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.     

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.     

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.     

The AcrAB-TolC efflux system of Salmonella enterica serovar Typhimurium plays a role in pathogenesis

The ability of an isogenic set of mutants of Salmonella enterica serovar Typhimurium L354 (SL1344) with defined deletions in genes encoding components of tripartite efflux pumps, including acrB, acrD, acrF and tolC, to colonize chickens was determined in competition with L354. In addition, the ability of L354 and each mutant to adhere to, and invade, human embryonic intestine cells and mouse monocyte macrophages was determined in vitro. The tolC and acrB knockout mutants were hyper-susceptible to a range of antibiotics, dyes and detergents; the tolC mutant was also more susceptible to acid pH and bile and grew more slowly than L354. Complementation of either gene ablated the phenotype. The tolC mutant poorly adhered to both cell types in vitro and was unable to invade macrophages. The acrB mutant adhered, but did not invade macrophages. In vivo, both the acrB mutant and the tolC mutant colonized poorly and did not persist in the avian gut, whereas the acrD and acrF mutant colonized and persisted as well as L354. These data indicate that the AcrAB-TolC system is important for the colonization of chickens by S. Typhimurium and that this system has a role in mediating adherence and uptake into target host cells.     

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.     

A new RNA polymerase sigma factor, σF is required for the late stages of morphological differentiation in Streptomyces spp.

A gene ( sigF ) encoding a new sigma factor was isolated from Streptomyces aureofaciens using a degenerate oligonucleotide probe designed from the GLI(KDNE)A motif lying within the well-conserved region 2.2 of the eubacterial σ⁷⁰ family. Homologues were present in other Streptomyces spp., and that of the genetically well-studied Streptomyces coelicolor A3(2) was also cloned. The nucleotide sequences of the two sigF genes were determined and shown to encode primary translation products of 287 ( S. coelicolor ) and 295 ( S. aureofaciens ) amino acid residues, both showing greatest similarity to σ^B of Bacillus subtilis . However, while σ^B is involved in stationary-phase gene expression and in the general stress response in B. subtilis , σ^F affects morphological differentiation in Streptomyces , Disruption of sigF did not affect vegetative growth but did cause a whi mutant phenotype. Microscopic examination showed that the sigF mutant produced spores that were smaller and deformed compared with those of the wild type, that the spore walls were thinner and sensitive to detergents and that in sigF mutant spores the chromosome failed to condense. σ^F is proposed to control the late stages of spore development in Streptomyces .     

The new vaccine strains (or variants) of Francisella tularensis

Abstract A colony (N83) of the vaccine strain of Francisella tularensis (15/10) and a strain (N268) isolated from a water sample in nature were revealed for susceptibility to cultivation at 42°C. Both strains had low virulence for white mice and were avirulent for guinea pigs but possessed high immunogenicity in these animals. The spontaneous mutant of vaccine strain 15/10 showed resistance to doxicycline and rifmanpicine (15/10 Dox ^r40 Rif ^r40). The obtained mutant had biological characteristics similar to the parent vaccine strain. It provided immunity in experimental animals when vaccination and antimicrobial agents were used in combination.     

MyD88 and IFN-alphabeta differentially control maturation of bystander but not Salmonella-associated dendritic cells or CD11cintCD11b+ cells during infection

The interface between dendritic cells (DCs) and T cells is critical to elicit effective immunity against pathogens. The maturation state of DCs determines the quality of the interaction and governs the type of response. DCs can be matured directly through activating Toll-like receptors (TLRs) or indirectly by cytokines. We explore the role of the TLR adaptor MyD88 on DC maturation during Salmonella infection. Using Salmonella expressing GFP, we also examine the phenotype and function of bacteria-associated DCs matured in the absence of bacteria-mediated TLR signalling. MyD88 was required for upregulation of CD80 on DCs during infection, whereas CD86 and CD40 were upregulated independently of MyD88, although requiring a higher bacterial burden in the MLN. MyD88-independent upregulation was mediated by IFN-αβ produced during infection. In infected MyD88^−/−IFN-αβR^−/− mice, which lack most bacteria-driven TLR signalling, indirect DC maturation was abolished. In contrast, DCs containing Salmonella upregulated co-stimulatory molecules independently of MyD88 and IFN-αβ, revealing a pathway of phenotypic maturation active in infected DCs. However, despite high co-stimulatory molecule expression, Salmonella -containing DCs from MyD88^−/− or MyD88^−/−IFN-αβR^−/− mice had a compromised capacity to activate T cells. Thus, bacterial stimulation of TLRs influences DC function at multiple levels that modulates their capacity to direct antibacterial immunity.     

Plasma esterase polymorphism in the Bank vole, Clethrionomys glareolus, in Britain

Three hundred and eighty-three Clethrionomys glareolus from 20 localities in England, Wales and Scotland were typed for plasma esterase and a genetic polymorphism was discovered. The esterase was named Es-1. Breeding tests suggested that three alleles were segregating: Es-1^o when homozygous results in complete absence of enzyme activity. The active alleles Es-1^f and Es-1^s code for enzyme variants which migrate more rapidly and less rapidly, respectively, under starch gel electrophoresis. Of these active alleles, Es-1^f is morc common in the north of Britain and Es-1⁸ in the south. A 23-month field study on two areas at Wicken Fen, Cambridgeshire, suggested that animals possessing Es-1^s survived less well at high population densities, perhaps through their being more likely to emigrate.     

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.     

Inheritance of Pathogenicity of Progeny from an F1 Culture of Melampsora lini

Abstract Races 1 and 400 of Melampsosau bni the causal agent of flax [Lmum usitatissimum]), were crossed and the resulting F² progeny evaluated on near-isogcnic fiax lines. The infection type of the parental cultures, the F¹² and the segregating f² progeny were used to evaluate the inheritance of pathogenicity. Races 1 and 400 as well as the F² and all the F² progeny were avirulent on lines N and P⁴ genes. This indicated both races to be homozygous avirulent. The F² and all the F² progeny were virulent on lines containing L9, M1, M4, and P. This indicated the F to be homuzygous for virulence at these loci. Segregation ratios of the F² cultures fit monogenic ratios on lines with host genes K. L2, L5, L6, L7, L11, M, M2, M3, N1, P1, P2, and P3. Race 1 was avirulent, race 400 virulent and tht F, avirulent on these lines. Several linkage groups were indicated including close linkage at A^p1, A^p2 and A^p3. Segregation on lines L, L3, M5, M6 and cultivars Flor, Dufferin, Linott, and Wishek fit a digenic model. The infection tvpes of the parents, the F¹ and the F², segregations could usually be explained by classical gene-for-gene interactions. Possible exceptions were segregations on L4, L8 and N2 which did not fit a 3: 1 ratio but did fit a 9: 7 ratio. Segregation on L 10 fit a 7: 9 ratio but line L10 may be temperature sensitive. Segregation on L1 indicated a single dominant gene for pathogenicity.     

Evidence that TolC is required for functioning of the Mar/AcrAB efflux pump of Escherichia coli.

A study examining the influence of TolC on AcrA, AcrR, and MarR1 mutants indicates that functional TolC is required for the operation of the AcrAB efflux system and for the expression of the Mar phenotype. That the effect of TolC on the AcrAB pump is not regulatory in nature is shown by studies measuring the influence of a tolC::Tn10 insertion mutation on the expression of an acrA::lacZ reporter fusion. These results are compatible with the hypothesis that TolC is a component of the AcrAB efflux complex.     

Salmonella typhimurium acrB-like gene: identification and role in resistance to biliary salts and detergents and in murine infection

Abstract Salmonella serotype typhimurium transpositional mutants altered in resistance to biliary salts and detergents were isolated previously. We have characterized further the LX1054 mutant strain, the most sensitive of them. The chromosomal DNA segment flanking transposon insertion was cloned and sequenced. The highest level of identity was found for the acrB (formerly acrE ) gene of Escherichia coli , a gene encoding a drug efflux pump of the Acr family. LX1054 exhibited a reduced capacity to colonize the intestinal tract. After passages in mice, the mutant strain lost the sensitive phenotype. In vitro, a resumption of growth appeared after 17 h of culture in medium with cholate or other tested biological or chemical detergents. Then, the acquired resistant phenotype seemed stable. The data suggested a role of S. typhimurium acrB -like gene in resistance to biliary salts and detergents and in mice intestinal colonization. However, the local and transient sensitivity observed in vivo, and the in vitro adaptations suggest that several detergent-resistance mechanisms operate in S. typhimurium .     

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.     

Identification and molecular characterization of a novel Salmonella enteritidis pathogenicity islet encoding an ABC transporter

Using a newly constructed minitransposon with a phoA reporter gene in a Salmonella enteritidis phoN mutant, we have identified an iron- and pH-inducible lipoprotein gene sfbA, which is a component of a novel ABC-type transporter system required for virulence. This gene is located on a 4 kb Salmonella-specific chromosomal segment, which constitutes a new pathogenicity islet. This islet encodes an outer membrane protein, OmpX, and contains the operon designated sfbABC (Salmonella ferric binding) encoding a putative periplasmic iron-binding lipoprotein SfbA, a nucleotide-binding ATPase SfbB and a cytoplasmic permease SfbC, as predicted by their characteristic signature sequences. Inactivation of the sfbA gene resulted in a mutant that is avirulent and induces protective immunity in BALB/c mice. The wild-type phenotype could be restored by in vivo complementation with the sfbABC operon. This novel transporter might be involved in iron uptake in Salmonella.