The surfactant of Legionella pneumophila Is secreted in a TolC-dependent manner and is antagonistic toward other Legionella species

When Legionella pneumophila grows on agar plates, it secretes a surfactant that promotes flagellum- and pilus-independent "sliding" motility. We isolated three mutants that were defective for surfactant. The first two had mutations in genes predicted to encode cytoplasmic enzymes involved in lipid metabolism. These genes mapped to two adjacent operons that we designated bbcABCDEF and bbcGHIJK. Backcrossing and complementation confirmed the importance of the bbc genes and suggested that the Legionella surfactant is lipid containing. The third mutant had an insertion in tolC. TolC is the outer membrane part of various trimolecular complexes involved in multidrug efflux and type I protein secretion. Complementation of the tolC mutant restored sliding motility. Mutants defective for an inner membrane partner of TolC also lacked a surfactant, confirming that TolC promotes surfactant secretion. L. pneumophila (lspF) mutants lacking type II protein secretion (T2S) are also impaired for a surfactant. When the tolC and lspF mutants were grown next to each other, the lsp mutant secreted surfactant, suggesting that TolC and T2S conjoin to mediate surfactant secretion, with one being the conduit for surfactant export and the other the exporter of a molecule that is required for induction or maturation of surfactant synthesis/secretion. Although the surfactant was not required for the extracellular growth, intracellular infection, and intrapulmonary survival of L. pneumophila, it exhibited antimicrobial activity toward seven other species of Legionella but not toward various non-Legionella species. These data suggest that the surfactant provides L. pneumophila with a selective advantage over other legionellae in the natural environment.     

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.     

The outer membrane protein TolC from Sinorhizobium meliloti affects protein secretion, polysaccharide biosynthesis, antimicrobial resistance, and symbiosis

Sinorhizobium meliloti is capable of establishing a symbiotic nitrogen fixation relationship with Medicago sativa. During this process, it must cope with diverse environments and has evolved different types of transport systems that help its propagation in the plant roots. TolC protein family members are the outer-membrane components of several transport systems involved in the export of diverse molecules, playing an important role in bacterial survival. In this work, we have characterized the protein TolC from S. meliloti 2011. An insertional mutation in the tolC gene strongly affected the resistance phenotype to antimicrobial agents and induced higher susceptibility to osmotic and oxidative stresses. Immunodetection experiments and comparison of the extracellular proteins present in the supernatant of the wild-type versus tolC mutant strains showed that the calcium-binding protein ExpE1, the endoglycanase ExsH, and the product of open reading frame SMc04171, a putative hemolysin-type calcium-binding protein, are secreted by a TolC-dependent secretion system. In the absence of TolC, neither succinoglycan nor galactoglucan were detected in the culture supernatant. Moreover, S. meliloti tolC mutant induced a reduced number of nonfixing nitrogen nodules in M. sativa roots. Taken together, our results confirm the importance of TolC in protein secretion, exopolysaccharide biosynthesis, antimicrobials resistance, and symbiosis.     

Salmonella enteritidis has a homologue of tolC that is required for virulence in BALB/c mice

The ability of Salmonella to invade tissue culture cells is correlated with virulence. Therefore, the tissue culture invasion model has been used extensively to study this process and to identify the bacterial genes involved and their products. Described here is the further characterization of a Salmonella enteritidis mutant (SM6T) originally identified as non-invasive for tissue culture cells. A chromosomal DNA fragment complementing this defect was cloned and sequenced. The derived protein sequence is 89% identical to TolC from Escherichia coli , an outer membrane protein required for the signal peptide-independent transport of α-haemolysin and colicin V. Therefore, sinA was renamed tolC and is referred to in this text as tolC ^s to distinguish it from tolC of E. coli TolC^s and TolC are functionally similar since tolC can complement the invasion-defective phenotype of a tolC^s mutant, and tolC^s is required for export of α-haemolysin by Salmonella . The tolC ^s mutant is avirulent for mice when administered by the oral route, suggesting that the gene is important for virulence. Further characterization of the tolC^s mutant indicated that like tolC mutants it is more sensitive than the wild-type strain to various detergents, antibiotics and dyes. This mutant is more sensitive to Triton X-100 only when associated with the monolayer, and the invasion-defective phenotype appears to be an artifact of this sensitivity. In addition, the tolC^s mutant is more sensitive to the bactericidal activity of human serum. Therefore, the avirulent phenotype could be the result of an inability to secrete a necessary virulence factor, or an increased sensitivity to complement and detergents as a result of a subtle alteration in the lipopolysaccharide (LPS) associated with tolC mutations.     

Antibiotic-sensitive TolC mutants and their suppressors

The TolC protein of Escherichia coli, through its interaction with AcrA and AcrB, is thought to form a continuous protein channel that expels inhibitors from the cell. Consequently, tolC null mutations display a hypersensitive phenotype. Here we report the isolation and characterization of tolC missense mutations that direct the synthesis of mutant TolC proteins partially disabled in their efflux role. All alterations, consisting of single amino acid substitutions, were localized within the periplasmic alpha-helical domain. In two mutants carrying an I106N or S350F substitution, the hypersensitivity phenotype may be in part due to aberrant TolC assembly. However, two other alterations, R367H and R390C, disrupted efflux function by affecting interactions among the helices surrounding TolC's periplasmic tunnel. Curiously, these two TolC mutants were sensitive to a large antibiotic, vancomycin, and exhibited a Dex(+) phenotype. These novel phenotypes of TolC(R367H) and TolC(R390C) were likely the result of a general influx of molecules through a constitutively open tunnel aperture, which normally widens only when TolC interacts with other proteins during substrate translocation. An intragenic suppressor alteration (T140A) was isolated from antibiotic-resistant revertants of the hypersensitive TolC(R367H) mutant. T140A also reversed, either fully (R390C) or partially (I106N and S350F), the hypersensitivity phenotype of other TolC mutants. Our data suggest that this global suppressor phenotype of T140A is the result of impeded antibiotic influx caused by tapering of the tunnel passage rather than by correcting individual mutational defects. Two extragenic suppressors of TolC(R367H), mapping in the regulatory region of acrAB, uncoupled the AcrR-mediated repression of the acrAB genes. The resulting overexpression of AcrAB reduced the hypersensitivity phenotype of all the TolC mutants. Similar results were obtained when the chromosomal acrR gene was deleted or the acrAB genes were expressed from a plasmid. Unlike the case for the intragenic suppressor T140A, the overexpression of AcrAB diminished hypersensitivity towards only erythromycin and novobiocin, which are substrates of the TolC-AcrAB efflux pump, but not towards vancomycin, which is not a substrate of this pump. This showed that the two types of suppressors produced their effects by fundamentally different means, as the intragenic suppressor decreased the general influx while extragenic suppressors increased the efflux of TolC-AcrAB pump-specific antibiotics.     

Involvement of the drug efflux protein TolC in mutagenicity induced by MNNG or Trp-P-2

In the development of mutation assay systems, a number of approaches have been performed with a particular view to improve sensitivity. The inhibition of mutagen-efflux from tester bacteria might lead to increased mutagenic activity as the concentration of mutagen increases inside the cell. In this study, we constructed a series of Escherichia coli CC strains lacking the TolC protein to determine if mutation is actually enhanced by the inhibition of mutagen reflux. TolC is an outer-membrane protein that forms part of an excretion system in E. coli. The frequency of induction of mutations by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) and ethyl methanesulfonate (EMS) were significantly higher in TolC-deficient strain KA796-1/CC102 than in TolC-proficient strains, especially that of MNNG was seven times higher and detected at lower doses than in the parent strain. In a KA796-1/CC108 TolC-deficient strain, mutation induced by Trp-P-2 was detected at significant levels, even at low doses that did not induce detectable levels of mutation in the parent strain KA796/CC108. When the wild-type E. coli tolC gene was introduced into a strain lacking the gene, TolC function was restored and the frequency of induction by MNNG became similar to that of the wild-type. In contrast, introduction of a mutant tolC gene did not complement the TolC deficiency and the frequency of MNNG-induced mutations remained high. These results suggest that some mutagens are excreted at least in part via the TolC system, and that the lack of functional TolC increases the susceptibility of bacteria to many mutagens.     

Isolation and characterization of Escherichia coli tolC mutants defective in secreting enzymatically active alpha-hemolysin.

This study describes the isolation and characterization of a unique class of TolC mutants that, under steady-state growth conditions, secreted normal levels of largely inactive alpha-hemolysin. Unlike the reduced activity in the culture supernatants, the cell-associated hemolytic activity in these mutants was identical to that in the parental strain, thus reflecting a normal intracellular toxin activation event. Treatment of the secreted toxin with guanidine hydrochloride significantly restored cytolytic activity, suggesting that the diminished activity may have been due to the aggregation or misfolding of the toxin molecules. Consistent with this notion, sedimentation and filtration analyses showed that alpha-hemolysin secreted from the mutant strain has a mass greater than that secreted from the parental strain. Experiments designed to monitor the time course of alpha-hemolysin release showed delayed appearance of toxin in the culture supernatant of the mutant strain, thus indicating a possible defect in alpha-hemolysin translocation or release. Eight different TolC substitutions displaying this toxin secretion defect were scattered throughout the protein, of which six localized in the periplasmically exposed alpha-helical domain, while the remaining two mapped within the outer membrane-embedded beta-barrel domain of TolC. A plausible model for the secretion of inactive alpha-hemolysin in these TolC mutants is discussed in the context of the recently determined three-dimensional structure of TolC.     

Genetic Analysis of the Colicin V Secretion Pathway

Colicin V (ColV) is peptide antibiotic secreted by Escherichia coli through a dedicated exporter composed of three proteins, CvaA, CvaB, and TolC. ColV secretion is independent of the E. coli general secretory pathway (Sec) but requires an N-terminal export signal specific for the CvaAB/TolC exporter. ColV secretion was characterized using genetic and biochemical methods. When the ColV N-terminal extension is replaced with the OmpA signal sequence, the Sec system can localize ColV to the periplasm. Periplasmic ColV is lethal to cells lacking the ColV immunity protein, Cvi. Based on this result, a genetic assay was designed to monitor for the presence of periplasmic ColV during normal CvaAB/TolC mediated secretion. Results indicate that low levels of ColV may be present in the periplasm during secretion. Precursor and mature ColV were also characterized from the wild-type system and in various exporter mutant backgrounds using immunoprecipitation. ColV processing is rapid in wild-type cells, and CvaA and CvaB are critical for processing to occur. In contrast, processing occurs normally, albeit more slowly, in a TolC mutant.     

Erwinia chrysanthemi tolC is involved in resistance to antimicrobial plant chemicals and is essential for phytopathogenesis

TolC is the outer-membrane component of several multidrug resistance (MDR) efflux pumps and plays an important role in the survival and virulence of many gram-negative bacterial animal pathogens. We have identified and characterized the outer-membrane protein-encoding gene tolC in the bacterial plant pathogen Erwinia chrysanthemi EC16. The gene was found to encode a 51-kDa protein with 70% identity to its Escherichia coli homologue. The E. chrysanthemi gene was able to functionally complement the E. coli tolC gene with respect to its role in MDR efflux pumps. A tolC mutant of E. chrysanthemi was found to be extremely sensitive to antimicrobial agents, including several plant-derived chemicals. This mutant was unable to grow in planta and its ability to cause plant tissue maceration was severely compromised. The tolC mutant was shown to be defective in the efflux of berberine, a model antimicrobial plant chemical. These results suggest that by conferring resistance to the antimicrobial compounds produced by plants, the E. chrysanthemi tolC plays an important role in the survival and colonization of the pathogen in plant tissue.     

Plasmid-associated bacteriocin production in a JK-type coryneform bacterium

Abstract The outer membrane of Escherichia coli K-12 has a variety of proteolytic activities. We were able to label several outer membrane proteins with [³H]diisopropylfluorophosphate (DFP). This suggests that they are serine proteases. The number of labelled proteins detected varied with the E. coli K-12 strain used. Strains bearing a tolC mutation, in addition, gave better labelling and/or had more labelled proteins. A previously described [³H]DFP-labelled outer membrane protein was shown not to be the TolC protein since it has a slightly lower M _r, it is not labelled more intensely in a TolC-overproducing strain, and it is still labelled in tolC mutant strains.     

Probing the in vivo dynamics of type I protein secretion complex association through sensitivity to detergents

The Serratia marcescens hemophore is secreted by a type I secretion system consisting of three proteins: a membrane ABC protein, an adaptor protein, and the TolC-like outer membrane protein. Assembly of these proteins is induced by substrate binding to the ABC protein. Here we show that a hemophore mutant lacking the last 14 C-terminal amino acids is not secreted but rather interacts with the ABC protein and promotes a stable multiprotein complex. Strains expressing the transporter and the mutant protein are sensitive to detergents (sodium dodecyl sulfate [SDS]). TolC is trapped in the transporter jammed by the truncated substrate and therefore is not present at sufficient concentrations to allow the efflux pumps to expel detergents. Using an SDS sensitivity assay, we showed that the hemophore interacts with the ABC protein via two nonoverlapping sites. We also demonstrated that the C-terminal peptide, which functions as an intramolecular signal sequence in the complete substrate, may also have intermolecular activity and triggers complex dissociation in vivo when it is provided as a distinct peptide. The SDS sensitivity test on plates enables workers to study type I secretion protein association and dissociation independent of the secretion process itself.     

The TolC protein of Escherichia coli serves as a cell-surface receptor for the newly characterized TLS bacteriophage

The TolC protein of Escherichia coli is implicated in a variety of diverse cellular functions, including antibiotic efflux and alpha-hemolysin secretion. An incidental role of TolC is to facilitate the entry of the bacteriophage TLS and colicin E1 into the bacterial cell. Despite the resolution of TolC's atomic structure, the roles of specific residues in its diverse functions are unknown. Here, we describe a genetic strategy for isolating missense tolC mutations that abolish the bacteriophage receptor activity of the TolC protein without influencing its role in antibiotic efflux. These spontaneous mutations affected two regions of the TolC protein and included base-pair substitutions, insertions, and deletions. Comparison of the TolC sequence with those of its homologues revealed two hypervariable stretches that were predicted to represent loops. Interestingly, all but one of the TolC alterations preventing phage binding were located in these two hypervariable regions, which are likely to be exposed on the cell surface. This was substantiated by the recently solved three-dimensional structure of TolC. Curiously, all the phage-resistant TolC mutants showed varying degrees of resistance to colicin E1, suggesting the involvement of overlapping regions of TolC in colicin E1 import and phage binding.The phage used in this study, TLS, was earlier reported as a strain of U3. However, we show here that, unlike the previously reported lipopolysaccharide-specific U3 phage, this phage displays a distinctly different host range and discrete morphological features and, in addition to utilizing TolC as receptor, it requires the inner core of a lipopolysaccharide.     

Secretion of the Rhizobium leguminosarum nodulation protein NodO by haemolysin-type systems

The Rhizobium leguminosarum biovar viciae nodulation protein NodO is partially homologous to haemolysin of Escherichia coli and, like haemolysin, is secreted into the growth medium. The NodO protein can be secreted by a strain of E. coli carrying the cloned nodO gene plus the haemolysin secretion genes hlyBD, in a process that also requires the outer membrane protein encoded by tolC. The related protease secretion genes, prtDEF, from Erwinia chrysanthemi also enable E. coli to secrete NodO. The Rhizobium genes encoding the proteins required for NodO secretion are unlinked to nodO and are unlike other nod genes, since they do not require flavonoids or NodO for their expression. Although proteins similar to NodO were not found in rhizobia other than R. leguminosarum bv. viciae, several rhizobia and an Agrobacterium strain containing the cloned nodO gene were found to have the ability to secrete NodO. These observations indicate that a wide range of the Rhizobiaceae have a protein secretion mechanism analogous to that which secretes haemolysin and related toxins and proteases in the ENterobacteriaceae.     

Escherichia coli outer membrane protein TolC is involved in production of the peptide antibiotic microcin J25

A Tn5 insertion in tolC eliminated microcin J25 production. The mutation had little effect on the expression of the microcin structural gene and presumably acted by blocking microcin secretion. The tolC mutants carrying multiple copies of the microcin genes were less immune to the microcin. TolC is thus likely a component of a microcin export complex containing the McjD immunity protein, an ABC exporter.     

Genetic evidence for functional interactions between TolC and AcrA proteins of a major antibiotic efflux pump of Escherichia coli

Genetic data have suggested that TolC, AcrA and AcrB constitute a major antibiotic efflux system in Escherichia coli. Through reversion analysis of an unstable and antibiotic-sensitive TolC mutant (TolCP246R,S350C), we isolated extragenic suppressors that mapped within the acrRAB loci. DNA sequence analysis revealed that 18 isolates contained 10 different missense mutations within the acrA gene, whereas a single isolate had a missense mutation within the acrR gene, which codes for the acrAB repressor. Besides reversing the hypersensitivity phenotype of TolCP246R,S350C, AcrA and AcrR alterations elevated the mutant TolC protein level, thus indicating that the mechanism of suppression involves the stabilization of an unstable mutant TolC protein. Eight of the 10 AcrA alterations were clustered in the 202-265 region of the mature protein, whereas the other two suppressors affected residues 30 and 146. Based on the recently solved crystal structure of MexA, an AcrA counterpart from Pseudomonas aeruginosa, the regions encompassing residues 30 and 202-265 constitute the alpha+beta-domain of AcrA (MexA), whereas that of 146 form the alpha-domain. The data suggest that residues of these two AcrA domains either directly or indirectly influence interactions with TolC. Curiously, the stability of three mutant AcrA proteins, bearing an L222Q, L222R or P265R substitution, became dependent on the presence of either wild-type or mutant TolC. This dependence of the mutant AcrA proteins on TolC further supported the notion of a direct physical interaction between these two proteins. Because a mutation in acrR or acrAB expression from a multicopy plasmid also suppressed the TolCP246R,S350C defects, it indicated that wild-type AcrA when produced in high levels presumably establishes similar interactions with the mutant TolC protein as do the suppressor forms of AcrA produced from the chromosomal copy. The AcrA-mediated suppression of mutant TolC phenotypes and the stabilization of mutant TolC protein were dependent on AcrB, reflecting the existence of a functional complex between TolC and AcrAB in vivo.