Functional complementation between bacterial MDR-like export systems: colicin V, alpha-hemolysin, and Erwinia protease.

The antibacterial protein Colicin V (ColV) is secreted from gram-negative bacteria by a signal sequence-independent pathway. The proteins that mediate the export of ColV share sequence similarities with components from other signal sequence-independent export systems such as those for alpha-hemolysin (Hly) and Erwinia protease (Prt). We report here that the intact HlyBD export system can export active ColV from Escherichia coli strains lacking the ColV export proteins CvaA and CvaB. The individual Hly export genes complement mutations in their respective ColV homologs, but do so at a lower efficiency. When CvaA or CvaB is expressed along with the intact HlyBD exporter, the Cva export protein interferes with export of ColV through the HlyBD system. Gene fusions and point mutations in the ColV structural gene were used to define signals in ColV recognized by the Hly exporter. An export signal in ColV recognized by HlyBD is localized to the amino-terminal 57 amino acids of the protein. In addition, mutations in the ColV export signal differentially affect export through CvaAB and HlyBD, suggesting differences in signal specificity between the Cva and Hly systems. The three Erwinia protease export proteins can also export active ColV, and interference is seen when CvaA or CvaB is expressed along with the intact Prt exporter. Functional complementation is not reciprocal; alpha-hemolysin is not exported through either the ColV system or the Prt system.     

Interactions of dedicated export membrane proteins of the colicin V secretion system: CvaA, a member of the membrane fusion protein family, interacts with CvaB and TolC.

The antibacterial peptide toxin colicin V uses a dedicated signal sequence-independent system for its secretion in Escherichia coli and requires the products of three genes, cvaA, cvaB, and tolC. As a member of the membrane fusion protein family, CvaA is supposed to form a bridge that connects the inner and outer membranes via interaction with CvaB and TolC, respectively. In this study, we investigated the possible interaction of these proteins. When CvaA or CvaB was absent, the corresponding amount of CvaB or CvaA, respectively, was decreased, and the amounts of both proteins were reduced when TolC was depleted. Translational lacZ fusions showed that TolC did not affect the synthesis of either CvaA-beta-galactosidase or CvaB-beta-galactosidase, and CvaA or CvaB did not affect the synthesis of CvaB-beta-galactosidase or CvaA-beta-galactosidase, respectively. However, the stabilities of CvaA and CvaB proteins were affected by the absence of one another and by that of TolC. The instability of CvaA was more severe in TolC-depleted cells than in CvaB-depleted cells. On the other hand, CvaB was less stable in the absence of CvaA than in the absence of TolC. In addition, using a cross-linking reagent, we showed that CvaA directly interacts with both CvaB and TolC proteins. Taken together, these data support the hypothesized structural role of CvaA in connecting CvaB and TolC.     

Characterization of in-frame proteins encoded by cvaA, an essential gene in the colicin V secretion system: CvaA* stabilizes CvaA to enhance secretion.

Colicin V (ColV), an antibacterial peptide toxin, uses a dedicated signal sequence-independent export system for its extracellular secretion in Escherichia coli. The products of at least three genes (a chromosomal tolC gene and two plasmid-born cvaA and cvaB genes) are involved in this process. To characterize the gene products, the cvaA gene was subcloned and expressed under the control of T7 RNA polymerase promoter. Two in-frame proteins, CvaA and CvaA*, were expressed and identified. DNA sequences predicted that both proteins have two potential translational initiation sites. N-terminal peptide sequencing showed that the translation of CvaA starts from a TTG, 11 amino acids upstream of the previously proposed ATG initiation site. CvaA* is translated from an upstream ATG. Expression of both CvaA and CvaA* was induced by the iron chelator 2,2'-dipyridyl, indicating that cvaA is negatively regulated at least partially by Fur. CvaA*-depleted cells were found to secrete less ColV, based on reduced activity in the supernatant, than did wild type, which was recovered by the addition of a plasmid producing CvaA*. Interestingly, CvaA*-depleted and wild-type cells had similar levels of intracellular ColV activity. Translational fusions showed that the syntheses of ColV and CvaA are not affected by CvaA* depletion. However, CvaA in CvaA*-depleted cells was less stable than that in wild-type cells, indicating that CvaA* may directly or indirectly affect the stability of CvaA. We conclude that CvaA* is not essential for ColV secretion but that it enhances the ColV secretion by stabilizing the CvaA protein.     

Nucleotide-dependent dimerization of the C-terminal domain of the ABC transporter CvaB in colicin V secretion

The cytoplasmic membrane proteins CvaB and CvaA and the outer membrane protein TolC constitute the bacteriocin colicin V secretion system in Escherichia coli. CvaB functions as an ATP-binding cassette transporter, and its C-terminal domain (CTD) contains typical motifs for the nucleotide-binding and Walker A and B sites and the ABC signature motif. To study the role of the CvaB CTD in the secretion of colicin V, a truncated construct of this domain was made and overexpressed. Different forms of the CvaB CTD were found during purification and identified as monomer, dimer, and oligomer forms by gel filtration and protein cross-linking. Nucleotide binding was shown to be critical for CvaB CTD dimerization. Oligomers could be converted to dimers by nucleotide triphosphate-Mg, and nucleotide release from dimers resulted in transient formation of monomers, followed by oligomerization and aggregation. Site-directed mutagenesis showed that the ABC signature motif was involved in the nucleotide-dependent dimerization. The spatial proximity of the Walker A site and the signature motif was shown by disulfide cross-linking a mixture of the A530C and L630C mutant proteins, while the A530C or L630C mutant protein did not dimerize on its own. Taken together, these results indicate that the CvaB CTD formed a nucleotide-dependent head-to-tail dimer.     

Topology analysis of the colicin V export protein CvaA in Escherichia coli.

The antibacterial protein toxin colicin V is secreted from Escherichia coli cells by a dedicated export system that is a member of the multicomponent ATP-binding cassette (ABC) transporter family. At least three proteins, CvaA, CvaB, and TolC, are required for secretion via this signal sequence-independent pathway. In this study, the subcellular location and transmembrane organization of membrane fusion protein CvaA were investigated. First, a series of CvaA-alkaline phosphatase (AP) protein fusions was constructed. Inner and outer membrane fractionations of cells bearing these fusions indicated that CvaA is inner membrane associated. To localize the fusion junctions, the relative activities of the fusion proteins, i.e., the amounts of phosphatase activity normalized to the rate of synthesis of each protein, as well as the stability of each fusion, were determined. These results indicated that all of the fusion junctions occur on the same side of the inner membrane. In addition, the relative activities were compared with that of native AP, and the protease accessibility of the AP moieties in spheroplasts and whole cells was analyzed. The results of these experiments suggested that the fusion junctions occur within periplasmic regions of CvA. We conclude that CvaA is an inner membrane protein with a single transmembrane domain near its N terminus; the large C-terminal region extends into the periplasm. This study demonstrates the application of AP fusion analysis to elucidate the topology of a membrane-associated protein having only a single transmembrane domain.     

Mutational Analysis of CvaA in the Highly Conserved Domain of the Membrane Fusion Protein Family

The antibacterial peptide toxin colicin V (ColV) uses a dedicated signal sequence-independent export system for its secretion in Escherichia coli that involves the products of three genes, cvaA, cvaB, and tolC in this process. As a member of the membrane fusion protein (MFP) family, the CvaA protein has been proposed to interact with an outer membrane protein TolC via its C-terminal hydrophobic domain. The importance of this domain, which is highly conserved throughout the members of MFP family, was analyzed by use of site-directed mutagenesis of missense or nonsense mutations with suppressors. All the nonsense mutations tested resulted in the loss of ColV secretion, indicating the importance of the C-terminus of CvaA, including the last 100 residue–hydrophilic domain. The missense mutations of several conserved amino acids have no drastic effects. On the other hand, when Glu-248, Ala-262, Thr-274, Leu-285, Gly-313, Ala-322, or Val-335 of CvaA protein was mutated, the secretion of ColV was greatly reduced in certain mutants. While some mutations resulted in structural instability, Glu-248 to Lys and Ala-322 to Gly proteins were relatively stable, but were not functional in ColV secretion. The results indicate that these conserved amino acids are important for the structure and functions of CvaA in the secretion of ColV. Received: 6 February 1999 / Accepted: 26 June 1999     

Genetic analysis of an MDR-like export system: the secretion of colicin V.

The extracellular secretion of the antibacterial toxin colicin V is mediated via a signal sequence independent process which requires the products of two linked genes: cvaA and cvaB. The nucleotide sequence of cvaB reveals that its product is a member of a subfamily of proteins, involved in the export of diverse molecules, found in both eukaryotes and prokaryotes. This group of proteins, here referred to as the 'MDR-like' subfamily, is characterized by the presence of a hydrophobic region followed by a highly conserved ATP binding fold. By constructing fusions between the structural gene for colicin V, cvaC, and a gene for alkaline phosphatase, phoA, lacking its signal sequence, it was determined that 39 codons in the N-terminus of cvaC contained the structural information to allow CvaC-PhoA fusion proteins to be efficiently translocated across the plasma membrane of Escherichia coli in a CvaA/CvaB dependent fashion. This result is consistent with the location of point mutations in the cvaC gene which yielded export deficient colicin V. The presence of the export signal at the N-terminus of CvaC contrasts with the observed C-terminal location of the export signal for hemolysin, which also utilizes an MDR-like protein for its secretion. It was also found that the CvaA component of the colicin V export system shows amino acid sequence similarities with another component involved in hemolysin export, HlyD. The role of the second component in these systems and the possibility that other members of the MDR-like subfamily will also have corresponding second components are discussed. A third component used in both colicin V and hemolysin extracellular secretion is the E. coli host outer membrane protein, TolC.     

The superoxide dismutase SodA is targeted to the periplasm in a SecA-dependent manner by a novel mechanism

The manganese/iron-type superoxide dismutase (SodA) of Rhizobium leguminosarum bv. viciae 3841 is exported to the periplasm of R. l. bv. viciae and Escherichia coli. However, it does not possess a hydrophobic cleaved N-terminal signal peptide typically present in soluble proteins exported by the Sec-dependent (Sec) pathway or the twin-arginine translocation (TAT) pathway. A tatC mutant of R. l. bv. viciae exported SodA to the periplasm, ruling out export of SodA as a complex with a TAT substrate as a chaperone. The export of SodA was unaffected in a secB mutant of E. coli, but its export from R. l. bv. viciae was inhibited by azide, an inhibitor of SecA ATPase activity. A temperature-sensitive secA mutant of E. coli was strongly reduced for SodA export. The 10 N-terminal amino acid residues of SodA were sufficient to target the reporter protein alkaline phosphatase to the periplasm. Our results demonstrate the export of a protein lacking a classical signal peptide to the periplasm by a SecA-dependent, but SecB-independent targeting mechanism. Export of the R. l. bv. viciae SodA to the periplasm was not limited to the genus Rhizobium, but was also observed in other proteobacteria.     

MacAB is involved in the secretion of Escherichia coli heat-stable enterotoxin II

The heat-stable enterotoxin (ST) produced by enterotoxigenic Escherichia coli is an extracellular peptide toxin that evokes watery diarrhea in the host. Two types of STs, STI and STII, have been found. Both STs are synthesized as precursor proteins and are then converted to the active forms with intramolecular disulfide bonds after being released into the periplasm. The active STs are finally translocated across the outer membrane through a tunnel made by TolC. However, it is unclear how the active STs formed in the periplasm are led to the TolC channel. Several transporters in the inner membrane and their periplasmic accessory proteins are known to combine with TolC and form a tripartite transport system. We therefore expect such transporters to also act as a partner with TolC to export STs from the periplasm to the exterior. In this study, we carried out pulse-chase experiments using E. coli BL21(DE3) mutants in which various transporter genes (acrAB, acrEF, emrAB, emrKY, mdtEF, macAB, and yojHI) had been knocked out and analyzed the secretion of STs in those strains. The results revealed that the extracellular secretion of STII was largely decreased in the macAB mutant and the toxin molecules were accumulated in the periplasm, although the secretion of STI was not affected in any mutant used in this study. The periplasmic stagnation of STII in the macAB mutant was restored by the introduction of pACYC184, containing the macAB gene, into the cell. These results indicate that MacAB, an ATP-binding cassette transporter of MacB and its accessory protein, MacA, participates in the translocation of STII from the periplasm to the exterior. Since it has been reported that MacAB cooperates with TolC, we propose that the MacAB-TolC system captures the periplasmic STII molecules and exports the toxin molecules to the exterior.     

The SecB chaperone is bifunctional in Serratia marcescens: SecB is involved in the Sec pathway and required for HasA secretion by the ABC transporter

HasA is the secreted hemophore of the heme acquisition system (Has) of Serratia marcescens. It is secreted by a specific ABC transporter apparatus composed of three proteins: HasD, an inner membrane ABC protein; HasE, another inner membrane protein; and HasF, a TolC homolog. Except for HasF, the structural genes of the Has system are encoded by an iron-regulated operon. In previous studies, this secretion system has been reconstituted in Escherichia coli, where it requires the presence of the SecB chaperone, the Sec pathway-dedicated chaperone. We cloned and inactivated the secB gene from S. marcescens. We show that S. marcescens SecB is 93% identical to E. coli SecB and complements the secretion defects of a secB mutant of E. coli for both the Sec and ABC pathways of HasA secretion. In S. marcescens, SecB inactivation affects translocation by the Sec pathway and abolishes HasA secretion. This demonstrates that S. marcescens SecB is the genuine chaperone for HasA secretion in S. marcescens. These results also demonstrate that S. marcescens SecB is bifunctional, as it is involved in two separate secretion pathways. We investigated the effects of secB point mutations in the reconstituted HasA secretion pathway by comparing the translocation of a Sec substrate in various mutants. Two different patterns of SecB residue effects were observed, suggesting that SecB functions may differ for the Sec and ABC pathways.     

Cys32 and His105 are the critical residues for the calcium-dependent cysteine proteolytic activity of CvaB, an ATP-binding cassette transporter

CvaB, a member of the ATP-binding cassette transporter superfamily, is the central membrane transporter of the colicin V secretion system in Escherichia coli. Cys32 and His105 in the N-terminal domain of CvaB were identified as critical residues for both colicin V secretion and cysteine proteolytic activity. By inhibiting degradation with N-ethylmaleimide and a mixture of protease inhibitors, a stable wild-type N-terminal domain (which showed cysteine protease activity when activated) was purified. Such protease activity was Ca2+- and concentration-dependent and could be inhibited by antipain, N-ethylmaleimide, EDTA, and EGTA. At low concentrations, the Ca2+ analogs Tb3+ and La3+ (but not Fe3+) significantly enhanced proteolytic activity, suggesting that the size of the cations is important for activity. Together with comparisons of the sequences of members of the cysteine protease family, these results indicate that Cys32 and His105 are the critical residues in the CvaB N-terminal domain for the calcium-dependent cysteine protease activity and secretion of colicin V.     

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

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

Bactericidal activity of colicin V is mediated by an inner membrane protein, SdaC, of Escherichia coli

Colicin V (ColV) is a peptide antibiotic that kills sensitive cells by disrupting their membrane potential once it gains access to the inner membrane from the periplasmic face. Recently, we constructed a translocation suicide probe, RR-ColV, that is translocated into the periplasm via the TAT pathway and thus kills the host cells. In this study, we obtained an RR-ColV-resistant mutant by using random Tn10 transposition mutagenesis. Sequencing analysis revealed that the mutant carried a Tn10 insertion in the sdaC (also called dcrA) gene, which is involved in serine uptake and is required for C1 phage adsorption. ColV activity was detected both in the cytoplasm and in the periplasm of this mutant, indicating that RR-ColV was translocated into the periplasm but failed to interact with the inner membrane. The sdaC::Tn10 mutant was resistant only to ColV and remained sensitive to colicins Ia, E3, and A. Most importantly, the sdaC::Tn10 mutant was killed when ColV was anchored to the periplasmic face of the inner membrane by fusion to EtpM, a type II integral membrane protein. Taken together, these results suggest that the SdaC/DcrA protein serves as a specific inner membrane receptor for ColV.     

A new genetic selection identifies essential residues in SecG, a component of the Escherichia coli protein export machinery.

The signal sequence of the murine serine protease inhibitor PAI-2 promotes alkaline phosphatase export to the E. coli periplasm. However, high level expression of this chimeric protein interferes with cell growth. Since most suppressors of this toxic phenotype map to secA and secY, growth arrest results from a defective interaction of the chimeric protein with the export machinery. We have characterized suppressors which map in secG, a newly defined gene of the export machinery. All single amino acid substitutions map to three adjacent codons. These secG mutants have a weak Sec phenotype, as determined by their effect on export mediated by wild-type and mutant signal sequences. Whilst a secG disruption allele also confers a weak Sec phenotype, it does not suppress the toxicity of the chimeric protein. This difference results from a selective effect of the secG suppressors on the kinetics of export mediated by the PAI-2 signal sequence. Using a malE signal sequence mutant, which has a Mal-phenotype in secG mutant strains, we have isolated extragenic Mal+ suppressors. Most suppressors map to secY, and several are allele-specific. Finally, SecG overexpression accelerates the kinetics of protein export, suggesting that there are two types of functional translocation complexes: with or without SecG.     

Influence of tat mutations on the ribose-binding protein translocation in Escherichia coli

Proteins are exported across the bacterial cytoplasmic membrane either as unfolded precursors via the Sec machinery or in folded conformation via the Tat system. The ribose-binding protein (RBP) of Escherichia coli is a Sec-pathway substrate. Intriguingly, it exhibits fast folding kinetics and its export is independent of SecB, a general chaperone protein dedicated for protein secretion. In this study, we found that the quantity of RBP was significantly reduced in the periplasm of tat mutants, which was restored by in trans expression of the tatABC genes. Pulse-chase experiments showed that significant amount of wild-type RBP was processed in a secY mutant in the presence of azide (SecA inhibitor), whereas the processing of a slow folding RBP derivative was almost completely blocked under the same conditions. These results would suggest that under the Sec-defective conditions the export of a portion of folded RBP could be rescued by the Tat system.     

Multidrug-exporting secondary transporters

The major cause of intrinsic drug resistance in Gram-negative bacteria is a resistance nodulation division type multidrug exporter, which couples with an outer membrane channel and a membrane fusion protein and exports drugs out of the cell, bypassing the periplasm; this process is driven by proton motive force. A recent crystal structure determination of a major resistance nodulation division type multidrug exporter, AcrB in Escherichia coli, greatly advances our understanding of the multidrug export mechanism. The most striking feature of the AcrB trimer is the presence of three vestibules open to the periplasm at the boundary between the periplasmic headpiece and the transmembrane region. Substrates can gain access to the central cavity from the periplasmic surface of the cytoplasmic membrane and are then actively transported through the extramembrane pore into the outer membrane channel TolC, via the funnel at the top of the AcrB headpiece.     

Substrate-triggered recruitment of the TolC channel-tunnel during type I export of hemolysin by Escherichia coli.

A defining event in type I export of hemolysin by Escherichia coli is the substrate-triggered recruitment of the TolC channel-tunnel by an inner membrane complex. This complex comprises a traffic ATPase (HlyB) and the 478 residue adaptor protein (HlyD), which contacts TolC during recruitment. HlyD has a large periplasmic domain (amino acid residues 81-478) linked by a single transmembrane helix to a small N-terminal cytosolic domain (1-59). Export was disabled by deletion of the ca 60 amino acid residue cytosolic domain of HlyD, even though the truncated HlyD (HlyDDelta45) was, like the wild-type, able to trimerise in the cytosolic membrane, and interact with the traffic ATPase. The mutant HlyB/HlyDDelta45 inner membrane complex engaged the hemolysin substrate, but this substrate-engaged complex failed to trigger recruitment of TolC. Further analyses showed that HlyDDelta45 was specifically unable to bind the substrate. The result suggests that substrate engagement by the traffic ATPase alone is insufficient to trigger TolC recruitment, and that substrate binding to the HlyD cytosolic domain is essential. Analysis of three further N-terminal deletion variants, HlyDDelta26, HlyDDelta26-45 and HlyDDelta34-38, indicated that an extreme N-terminal amphipathic helix and a cytosolic cluster of charged residues are central to the cytosolic domain function. The cytosolic amphipathic helix was not essential for substrate engagement or TolC recruitment, but export was impaired without it. In contrast, when the charged amino acid residues were deleted, the substrate was still engaged by HlyD but engagement was unproductive, i.e. TolC recruitment was not triggered. Our results are compatible with the HlyD cytosolic domain mediating transduction of the substrate binding signal directly, presumably to the HlyD periplasmic domain, to trigger recruitment of TolC and assemble the type I export complex.     

Signal recognition particle-dependent inner membrane targeting of the PulG Pseudopilin component of a type II secretion system

The pseudopilin PulG is an essential component of the pullulanase-specific type II secretion system from Klebsiella oxytoca. PulG is the major subunit of a short, thin-filament pseudopilus, which presumably elongates and retracts in the periplasm, acting as a dynamic piston to promote pullulanase secretion. It has a signal sequence-like N-terminal segment that, according to studies with green and red fluorescent protein chimeras, anchors unassembled PulG in the inner membrane. We analyzed the early steps of PulG inner membrane targeting and insertion in Escherichia coli derivatives defective in different protein targeting and export factors. The beta-galactosidase activity in strains producing a PulG-LacZ hybrid protein increased substantially when the dsbA, dsbB, or all sec genes tested except secB were compromised by mutations. To facilitate analysis of native PulG membrane insertion, a leader peptidase cleavage site was engineered downstream from the N-terminal transmembrane segment (PrePulG*). Unprocessed PrePulG* was detected in strains carrying mutations in secA, secY, secE, and secD genes, including some novel alleles of secY and secD. Furthermore, depletion of the Ffh component of the signal recognition particle (SRP) completely abolished PrePulG* processing, without affecting the Sec-dependent export of periplasmic MalE and RbsB proteins. Thus, PulG is cotranslationally targeted to the inner membrane Sec translocase by SRP.