Phage-resistant Mutants Research Articles

Activation and Transfer of the Chromosomal Phage Resistance Mechanism AbiV in Lactococcus lactis

AbiV is a chromosomally encoded phage resistance mechanism that is silent in the wild-type phage-sensitive strain Lactococcus lactis subsp. cremoris MG1363. Spontaneous phage-resistant mutants of L. lactis MG1363 were analyzed by reverse transcriptase PCR and shown to express AbiV. This expression was related to a reorganization in the upstream region of abiV. Transfer of abiV between two lactococcal strains, most likely by conjugation, was also demonstrated. To our knowledge, this is the first report of natural transfer of a chromosomally encoded phage resistance mechanism.

EFFECTS OF TEMPERATURE ON THE FITNESS COST OF RESISTANCE TO BACTERIOPHAGE T4 INESCHERICHIA COLI.

Resistance to predation, herbivory, or disease often comes at a cost such that resistant genotypes are competitively inferior to their sensitive counterparts in the absence of predators, herbivores, or pathogens. The effects of this trade-off on natural populations depend on its sensitivity to environmental changes. We used Escherichia coli and bacteriophage T4 as a model predator/prey system to study the effects of temperature on the cost of resistance. An array of independent T4-resistant mutants, derived from a single ancestral strain of E. coli B, had a mean reduction in competitive fitness that depended strongly on environmental temperature; the cost of resistance generally increased with temperature. Genetic variance for fitness among phage-resistant mutants also depended on temperature; however, genetic variance increased at high and low thermal extremes. These results suggest that temperature is likely to be an important determinant of the consequences of predation in natural communities. We also discuss the underlying mechanistic basis for the cost of resistance in this system and its interaction with temperature.

Isolation and Symbiotic Characteristics of Two Tn5-Derived Phage-Resistant Bradyrhizobium japonicum Strains that Nodulate Soybean

Using transponson Tn5 mutagenesis, two transconjugants of Bradyrhizobium japonicum with the properties of both phage resistance and ability to induce nodulation were isolated at the frequency of 0.02%. These transconjugants were tested for their symbiotic performance on soybean cv. JS335 under greenhouse and field conditions. Both phage-resistant mutants induced nodules (nod (+)), but the transconjugant B. japonicum E13 was ineffective in nitrogen fixation (fix (-)). Rhizobiophage presence in the inoculum of phage-resistant mutants did not influence the symbiotic effectiveness. The mixture of wild strain and phage in the inoculum caused reduced symbiotic performance under controlled conditions, while under a field environment phage (100 and 500 mul of approximately 10(8) particles ml(-1)) presence did not have any recognizable effect on increased nodule dry weight, nitrogenase activity, or foliar N(2) content. On the basis of restriction fragment length polymorphism analysis, phage-sensitive, less effective, homologous bradyrhizobia belonging to B. japonicum were detected in root nodules of both inoculated and uninoculated plants. Inoculation of a higher concentration of phage in the inoculum significantly reduced the symbiotic performance, while the lower concentration of phage did not show any effect on phage-susceptible, less effective, homologous bradyrhizobia or, thus, symbiotic efficiency under field conditions. The phage-resistant mutant B. japonicum A49 showed effective symbiosis as efficient as that of the wild strain. Inoculation of phage-resistant mutants with lytic phage may reduce the occupancy of phage-susceptible, ineffective/less effective/mediocre homologous bradyrhizobia strains under natural complex soil conditions.

A Listeria monocytogenes mutant defective in bacteriophage attachment is attenuated in orally inoculated mice and impaired in enterocyte intracellular growth.

A Listeria monocytogenes bacteriophage was used to identify a phage-resistant Tn917 insertion mutant of the mouse-virulent listerial strain F6214-1. The mutant was attenuated when it was inoculated orally into female A/J mice and failed to replicate efficiently in cultured mouse enterocytes. Phage binding studies indicated that the mutant had a cell surface alteration that precluded phage attachment. All phenotypes associated with the mutation could be complemented in trans by a single open reading frame (ORF) that corresponded to the ORF interrupted by the Tn917 insertion. The complementation effected was, in all cases, at a level indistinguishable from that of the parent. The Tn917 insertion interrupted a gene that is predicted to encode a group 2 glycosyl transferase (provisionally designated glcV). A similar glcV gene is present in Listeria welshimeri and Listeria innocua and in some serotypes of L. monocytogenes. We speculate that the loss of the glcV product results in a defective phage receptor and that this alteration coincidentally influences a feature of the normal host-pathogen interaction required for virulence. Interestingly, the glcV lesion, while preventing phage attachment, did not alter the mutant's ability to bind to cultured mouse enterocyte monolayers. Rather, the mutation appeared to alter a subsequent step in intracellular replication measured by a reduction in plaque-forming efficiency and plaque size. In vivo, the mutant was undetectable in the liver and spleen 48 h after oral inoculation. The mutation is significant in part because it is one of the few that produce attenuation when the mutant is delivered orally.

Stochastic Receptor Expression Allows Sensitive Bacteria to Evade Phage Attack. Part I: Experiments

It has long been suspected that population heterogeneity, either at a genetic level or at a protein level, can improve the fitness of an organism under a variety of environmental stresses. However, quantitative measurements to substantiate such a hypothesis turn out to be rather difficult and have rarely been performed. Herein, we examine the effect of expression heterogeneity of λ-phage receptors on the response of an Escherichia coli population to attack by a high concentration of λ-phage. The distribution of the phage receptors in the population was characterized by flow cytometry, and the same bacterial population was then subjected to different phage pressures. We show that a minority population of bacteria that produces the receptor slowly and at low levels determines the long-term survivability of the bacterial population and that phage-resistant mutants can be efficiently isolated only when the persistent phage pressure >10 10 viruses/cm 3 is present. Below this phage pressure, persistors instead of mutants are dominant in the population.

Stochastic Receptor Expression Allows Sensitive Bacteria to Evade Phage Attack. Part II: Theoretical Analyses

Stochastic gene expression in bacteria can create a diverse protein distribution. Most of the current studies have focused on fluctuations around the mean, which constitutes the majority of a bacterial population. However, when the bacterial population is subject to a severe selection pressure, it is the properties of the minority cells that determine the fate of the population. The central question is whether phenotype heterogeneity, such as a spread in the expression level of a critical protein, is sufficient to account for the persistence of the bacteria under the selection. A related question is how long such persistence can last before genetic mutation becomes significant. In this work, survival statistics of a bacterial population with a diverse phage-receptor number distribution is theoretically investigated when the cells are subject to phage pressures. The calculations are compared with our experimental observations presented in Part I in this issue. The fundamental basis of our analysis is the Berg-Purcell theoretical result for the reaction rate between a phage particle and a bacterium with a discrete number of receptors, and the observation that most phage-resistant mutants isolated in laboratory cultures are defective in phage binding. It is shown that a heterogeneous bacterial population is significantly more fit compared to a homogeneous population when confronting a phage attack.

Study of the diversity in a group of phages of Pseudomonas aeruginosa species PB1 (Myoviridae) and their behavior in adsorbtion-resistant bacterial mutants

A group of 12 Pseudomonas aeruginosa virulent bacteriophages of different origin scored with regard to the plaque phenotype are assigned to PB1-like species based on the similarity in respect to morphology of particles and high DNA homology. Phages differ in restriction profile and the set of capsid major proteins. For the purpose of studying adsorption properties of these phages, 20 random spontaneous mutants of P. aeruginosa PAO1 with the disturbed adsorption placed in two groups were isolated. Mutants of the first group completely lost the ability to adsorb all phages of this species. It is assumed that their adsorption receptors are functionally inactive or lost at all, because the attempt to isolate phage mutants or detect natural phages of PB1 species capable of overcoming resistance of these bacteria failed. The second group includes five bacterial mutants resistant to the majority of phages belonging to species PB1, These mutants maintain the vigorous growth of phage SN and poor growth of phage 9/3, which forms turbid plaques with low efficiency of plating. In the background of weak growth, phage 9/3 yields plaques that grew well. The examination of the progeny of phage 9/3, which can grow on these bacteria, showed that its DNA differed from DNA of the original phage 9/3 by restriction profile and is identical to DNA of phage PB1 with regard to this trait. Data supported a suggestion that this phage variant resulted from recombination of phage 9/3 DNA with the locus of P. aeruginosa PAO1 genome encoding the bacteriocinogenic factor R. However, this variant of phage 9/3 did not manifest the ability to grow on phage-resistant mutants of the first group. Possible reasons for the difference between phages 9/3 or SN and the remaining phages of PB1 species are discussed. A preliminary formal scheme of the modular structure for adsorption receptors on the surface of P. aeruginosa PAO1 bacteria was constructed based on the analysis of growth of some other phage species on adsorption mutants of the first type.

Interaction of RNA-containing bacteriophages with host cell: MS2-induced mutants of E. coli and the occurrence of DNA-containing derivatives of the bacteriophage MS2

Sensitive cells of Escherichia coli AB 259 Hfr 3000 infected with RNA-containing phage MS2 produce phage particles and simultaneously continue to divide, thereby segregating sensitive cells capable of sustaining new cycles of infection. Multiplication of phage in sensitive cells gives rise to phage-resistant forms in the progeny of these cells. It is shown that this phenomenon is due not to selection of pre-existing phage-resistant mutants, but is instead the result of interaction between the phage and the cell. Unlike ordinary spontaneous or chemically induced E. coli mutants, MS2-induced phage-resistant cells are genetically unstable forms. In the course of reproduction they segregate new MS2-resistant forms with more highly expressed variations in the region encoded by the sex factors. Cells of the two final forms of MS2-induced mutants also produce a new type of phage. This new type constitutes DNA-containing forms which, however, are neutralized by anti-MS2-serum. The segregation of these forms serves to confirm that the genetic substance of the RNA-containing bacteriophage is capable of being expressed as a component of the DNA-containing structure.

Spontaneous Lactobacillus delbrueckii phage-resistant mutants with acquired bile tolerance

Three commercial phage-sensitive strains of Lactobacillus delbrueckii (strains Ab 1, YSD V and Ib 3) and four spontaneous phage-resistant mutants (strains A 7, A 17, V 2 and I 39) isolated from them, all with a probiotic potential previously demonstrated were studied for their tolerance of bile salts (ox gall). Minimal Inhibitory Concentrations (MICs) ranged from 0.30% to 0.35% (w/v) of ox gall. These strains were exposed to gradually increasing concentrations of ox gall with the aim of isolating bile resistant derivatives. Stable derivatives able to tolerate up to 0.9% of ox gall were obtained from L. delbrueckii Ab 1, as well as from its spontaneous phage-resistant mutants A 7 and A 17. Random Amplified Polymorphic DNA (RAPD-PCR) analysis revealed a strong genetic homology between the ox gall-tolerant derivatives and their respective non-adapted original strains. These derivatives maintained, in general, the phage resistance phenotype of the non-adapted strains, with only one exception (phage-resistant mutant A 7). After progressive ox gall adaptation, the phage-resistant mutant A 7 also exhibited progressive reversion of the phage resistance phenotype. The derivative with the highest ox gall-acquired tolerance (A 7 0.9) became sensitive to the phage, but derivatives with low (A 7 0.3) and intermediate (A 7 0.6) ox gall-acquired tolerance retained phage resistance. The technological properties of ox gall derivatives were comparable to those of their respective parent strains. However, the cells of the former were smaller than those of the original strains. Finally, the tolerant derivatives grew faster in the presence of ox gall than the parent strains. Our results demonstrated that it was possible to obtain, by a natural selection strategy, probiotic strains with acquired ox gall-tolerance from three ( L. delbrueckii Ab 1 and their phage-resistant mutants A 7 and A 17) of seven tested strains. Since such derivatives keep both phage resistance and other useful technological properties, they could be used for production of functional foods.

Host–parasite interactions in closed and open microbial cultivation system

The study addresses interaction of bacteria and phages in the host–parasite system in batch and continuous cultures. The study system consists of the auxotrophic strain of Brevibacterium – Brevibacterium sp. 22L – and the bacteriophage of Brevibacterium sp., isolated from the soil by the enrichment method. 1. Closed system. In the investigation of the relationship between the time of bacterial lysis and multiplicity of phage infection it has been found that at a lower phage amount per cell it takes a longer time for the lysis of the culture to become discernible. Another important factor determining cytolysis in liquid medium is the physiological state of bacterial population. Specific growth rate of bacteria at the moment of phage infection has been chosen as an indicator of the physiological state of bacteria. It has been shown that the shortest latent period and the largest output of the phage are observed during the logarithmic growth phase of bacteria grown under favorable nutrient conditions. In the stationary phase, bacterial cells become “a bad host” for the phage, whose reproduction rate decreases, and the lysis either slows down significantly or does not occur at all. 2. Open system. It has been found that in continuous culture, the components of the host–parasite system can coexist over a long period of time. After phage infection, the sizes of the both populations vary for some time and then the density of the host population reaches the level close to that of the uninfected culture. The phage population copies the variations in the density of the host population, but in antiphase. It has been proven that the bacterium becomes resistant to the phage rather soon. It has been supposed that primary resistance is of physiological origin, because the percentage of cells that have survived lysis – about 0.2% of the initial bacterial population – is too high for phage-resistant mutants. Bacteria and phages cultured over extended periods of time in the host–parasite system have been found to co-evolve. The stressful effect of the phage causes development of bacteria resistant to this phage, then a mutant phage capable of lysing these bacteria evolves, etc.

Probiotic potential of Lactobacillus delbrueckii strains and their phage resistant mutants

Three commercial phage sensitive strains of Lactobacillus delbrueckii and spontaneous phage resistant mutants isolated from them were studied by focusing on their biological and probiotic features. After incubation in a simulated gastric solution (pH 2.0), viable cell counts decreased moderately, but a protective effect on strains was observed when milk was added. A limited resistance to bile was observed, while most of the strains tolerated lysozyme, and grew in the presence of bile salts and fermented prebiotics. Some strains showed high hydrophobicity values and β-galactosidase activity. The strong antibacterial activity displayed toward pathogens was due to the production of lactic acid. Sensitive strains and their phage resistant variants were able to adhere to Caco-2/TC-7 monolayers and significantly inhibited the invasion of Salmonella enterica serovar Enteritidis into Caco-2/TC-7 cells. All strains tested showed potential probiotic features, suggesting a potential probiotic role of L. delbrueckii.

Characterization of spontaneous phage-resistant variants of Streptococcus thermophilus by randomly amplified polymorphic DNA analysis and identification of phage-resistance mechanisms

A total of 100 spontaneous phage-resistant mutants isolated from nine commercial Streptococcus thermophilus strains were characterized preliminarily by randomly amplified polymorphic DNA (RAPD) and the nature of their phage-resistance mechanisms was investigated. Only for mutants isolated from one strain, free phages were detected in their culture supernatants when these were titrated on the sensitive strain, suggesting that the mutants could have acquired the resistance phenotype by integrating the phage in their genomes (lysogeny). Adsorption interference was observed in the derivatives isolated from two strains. For mutants isolated from two other strains, restriction–modification (R–M) type systems were detected. In one of these cases, R–M was probably combined with another intracellular anti-phage system. In most cases, the molecular profiles (RAPD fingerprints) obtained with four arbitrary primers showed a high similarity among parent strains and their respective phage-resistant mutants. Some of these mutants were identified as potentially improved strains for industrial use.

Phage-resistant mutants of Lactobacillus delbrueckii may have functional properties that differ from those of parent strains

Three commercial phage sensitive Lactobacillus delbrueckii strains (identified as Ab 1, YSD V and Ib 3), and four spontaneous phage-resistant mutants isolated from them were tested for their capacity to activate the gut mucosal immune response in mice, as indicated by the numbers of IgA-producing cells. Random Amplified Polymorphic DNA (RAPD) analysis revealed a strong genetic homology between the sensitive strains and their respective derivatives. The phage-resistant mutants exhibited high levels of phage resistance, elevated stability of this phenotype and technological properties comparable to those of their respective parent strains. The tolerance to acidic conditions, bile salts and lysozyme was strain dependent and total cell viability losses as a result of exposure to all three stresses ranged from 2.0 to 3.7 log units. All the strains were highly resistant to a simulated gastric solution of pH 3, while significant additional losses in cell viability were observed when acid treated cells were exposed to bile salts and lysozyme. BALB/c mice received pure cultures of Lb. delbrueckii sensitive and phage-resistant strains for 2, 5 or 7 consecutive days. The ability of the parent strains to activate the small intestine immune response was preserved or enhanced in phage-resistant mutants. The maximal proliferation of IgA + cells was observed at day 5 or 7, depending on the strain. Mutants isolated in this study using natural selection strategies had improved phage resistance, adequate technological properties and satisfactory gut mucosal immunostimulation ability, and so would be good candidates for industrial applications in functional foods.

Characterization of spontaneous phage-resistant derivatives of Lactobacillus delbrueckii commercial strains

A total of 44 spontaneous phage-resistant mutants were isolated from three commercial Lactobacillus delbrueckii strains by secondary culture and agar plate methods. Phenotypic characteristics related to their phage-resistance capacities, i.e. plaquing efficiency, phage-resistance stability, lysogeny and adsorption rates were determined. The morphological, biochemical (sugar fermentation patterns) and technological (acidifying and proteolytic activities and acidification kinetics) properties of mutants were also studied. Amplification and restriction analysis of the 16S rRNA gene (PCR-ARDRA) was applied to confirm strain identity at the subspecies level. Random amplification of polymorphic DNA (RAPD-PCR) was used to determine genetic diversity among the isolates and their respective parent strains. The secondary culture method was the most useful for obtaining phage-resistant mutants. Phage resistance stability was a variable property among the isolates, but a high level of resistance was exhibited as quantified by the efficiency of plaquing. Furthermore, a total absence of spontaneous lysogeny was demonstrated. Adsorption rates were heterogeneously distributed among the three groups of mutants. All mutants isolated from two sensitive strains were similar to them with respect to technological properties. Two groups of mutants with distinctive technological properties were isolated from the other sensitive strain. PCR-ARDRA revealed that two out of three sensitive strains identified commercially as Lb. delbrueckii subsp. bulgaricus were actually Lb. delbrueckii subsp. lactis. Some of the phage-resistant mutants that were obtained might be used in culture rotation programs without regulatory restrictions when commercial strains become sensitive to phages present in industrial environments.

Spontaneous phage-resistant mutants of Streptococcus thermophilus: Isolation and technological characteristics

Spontaneous phage-resistant mutants were isolated from 12 commercial Streptococcus thermophilus strains by secondary-culture method. They were characterized by cell and colony morphology, carbohydrate fermentation patterns, phage-resistance stability, efficiency of plaquing (EOP), acidifying and proteolytic powers and milk acidification kinetics. Only 22.6% ( n = 100 ) of isolates proved to be true phage-resistant mutants. Phage resistance stability was a variable parameter among the mutants isolated, while EOP values were mostly very low (<10 −7). Acidifying and proteolytic powers did not differ ( p > 0.05 ) between phage-sensitive strains and their respective phage-resistant derivatives. Groups of mutants were found showing a milk acidifying activity which was slower or faster than that observed for the parent strain. Some mutants exhibiting high phage resistance and good technological capacities could be used as improved strains for industrial processes.

Determinants of Pseudomonas putida WCS358 involved in inducing systemic resistance in plants

SUMMARY Pseudomonas putida WCS358 is a plant growth-promoting rhizobacterium originally isolated from the rhizosphere of potato. It can suppress soil-borne plant diseases by siderophore-mediated competition for iron, but it has also been reported to result in induced systemic resistance (ISR) in Arabidopsis thaliana. Bacterial determinants of this strain involved in inducing systemic resistance in Arabidopsis were investigated using a Tn5 transposon mutant defective in biosynthesis of the fluorescent siderophore pseudobactin, a non-motile Tn5 mutant lacking flagella, and a spontaneous phage-resistant mutant lacking the O-antigenic side chain of the lipopolysaccharides (LPS). When using Pseudomonas syringae pv. tomato as the challenging pathogen, purified pseudobactin, flagella and LPS all triggered ISR. However, the mutants were all as effective as the parental strain, suggesting redundancy in ISR-triggering traits in WCS358. The Botrytis cinerea-tomato, B. cinerea-bean and Colletotrichum lindemuthianum-bean model systems were used to test further the potential of P. putida WCS358 to induce ISR. Strain WCS358 significantly reduced disease development in all three systems, indicating that also on tomato and bean WCS358 can trigger ISR. In both tomato and bean, the LPS mutant had lost the ability to induce resistance, whereas the flagella mutant was still effective. In bean, the pseudobactin mutant was still effective, whereas this mutant has lost its effectivity in tomato. In both bean and tomato, flagella isolated from the parental strain were not effective, whereas LPS or pseudobactin did induce systemic resistance.

Selection of bacteriophage-resistant mutants of Streptococcus thermophilus

Phage-resistant mutants have been isolated from Streptococcus thermophilus. Selection was carried out using anti-phage antibodies or Hoechst 33258-labelled phages. Two mutants out of eight tested displayed reduced acidifying capacity. Selection of the bacteria that extruded more rapidly the fluorochrome 5-6-carboxyfluorescein diacetate (CFDA) restored the acidifying capacity of these two mutants to the level of the parental strains. Mutants displaying phage resistance and good acidifying capacity were obtained in 4–5 days. New phages that are able to overcome the protection mechanisms of the existing bacteria arise continually in the dairy environment. The procedures described here permit to replace promptly the starter culture susceptible to newly emerged phages with a resistant one.

Rapid selection of phage-resistant mutants in Streptococcus thermophilus by immunoselection and cell sorting

Immunoselection and flow cytometry allowed the isolation from Streptococcus thermophilus strain Str31 of double mutants displaying resistance to the phage φ31 and good acid production. Strain Str31 is very sensitive to phage φ31. This phage–host system seemed therefore particularly suitable to test the validity of the selection method adopted in this study. Mutants were stable with respect to both characters. The isolation of the double mutants required 4 to 5 days. The approach does not involve genetic manipulations and can therefore be an alternative to genetic engineering when this technology cannot be applied.

Use of Bacteriophage Ba1 To Identify Properties Associated withBordetella aviumVirulence

Bordetella avium causes bordetellosis, an upper respiratory disease of birds. Commercially raised turkeys are particularly susceptible. We report here on the use of a recently described B. avium bacteriophage, Ba1, as a tool for investigating the effects of lysogeny and phage resistance on virulence. We found that lysogeny had no effect on any of the in vivo or in vitro measurements of virulence we employed. However, two-thirds (six of nine) spontaneous phage-resistant mutants of our virulent laboratory strain, 197N, were attenuated. Phage resistance was associated, in all cases, with an inability of the mutants to bind phage. Further tests of the mutants revealed that all had increased sensitivities to surfactants, and increased amounts of incomplete (O-antigen-deficient) lipopolysaccharide (LPS) compared to 197N. Hot phenol-water-extracted 197N LPS inactivated phage in a specific and dose-dependent manner. Acid hydrolysis and removal of lipid A had little effect upon the ability of isolated LPS to inactivate Ba1, suggesting that the core region and possibly the O antigen were required for phage binding. All of the mutants, with one exception, were significantly more sensitive to naive turkey serum and, without exception, significantly less able to bind to tracheal rings in vitro than 197N. Interestingly, the three phage-resistant mutants that remained virulent appeared to be O antigen deficient and were among the mutants that were the most serum sensitive and least able to bind turkey tracheal rings in vitro. This observation allowed us to conclude that even severe defects in tracheal ring binding and serum resistance manifested in vitro were not necessarily indicative of attenuation and that complete LPS may not be required for virulence.

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 α-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.