in Escherichia coli although some differences exist in the siderophores used. A TonB function was identified which in a tonB mutant was complemented by the E. coli tonB gene. Three outer membrane receptors for siderophores were cloned and sequenced: FoxA for ferrioxamine B and E, FcuA for ferrichrome and HemR for heme uptake. In addition, two receptors were identified by mutants: CccA for catechol cephalosporins and FyuA for yersiniabactin, the siderophore produced by virulent yersiniae. In addition, the FyuA protein is assumed to be the pesticin receptor.

Bacterial Proteins↗

PubMed1993

The TonB-dependent ferrichrome receptor FcuA of Yersinia enterocolitica: evidence against a strict co-evolution of receptor structure and substrate specificity.

A Yersinia enterocolitica receptor mutant was isolated which is impaired in ferrichrome uptake. The receptor-encoding gene fcuA was cloned in Escherichia coli K-12. A fcuA mutant of Y. enterocolitica could be complemented by the cloned DNA fragment. The FcuA-encoding region was sequenced and an open reading frame encoding 758 amino acids including a signal sequence of 36 amino acids was found. FcuA shared 34.6% amino acid sequence homology with FatA, the anguibactin receptor of Vibrio anguillarum, but only 20.6% homology with FhuA, the ferrichrome receptor of E. coli. Since the structure of anguibactin differs strongly from that of ferrichrome there seems to be no co-evolution of receptor structure and substrate specificity. The ferrichrome receptors FcuA from Y. enterocolitica and FhuA from E. coli had slightly different substrate specificities. In contrast to FhuA from E. coli, FcuA from Y. enterocolitica was more stereoselective and failed to transport enantio ferrichrome. Three additional ferrichrome receptors were cloned from Pantoea agglomerans (formerly Erwinia herbicola), Salmonella paratyphi B and Salmonella typhimurium. Their substrate specificity was similar but not identical.

Amino Acid Sequence↗

PubMed1993

Amino acid replacements in the Serratia marcescens haemolysin ShIA define sites involved in activation and secretion.

The haemolysin of Serratia marcescens (ShIA) is translocated through the cytoplasmic membrane by the signal peptide-dependent export apparatus. Translocation across the outer membrane (secretion) is mediated by the ShIB protein. Only the secreted form of ShIA is haemolytic. ShIB also converts in vitro inactive ShIA (ShIA*), synthesized in the absence of ShIB, into the haemolytic form (a process termed activation). To define regions in ShIA involved in both processes, ShIA derivatives were isolated and tested for secretion and activation. Analysis of C-terminally truncated proteins (ShIA) assigned the secretion signal to the amino-terminal 238 residues of ShIA. Trypsin cleavage of a secreted ShIA' derivative yielded a 15 kDa N-terminal fragment, by which a haemolytically inactive ShIA* protein could be activated in vitro. It is suggested that the haemolysin activation site is located in this N-terminal fragment. Replacement of asparagine-69 and asparagine-109 by isoleucine yielded inactive haemolysin derivatives. Both asparagine residues are part of two short sequence motifs, reading Ala-Asn-Pro-Asn, which are critical to both activation and secretion. These point mutants as well as N-terminal deletion derivatives which were not activated by ShIB were activated by adding a non-haemolytic N-terminal fragment synthesized in an ShIB+ strain (complementation). Apparently the activated N-terminal fragment substituted for the missing activation of the ShIA derivatives and directed them into the erythrocyte membrane, where they formed pores. It is concluded that activation is only required for initiation of pore formation, and that in vivo activation and secretion are tightly coupled processes. Complementation may also indicate that haemolysin oligomers form the pores.

Amino Acid Sequence↗

PubMed1993

Evolutionary relationship of uptake systems for biopolymers in Escherichia coli: cross-complementation between the TonB-ExbB-ExbD and the TolA-TolQ-TolR proteins.

Escherichia coli possesses two energy-coupled import systems through which substances of low concentration and of a size too large to permit diffusion through the porins are translocated across the outer membrane. Group B colicins, ferric siderophores and vitamin B12 are taken up via the TonB-ExbB-ExbD, group A colicins via the TolA-TolQ-TolR system. Cross-complementation between the two systems was demonstrated in that tolQ tolR mutants transformed with plasmids carrying exbB exbD became sensitive to group A colicins, and exbB exbD mutants transformed with plasmid-encoded tolQ tolR became sensitive to group B colicins. TolQ-TolR interacted through TonB, and ExbB-ExbD interacted through TolA with the outer membrane receptors and colicins. Activity of ExbB ExbD via TolA was higher in cells lacking TonB, and activity of TolQ TolR via TonB was increased when TolA was missing. The very distinct TolA and TonB proteins mediate exclusive interaction with group A and group B receptors, respectively. ExbB-TolR and ExbD-TolQ mixtures showed little if any complementation of exbB exbD and tolQ tolR mutants indicating coevolution of ExbB with ExbD and TolQ with TolR. Sequence homology and mutual functional substitution of ExbB-ExbD and TolQ-TolR suggest the evolution of the two import systems from a single import system.

Bacterial Proteins↗

PubMed1993

Activity domains of the TonB protein.

Escherichia coli and related Gram-negative bacteria contain an energy-coupled transport system through the outer membrane which consists of the proteins TonB, ExbB, ExbD anchored in the cytoplasmic membrane and receptors in the outer membrane. Differences in the activities of the Escherichia coli and the Serratia marcescens TonB proteins were used to identify TonB functional domains. In E.coli TonB segments were replaced by equivalent fragments of S. marcescens TonB and the activities of the resulting chimaeric proteins were determined. In addition, E. coli TonB was truncated at the C-terminal end, and point mutants were generated using bisulphite. From the results obtained we draw the following conclusions: an important site of interaction between TonB and ExbB is located in the N-terminal region of TonB within or close to the cytoplasmic membrane since an N-terminal 44-residue fragment of TonB was stabilized by ExbB and interfered with wild-type TonB activity. In addition, the activity of a TonB derivative in which histidine residue 20 was replaced by arginine was strongly reduced, and a double mutant containing arginine-7 to histidine and alanine-22 to threonine substitutions displayed an impaired uptake of ferrichrome. Furthermore, the domain around residue 160 is involved in TonB activity. S. marcescens TonB segments of this region in E. coli TonB conferred S. marcescens TonB activities, and E. coli TonB point mutants displayed strongly impaired activities for the uptake of colicin B and M and ferric siderophores. Plasmid-encoded tonB mutants of this region showed negative complementation of chromosomal wild-type tonB, and certain tonB mutants suppressed colicin B TonB-box mutants. Uptake of colicins required different domains in TonB, for colicin B and M around residue 160 and for colicin Ia, a domain closer to the C-terminal end. Tandem duplication of the E. coli (EP)X(KP) region by insertion of the S. marcescens (EP)X(KP) region (38 residues) and replacement of lysine residue 91 by glutamate did not alter TonB activity so that no evidence was obtained for this region to be implicated in receptor binding. The aberrant electrophoretic mobility of TonB was caused by the proline-rich sequence since its removal resulted in a normal mobility.

Amino Acid Sequence↗

PubMed1993

Membrane topologies of the TolQ and TolR proteins of Escherichia coli: inactivation of TolQ by a missense mutation in the proposed first transmembrane segment.

The TolQ and TolR proteins of Escherichia coli are required for the uptake of group A colicins and for infection by filamentous phages. Their topology in the cytoplasmic membrane was determined by cleavage with aminopeptidase K, proteinase K, and trypsin in spheroplasts and cell lysates. From the results obtained, it is proposed that the N terminus of TolQ is located in the periplasm and that it contains three transmembrane segments (residues 9 to 36, 127 to 159, and 162 to 191), a small periplasmic loop, and two large portions in the cytoplasm. The N terminus of TolR is located in the cytoplasm and is followed by a transmembrane segment (residues 21 to 40), and the remainder of the protein is located in the periplasm. A tolQ mutant, which rendered cells resistant to group A colicins and sensitive to cholate, had alanine 13 replaced by glycine and was lacking serine 14 in the first transmembrane segment. The membrane topologies of TolQ and TolR are similar to those proposed for ExbB and ExbD, respectively, which is consistent with the partial functional substitution between ExbB and TolQ and between ExbD and TolR. The amino acid sequences of these proteins display the highest homology in the transmembrane segments, which indicates that the membrane-spanning regions play an important role in the activities of the proteins.

Amino Acid Sequence↗

PubMed1993

Insertion derivatives containing segments of up to 16 amino acids identify surface- and periplasm-exposed regions of the FhuA outer membrane receptor of Escherichia coli K-12.

The FhuA receptor in the outer membrane of Escherichia coli K-12 is involved in the uptake of ferrichrome, colicin M, and the antibiotic albomycin and in infection by phages T1, T5, and phi 80. Fragments of up to 16 amino acid residues were inserted into FhuA and used to determine FhuA active sites and FhuA topology in the outer membrane. For this purpose antibiotic resistance boxes flanked by symmetric polylinkers were inserted into fhuA and subsequently partially deleted. Additional in-frame insertions were generated by mutagenesis with transposon Tn1725. The 68 FhuA protein derivatives examined contained segments of 4, 8, 12, 16, and 22 additional amino acid residues at 34 different locations from residues 5 to 646 of the mature protein. Most of the FhuA derivatives were found in normal amounts in the outer membrane fraction. Half of these were fully active toward all ligands, demonstrating proper insertion into the outer membrane. Seven of the 12- and 16-amino-acid-insertion derivatives (at residues 378, 402, 405, 415, 417, 456, and 646) were active toward all of the ligands and could be cleaved by subtilisin in whole cells, suggesting a surface location of the extra loops at sites which did not affect FhuA function. Two mutants were sensitive to subtilisin (insertions at residues 511 and 321) but displayed a strongly reduced sensitivity to colicin M and to phages phi 80 and T1. Four of the insertion derivatives (at residues 162, 223, 369, and 531) were cleaved only in spheroplasts and probably form loops at the periplasmic side of the outer membrane. The number and size of the proteolytic fragments indicate cleavage at or close to the sites of insertion, which has been proved for five insertions by amino acid sequencing. Most mutants with functional defects were affected in their sensitivity to all ligands, yet frequently to different degrees. Some mutants showed a specifically altered sensitivity to a few ligands; for example, mutant 511-04 was partially resistant only to colicin M, mutant 241-04 was reduced in ferrichrome and albomycin uptake and showed a reduced colicin M sensitivity, and mutant 321-04 was fully resistant to phage T1 and partially resistant to phage phi 80. The altered residues define preferential binding sites for these ligands. Insertions of 4 to 16 residues at positions 69, 70, 402, 530, 564, and 572 resulted in strongly reduced amounts of FhuA in the outer membrane fraction, varying in function from fully active to inactive. These results provide the basis for a model of FhuA organization in the outer membrane.

Amino Acid Sequence↗