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TonB is an essential component of an energy-generating system that powers active transport across the outer membrane (OM) of compounds that are too large or too scarce to diffuse through porins. The TonB-dependent OM transport proteins (TBDTs) consist of β barrels forming pores that are closed by plugs. The binding of TonB to TBDTs elicits plug movement, which opens the pores and enables nutrient translocation from the cell surface into the periplasm. TonB is also involved in the uptake of certain proteins, particularly toxins, through OM proteins that differ structurally from TBDTs. TonB binds to a sequence of five residues, designated as the TonB box, which is conserved in all TBDTs. Energy from the proton motive force (pmf) of the cytoplasmic membrane is transmitted to TonB by two proteins, ExbB and ExbD. These proteins form an energy-transmitting protein complex consisting of five ExbB proteins, forming a pore that encloses the ExbD dimer. This review discusses the structural changes that occur in TBDTs upon interaction with TonB, as well as the interaction of ExbB-ExbD with TonB, which is required to transmit the energy of the pmf and thereby open TBDT pores. TonB facilitates import of a wide range of substrates., (© 2024 The Author(s). Molecular Microbiology published by John Wiley & Sons Ltd.)

Chaperones facilitate correct folding of newly synthesized proteins. We show here that the periplasmic FkpA chaperone is required for killing Escherichia coli by colicin M entering cells from the outside. Highly active colicin M preparations were inactive against fkpA mutant cells; 10(4)-fold dilutions killed fkpA(+) cells. Three previously isolated spontaneous mutants tolerant to colicin M carried a stop codon or an IS1 insertion in the peptidyl-prolyl-cis-trans-isomerase (PPIase) domain (C-domain) of FkpA, which resulted in deletion of the domain. A randomly generated mutant carried a G148D mutation in the C-domain. A temperature-sensitive mutant tolerant to colicin M carried a Y25N mutation in the FkpA N-domain. Mutants transformed with wild-type fkpA were colicin M-sensitive. Isolated FkpA-His reduced colicin M-His cleavage by proteinase K and renatured denatured colicin M-His in vitro; renaturation was prevented by the PPIase inhibitor FK506. In both assays, periplasmic SurA-His had no effect. No other tested periplasmic chaperone could activate colicin M. Among the tested colicins, only colicin M required FkpA for activity. Colicin M bound to cells via FhuA was inactivated by trypsin; unbound colicin M retained activity. We propose that colicin M unfolds during import across the outer membrane, FkpA specifically assists in folding colicin M into an active toxin in the periplasm and PPIase is essential for colicin M activity. Colicin M is a suitable tool for the isolation of FkpA mutants used to elucidate the functions of the FkpA N- and C-domains.

CdlSt1, a DNA insertion of 1975 bp, was identified within tcdA-C34, the enterotoxin gene of the Clostridium difficile isolate C34. Located in the catalytic domain A1-C34, Cd/St1 combines features of two genetic elements. Within the first 434 nt structures characteristic for group I introns were found; encoding the two transposase-like proteins tlpA and tlpB nucleotides 435-1975 represent the remainder of a IS605-like insertion element. We show that the entire CdlSt1 is accurately spliced from tcdA-C34 primary transcripts and that purified TcdA-C34 toxin is of regular size and catalytic activity. A search for CdlSt1-related sequences demonstrates that the element is widespread in toxinogenic and non-toxinogenic C. difficile strains, indicating the mobility of CdlSt1. In strain C34, we characterize 10 CdlSt1 variants; all are highly homologous to CdlSt1 (> 93% identity), integrated in bacterial open reading frames (ORFs), show the typical composite structure of CdlSt1 and are precisely spliced from their primary transcripts. CdlSt1-like chimeric ribozymes appear to combine the invasiveness of an insertion element with the splicing ability of a group I intron, rendering transposition harmless for the interrupted gene.

FhuA in the outer membrane of Escherichia coli serves as a transporter for ferrichrome, the antibiotics albomycin and rifamycin CGP4832, colicin M, and as receptor for phages T1, T5 and phi80. The previously determined crystal structure reveals that residues 160-714 of the mature protein form a beta-barrel that is closed from the periplasmic side by the globular N-proximal fragment, residues 1-159, designated the cork. In this study, deletion of the cork resulted in a stable protein, FhuADelta5-160, that was incorporated in the outer membrane. Cells that synthesized FhuADelta5-160 displayed a higher sensitivity to large antibiotics such as erythromycin, rifamycin, bacitracin and vancomycin, and grew on maltotetraose and maltopentaose in the absence of LamB. Higher concentrations of ferrichrome supported growth of a tonB mutant that synthesized FhuADelta5-160. These results demonstrate non-specific diffusion of compounds across the outer membrane of cells that synthesize FhuADelta5-160. However, growth of a FhuADelta5-160 tonB wild-type strain occurred at low ferrichrome concentrations, and ferrichrome was transported at about 45% of the FhuA wild-type rate despite the lack of ferrichrome binding sites provided by the cork. FhuADelta5-160 conferred sensitivity to the phages and colicin M at levels similar to that of wild-type FhuA, and to albomycin and rifamycin CGP 4832. The activity of FhuADelta5-160 depended on TonB, although the mutant lacks the TonB box (residues 7-11) previously implicated in the interaction of FhuA with TonB. CCCP inhibited tonB-dependent transport of ferrichrome through FhuADelta5-160. FhuADelta5-160 still functions as a specific transporter, and sites in addition to the TonB box are involved in the TonB-mediated response of FhuA to the proton gradient of the cytoplasmic membrane. It is proposed that TonB interacts with the TonB box of FhuA and with the beta-barrel to release ferrichrome from the FhuA binding sites and to open the channel in FhuA. For transport of ferrichrome through the open channel of FhuADelta5-160, interaction of TonB with the beta-barrel is sufficient to release ferrichrome from the residual binding sites at the beta-barrel and to induce the active conformation of the L4 loop at the cell surface for infection by the TonB-dependent phages T1 and phi80.

The ShlB protein in the outer membrane of Serratia marcescens is the only protein known to be involved in secretion of the ShlA protein across the outer membrane. At the same time, ShlB converts ShlA into a haemolytic and a cytolytic toxin. Surface-exposed residues of ShlB were determined by reaction of an M2 monoclonal antibody with the M2 epitope DYKDDDDK inserted at 25 sites along the entire ShlB polypeptide. The antibody bound to the M2 epitope at 17 sites in intact cells, which indicated surface exposure of the epitope, and to 23 sites in isolated outer membranes. Two insertion mutants contained no ShlB(M2) protein in the outer membrane. The ShlB derivatives activated and/or secreted ShlA. To gain insights into the secretion mechanism, we studied whether highly purified ShlB and ShlB deletion derivatives formed pores in artificial lipid bilayer membranes. Wild-type ShlB formed channels with very low single channel conductance that rarely assumed an open channel configuration. In contrast, open channels with a considerably higher single channel conductance were observed with the deletion mutants ShlB(Delta65-186), ShlB(Delta87-153), and ShlB(Delta126-200). ShlB(Delta126-200) frequently formed permanently open channels, whereas the conductance caused by ShlB(Delta65-186) and ShlB(Delta87-153) did not assume a stationary value, but fluctuated rapidly between open and closed configurations. The results demonstrate the orientation of large portions of ShlB in the outer membrane and suggest that ShlB may function as a specialized pore through which ShlA is secreted.

The Escherichia coli iron transport system via ferrichrome belongs to the group of ATP-dependent transporters that are widely distributed in prokaryotes and eukaryotes. Transport across the cytoplasmic membrane is mediated by three proteins: FhuD in the periplasm, FhuB in the cytoplasmic membrane and FhuC (ATPase) associated with the inside of the cytoplasmic membrane. Interaction of FhuD with FhuB was studied in vitro with biotinylated synthetic 10 residue and 20-24 residue peptides of FhuB by determining the activity of beta-galactosidase linked to the peptides via streptavidin. Peptides identical in sequence to only one of the four periplasmic loops (loop 2), predicted by a transmembrane model of FhuB, and peptides representing a transmembrane segment and part of the adjacent cytoplasmic loop 7 of FhuB bound to FhuD. Decapeptides were transferred into the periplasm of cells through a FhuA deletion derivative that forms permanently open channels three times as large as the porins in the outer membrane. FhuB peptides that bound to FhuD inhibited ferrichrome transport, while peptides that did not bind to FhuD did not affect transport. These data led us to propose that the periplasmic FhuD interacts with a transmembrane region and the cytoplasmic segment 7 of FhuB. The transmembrane region may be part of a pore through which a portion of FhuD inserts into the cytoplasmic membrane during transport. The cytoplasmic segment 7 of FhuB contains the conserved amino acid sequence EAA...G (in FhuB DTA ...G) found in ABC transporters, which is predicted to interact with the cytoplasmic FhuC ATPase. Triggering of ATP hydrolysis by substrate-loaded FhuD may occur by physical interaction between FhuD and FhuC, which bind close to each other on loop 7. Although FhuB consists of two homologous halves, FhuB(N) and FhuB(C), the sites identified for FhuD-mediated ferrichrome transport are asymmetrically arranged.

The cytolytic and haemolytic activity of Serratia marcescens is determined by the ShlA protein, which is secreted across the outer membrane with the aid of the ShlB protein. In the absence of ShlB, inactive ShlA* remains in the periplasm of Escherichia col transformed with an shlA-encoding plasmid, which indicates that ShlB converts ShlA* to active ShlA. ShlA* in a periplasmic extract and partially purified ShlA* were activated in vitro by partially purified ShlB. When both proteins were highly purified, ShlA* was only activated by ShlB when phosphatidylethanolamine (PE) or phosphatidylserine was added to the assay, while phosphatidylglycerol contributed little to ShlA* activation. Lyso-PE, cardiolipin, phosphatidylcholine, phosphatidic acid, lipopolysaccharide and various detergents could not substitute for PE. Although radioactively labelled PE was so tightly associated with ShlA that it remained bound to ShlA after heating and SDS-PAGE, it was not covalently linked to ShlA as PE could be removed by thin-layer chromatography with organic solvents. The number of PE molecules associated per molecule of ShlA was 3.9 +/- 2.2. Active ShlA was inactivated by treatment with phospholipase A2, which indicated that PE is also required for ShlA activity. ShlA-255 (containing the 255 N-terminal amino acids of ShlA) reversibly complemented ShlA* to active ShlA and was inactivated by phospholipase A2, which demonstrated that PE binds to the N-terminal portion of ShlA; this region has previously been found to be involved in ShlA secretion and activation. Electrospray mass spectroscopy of ShlA-255 determined a molar mass that corresponded to that of unmodified ShlA-255. An E. coli mutant that synthesized only minute amounts of PE did not secrete ShlA but contained residual cell-bound haemolytic activity. Since PE binds strongly to ShlA* in the absence of ShlB without converting ShlA* to haemolytic ShlA, ShlB presumably imposes a conformation on ShlA that brings PE into a position to mediate interaction of the hydrophilic haemolysin with the lipid bilayer of the eukaryotic membrane.

Ferric citrate induces transcription of the ferric citrate transport genes fecABCDE without entering the cells of Escherichia coli K-12. Point mutants of the outer membrane-receptor protein FecA are affected in induction independent of the FecA transport activity, suggesting that FecA is directly involved in induction. Alignment of FecA with the other ferric siderophore receptors of E. coli reveals an N-terminal extension in FecA that is not found in the receptors whose synthesis is not induced by their cognate ferric siderophores. In this study, we show that excision of the N-terminal region abolished the inducing activity of FecA, but retained its transport activity. Overproduction of the N-terminal FecA fragment inhibited FecA-dependent induction, but not transport. Constitutive expression caused by C-terminally truncated FecR derivatives was not inhibited by the N-terminal FecA fragment. The N-terminal region of FecA was localized in the periplasm, which indicates that FecA probably interacts with FecR, which is involved in signal transduction across the cytoplasmic membrane. Transcription initiation of the fec transport genes required the Ton system, consisting of TonB, ExbB, and ExbD, and was inhibited by carbonylcyanide-m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP), which dissipate the electrochemical potential of the cytoplasmic membrane. fec transcription of mutant fecA4, which displays constitutive fec transcription in the absence of TonB, was not affected by CCCP. The data support a model that proposes initiation of fec transport gene transcription by binding of ferric citrate to FecA. The transcription initiation signal is transferred across the outer membrane through the activity of the Ton system at the expense of the electrochemical potential of the cytoplasmic membrane. The N-terminus of FecA interacts in the periplasm with the C-terminus of FecR, through which the signal is transferred across the cytoplasmic membrane into the cytoplasm, where it increases the activity of the sigma factor Fecl, which then directs the RNA polymerase to the fec promoter upstream of fecA.

Transcription of the ferric citrate transport system of Escherichia coli K-12 is repressed by Fe(2+)-Fur and activated by ferric citrate. Ferric citrate does not have to enter the cytoplasm; it initiates a signal transduction mechanism by binding to the outer membrane receptor FecA. Presumably, a conformational change is transmitted in a TonB-dependent manner to the FecR protein. FecR activates FecI, and FecI activates transcription of the fecABCDE transport genes. In this communication, FecI was isolated after cloning fecI downstream of an ideal ribosome-binding site. Overexpressed FecI formed inclusion bodies which were solubilized and purified in active form using a mild detergent. FecI, in conjunction with RNA polymerase core enzyme, directed transcription from the fecA promoter in an in vitro run-off transcription assay. Furthermore, FecI retarded the electrophoretic mobility of a specific 75 bp DNA fragment located upstream of fecA. An in vivo competition experiment between the fecA promoters of wild-type and mutant strains identified the nucleotide positions 2747, 2749, 2751 and 2753, located within the 75 bp fragment, as important for FecI-induced transcription. Mobility band shift of fecA promoter DNA caused by cell lysates required growth of cells in the presence of ferric citrate and expression of FecA, FecI and FecR. These data support the previous assignment of FecI, based on sequence homologies, to a new subfamily of eubacterial RNA polymerase sigma 70 factors that respond to extra-cytoplasmic stimuli and regulate extracytoplasmic functions.

Uptake of a new colicin, colicin 10 (Col10), into cells of Escherichia coli required TonB, ExbBD (Ton system), but its cognate receptor, Tsx, functioned independently of Ton and TolQRAB (Tol system). Uptake of Col10 also required TolC which is unique for a Ton-coupled translocation through the outer membrane. A 2470 bp DNA fragment from the natural plasmid pCol10 encoding the Col10 activity (cta), immunity (cti) and lysis (ctl) genes was sequenced. The Cta, Cti and Ctl proteins, as deduced from the nucleotide sequences, consisted of 490 (M(r) 53,342), 96 (M(r) 11,586) and 43 (M(r) 4484) amino acid residues, respectively. Col10 (Cta) was highly homologous to colicin E1 in two regions which determined the common TolC requirement for uptake and the pore-forming activity. Col10 and E1 differed entirely in the regions which are predicted to determine the Ton dependence of Col10 and the Tol dependence of E1, and binding to the receptors Tsx and BtuB, respectively. The region responsible for the Ton-dependent uptake of Col10 was localized in the sequence ranging from residues 1 to 43 (Ton region), and the region responsible for the Tol-dependent uptake of colicin E1 extended from residues 1 to 34 (Tol region). Each Tol-dependent colicin contained a pentapeptide homologous to the sequence DGSGS in the Tol region of E1 which is proposed to be implicated in Tol-dependent uptake (TolA box). After the exchange of the Ton and the Tol regions between Col10 and E1, the Col10-E1 fusion protein was carried into cells via the Ton system and BtuB, whereas the E1-Col10 fusion protein was imported via the Tol system and Tsx. Although the immunity proteins of Col10 and E1 displayed a low homology, Cti conferred full immunity to E1, in contrast to the immunity protein of E1 which did not protect cells against Col10. It is proposed that Col10 belongs to the colicin E1, Ia, Ib group as opposed to the colicin A, B, N group of pore-forming colicins. Col10 consists of 4 domains of which two are very similar and two are very different to E1, supporting our previous proposal that colicins evolved by recombination of DNA fragments which encode uptake and activity domains.

Citrate-dependent Fe3+ transport into Escherichia coli K-12 is induced by iron and citrate. The inducer is probably ferric dicitrate which does not have to be taken up into the cytoplasm to induce transcription of the fec transport genes. Two regulatory genes, fecI and fecR, located upstream of the fecABCDE transport genes, are required for induction. We report that in vivo the chromosomally encoded FecI protein activates transcription of the fecA and fecB transport genes in response to ferric citrate and the FecR protein. Cells expressing chromosomally and plasmid-encoded truncated FecR derivatives no longer responded to ferric citrate and expressed the fec transport genes constitutively. The smallest active FecR derivative contained 59 amino acid residues as compared to the 317 residues of wild-type FecR. Constitutive induction was lower than induction of the FecR wild-type strain by ferric citrate. It is concluded that the N-terminal portion of FecR activates FecI and that the C-terminal portion of FecR responds to ferric citrate. Transcription of the fec transport genes is positively regulated by FecI and FecR and negatively regulated by the Fe2(+)-Fur repressor. Transcription activation and repression may occur independently of each other.

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.

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.

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.

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.

The TonB protein plays a key role in the energy-coupled transport of iron siderophores, of vitamin B12, and of colicins of the B-group across the outer membrane of Escherichia coli. In order to obtain more data about which of its particular amino acid sequences are necessary for TonB function, we have cloned and sequenced the tonB gene of Serratia marcescens. The nucleotide sequence predicts an amino acid sequence of 247 residues (Mr 27,389), which is unusually proline-rich and contains the tandem sequences (Glu-Pro)5 and (Lys-Pro)5. In contrast to the TonB proteins of E. coli and Salmonella typhimurium, translation of the S. marcescens TonB protein starts at the first methionine residue of the open reading frame, which is the only amino acid removed during TonB maturation and export. Only the N-terminal sequence is hydrophobic, suggesting its involvement in anchoring the TonB protein to the cytoplasmic membrane. The S. marcescens tonB gene complemented an E. coli tonB mutant with regard to uptake of iron siderophores, and sensitivity to phages T1 and phi 80, and to colicins B and M. However, an E. coli tonB mutant transformed with the S. marcescens tonB gene remained resistant to colicins Ia and Ib, to colicin B derivatives carrying the amino acid replacements Val/Ala and Val/Gly at position 20 in the TonB box, and they exhibited a tenfold lower activity with colicin D. In addition, the S. marcescens TonB protein did not restore T1 sensitivity of an E. coli exbB tolQ double mutant, as has been found for the overexpressed E. coli TonB protein, indicating a lower activity of the S. marcescens TonB protein. Although the S. marcescens TonB protein was less prone to proteolytic degradation, it was stabilized in E. coli by the ExbBD proteins. In E. coli, TonB activity of S. marcescens depended either on the ExbBD or the TolQR activities.

Colicin M (Cma) displays a unique mode of action in that it inhibits peptidoglycan and lipopolysaccharide biosynthesis through interference with bactoprenyl phosphate recycling. Protection of Cma-producing cells by the immunity protein (Cmi) was studied. The amount of Cmi determined the degree of inhibition of in vitro peptidoglycan synthesis by Cma. In cells, immunity breakdown could be achieved by overexpression of the Cma uptake system. Full immunity was restored after raising the cmi gene copy number. In sphaeroplasts, Cmi was degraded by trypsin, but this could be prevented by the addition of Cma. The N-terminal end includes the only hydrophobic amino acid sequence of Cmi, suggesting a function in anchoring of Cmi in the cytoplasmic membrane. It is proposed that Cmi does not act catalytically but binds Cma at the periplasmic face of the cytoplasmic membrane, thereby resulting in Cma inactivation. Two other possible modes of colicin M immunity, interference of Cmi with the uptake of Cma, and interaction of Cmi with the target of Cma, were ruled out by the data.

The pore-forming colicin B is taken up into Escherichia coli by a receptor and TonB-dependent process. The receptor and colicin B both contain a similar amino acid sequence, close to the N-terminal end, termed the TonB box. Point mutations were introduced into the TonB-box region of the colicin B structural gene cba resulting in colicin B derivatives which were partially or totally inactive against E. coli cells. All derivatives still bound to the receptor. An inactive derivative killed cells when translocated across the outer membrane by osmotic shock treatment, and formed pores in planar lipid bilayer membranes identical to the wild-type colicin. Some of the mutations were partially suppressed by mutations in the tonB structural gene. It was concluded that the TonB-box mutations define a region that is involved in the uptake of colicin B across the outer membrane.

The fhuE gene of Escherichia coli codes for an outer-membrane receptor protein required for the uptake of iron(III) via coprogen, ferrioxamine B and rhodotorulic acid. The amino acid sequence, deduced from the nucleotide sequence, consisted of 729 residues. The mature form, composed of 693 residues, has a calculated molecular weight of 77,453, which agrees with the molecular weight of 76,000 determined by polyacrylamide gel electrophoresis. The FhuE protein contains four regions of homology with other TonB-dependent receptors. A valine to proline exchange in the 'TonB box' abolished transport activity. Phenotypic revertants with substitutions of arginine, glutamine, or leucine at the valine position exhibited increasing iron-coprogen transport rates. Point mutations resulting in the replacement of glycine (127) in the second homology region with either alanine, aspartate, valine, asparagine or histidine exhibited decreased transport rates (listed in descending order). A truncated FhuE protein lacking 24 amino acids at the C-terminal end was exported to the periplasm but failed to be inserted into the outer membrane.

The haemolytic activity of Serratia marcescens is determined by two proteins, ShlA and ShlB. ShlA integrates into the erythrocyte membrane and causes osmotic lysis through channel formation. The conformation of ShlA and its interaction with erythrocyte membranes were studied by determining the cleavage of ShlA by added trypsin. Our results suggest that the conformation of inactive ShlA (from an ShlB- strain) differs from the active ShlA, and that in a hydrophobic environment (detergent or membrane) active ShlA assumes a conformation distinct from that in buffer. Only active haemolysin adsorbed to erythrocytes. ShlA was firmly integrated into the erythrocyte membrane since it was only released under conditions which also dissolved the integral erythrocyte membrane proteins. Moreover, ShlA integrated into 'ghosts' remained there and was not haemolytic when incubated with erythrocytes. From the trypsin cleavage pattern obtained with haemolysin and C-terminally truncated, but still active, haemolysin derivatives integrated into erythrocytes, and sealed and unsealed erythrocyte 'ghosts', we conclude that ShlA is preferentially cleaved by trypsin at a few sites but only from the inside of the erythrocyte. Haemolysin in the erythrocyte membrane forms a water-filled channel and is resistant to trypsin and other proteases.

The nucleotide sequence of a 2.4 kb Dral-EcoRV fragment of pColD-CA23 DNA was determined. The segment of DNA contained the colicin D structural gene (cda) and the colicin D immunity gene (cdi). From the nucleotide sequence it was deduced that colicin D had a molecular weight of 74,683 D and that the immunity protein had a molecular weight of 10,057 D. The amino-terminal portion of colicin D was found to be 96% homologous with the same region of colicin B. Both colicins share the same cell-surface receptor, FepA, and require the TonB protein for uptake. A putative TonB box pentapeptide sequence was identified in the amino terminus of the colicin D protein sequence. Since colicin D inhibits protein synthesis, it was unexpected that no homology was found between the carboxy-terminal part of this colicin and that of the protein synthesis inhibiting colicin E3 and cloacin DF13. This could indicate that colicin D does not function in the same manner as the latter two bacteriocins. The observed homology with colicin B supports the domain structure concept of colicin organization. The structural organization of the colicin operon is discussed. The extensive amino-terminal homology between colicins D and B, and the strong carboxy-terminal homology between colicins B, A, and N suggest an evolutionary assembly of colicin genes from a few DNA fragments which encode the functional domains responsible for colicin activity and uptake.