Role of the PhoP-PhoQ system in the virulence of Erwinia chrysanthemi strain 3937: involvement in sensitivity to plant antimicrobial peptides, survival at acid Hh, and regulation of pectolytic enzymes

Erwinia chrysanthemi is a phytopathogenic bacterium that causes soft-rot diseases in a broad number of crops. The PhoP-PhoQ system is a key factor in pathogenicity of several bacteria and is involved in the bacterial resistance to different factors, including acid stress. Since E. chrysanthemi is confronted by acid pH during pathogenesis, we have studied the role of this system in the virulence of this bacterium. In this work, we have isolated and characterized the phoP and phoQ mutants of E. chrysanthemi strain 3937. It was found that: (i) they were not altered in their growth at acid pH; (ii) the phoQ mutant showed diminished ability to survive at acid pH; (iii) susceptibility to the antimicrobial peptide thionin was increased; (iv) the virulence of the phoQ mutant was diminished at low and high magnesium concentrations, whereas the virulence of the phoP was diminished only at low magnesium concentrations; (v) in planta Pel activity of both mutant strains was drastically reduced; and (vi) both mutants lagged behind the wild type in their capacity to change the apoplastic pH. These results suggest that the PhoP-PhoQ system plays a role in the virulence of this bacterium in plant tissues, although it does not contribute to bacterial growth at acid pH.     

The role of several multidrug resistance systems in Erwinia chrysanthemi pathogenesis

The role of several multidrug resistance (MDR) systems in the pathogenicity of Erwinia chrysanthemi 3937 was analyzed. Using the blast algorithm, we have identified several MDR systems in the E. chrysanthemi genome and selected two acridine resistance (Acr)-like systems, two Emr-like systems, and one member of the major facilitator super-family family to characterize. We generated mutants in genes encoding for these systems and analyzed the virulence of the mutant strains in different hosts and their susceptibility to antibiotics, detergents, dyes, and plant compounds. We have observed that the mutant strains are differentially affected in their virulence in different hosts and that the susceptibility to toxic substances is also differential. Both Acr systems seem to be implicated in the resistance to the plant antimicrobial peptide thionin. Similarly, the emr1AB mutant is unable to grow in the presence of the potato protein tuber extract and shows a decreased virulence in this tissue. These results indicate that the function of these systems in plants could be related to the specificity to extrude a toxic compound that is present in a given host.     

Pectate lyase gene regulatory mutants of Erwinia chrysanthemi.

The pelB gene, which encodes one of the five pectate lyase isoenzymes of Erwinia chrysanthemi 3937, was mutagenized with a mini-Mu transposable element that can form gene fusions to the neomycin phosphotransferase-encoding region. Secondary mutants resistant to kanamycin in the absence of polygalacturonate, an inducer of wild-type pectate lyase activities, were selected. Such mutants produced other pectate lyase isoenzymes in the absence of the inducer.     

Identification of plant-inducible genes in Erwinia chrysanthemi 3937.

We present a method for identifying plant-inducible genes of Erwinia chrysanthemi 3937. Mutagenesis was done with the Mu dIIPR3 transposon, which carries a promoterless neomycin phosphotransferase gene (nptI), so upon insertion, the truncated gene can fuse to E. chrysanthemi promoters. Mutants containing insertions in plant-inducible genes were selected for their sensitivity to kanamycin on minimal plates and for their acquired resistance to this antibiotic when an S. ionantha plant extract was added to kanamycin minimal plates. The selection allowed the identification of E. chrysanthemi promoters inducible by host factors present in the S. ionantha plant extract. Using this method, we isolated 30 mutants and characterized 10 of them. Two mutants were defective in cation uptake, one was defective in the galacturonate degradation pathway, and another was altered in the production of the acidic pectate lyase. The functions of the other mutated genes are still unknown, but we show that most of them are involved in pathogenicity.     

Isolation and analysis of Pseudomonas aeruginosa PAO mutants resistant to nonlysogenic bacteriophages

Nonlysogenizing Pseudomonas aeruginosa PAO bacteriophages were studied. According to morphology of the plaques, they were distributed into three groups: phi k, phi m and phi mn. The mutants of P. aeruginosa PAO resistant to these bacteriophages were selected. On the basis of cris-cross resistance analysis of the mutants, a formal scheme of the receptor sites on the P. aeruginosa PAO bacterial cell surface is drawn. It is shown that bacteriophages phi k and phi m use different receptors for their adsorption. The receptors of phi m and phi mn phages are specifically interconnected. Thus, the receptor for phi k phages is connected with the receptor for phage phi 11. It appears that the receptor for bacteriophage E79 is identical to those of phi m phages. The phi m receptor is of a composite structure: it includes two different receptors used by phi mn phages.     

Bacteriocin-resistant mutants of Erwinia chrysanthemi: possible involvement of iron acquisition in phytopathogenicity.

A series of bacteriocin-resistant mutants of Erwinia chrysanthemi 3937JRH were unable to elicit soft-rot symptoms on saintpaulia plants. The loss of pathogenicity was correlated with the disappearance of one to three outer membrane polypeptides (molecular weights, about 80,000 to 90,000) whose production in wild-type strains was greatly enhanced under iron-limited growth conditions. The mutants did not exhibit altered extracellular pectinolytic or cellulolytic activities.     

hrp genes of Erwinia chrysanthemi 3937 are important virulence factors

We developed improved virulence assays for Erwinia chrysanthemi 3937 on African violet varieties and devised a new method for the construction of precise bacterial gene knockouts. These methods were tested by constructing mutations in genes suspected to be involved with plant interactions. The virulence of the hrpG and hrcC mutant strains (both gene products presumed to be involved in protein secretion) was greatly reduced on leaves of semitolerant African violet varieties. An hrpN mutant strain produced delayed symptoms on African violet leaves and an hrpN delta pel (delta pel = five major pectate lyase genes deleted) double mutant was nonpathogenic. The hrcC and hrpG mutants did not produce a rapid hypersensitive response (HR) in tobacco, unlike the wild-type bacterium, and the hrpN mutant gave a reduced HR. The results, therefore, establish the importance of hrp genes in the virulence of E. chrysanthemi and their ability to elicit HR on nonhosts. The data also suggest that other effector proteins secreted by the Hrp system are required for full virulence and HR elicitation.     

Identification of the lipopolysaccharide core region as the receptor site for a cytotoxin-converting phage, phi CTX, of Pseudomonas aeruginosa.

A temperate phage, phi CTX, is a cytotoxin-converting phage of Pseudomonas aeruginosa. In this study, we characterized the lipopolysaccharide (LPS) structures of phi CTX-resistant mutants derived from phi CTX-sensitive strains. phi CTX infectivity was neutralized by LPS preparations derived from sensitive strains but not by those from resistant strains. phi CTX-resistant mutants had lower-molecular-weight rough (R)-type LPS than the parental strains and lacked the reactivity of some anti-LPS core monoclonal antibodies. Some LPS core components were lacking or significantly decreased in the resistant mutants. These results suggested that a receptor site of the cytotoxin-converting phage phi CTX was the LPS core region and that especially L-rhamnose and D-glucose residues in the outer core were involved in phage binding. The host range of phi CTX was nearly O-serotype dependent, probably because of the diversity of the LPS core structure among P. aeruginosa strains. phi CTX bound to most strains of Homma serotypes A, G, and I but not to strains of serotypes B and E. Furthermore, we found that a genetic locus specifying phi CTX sensitivity (and consequently participating in the biosynthesis of part of the LPS core) existed in or near the locus participating in the determination of O-serotype specificity (somA), which has been mapped between leu-10 and eda-9001. phi CTX, as well as anti-LPS core monoclonal antibodies, will be a good tool for structural characterization of the P. aeruginosa LPS core region.     

Dynamics of the interaction between Pseudomonas aeruginosa bacteriophage phimF81 and host bacteria]

The population interactions of Pseudomonas aeruginosa virulent bacteriophage phi mF81 with host bacterial cells were studied in dynamics under the conditions of continuous cultivation in the chemostat regime with glucose limitation. It was detected that a maintenance of the bacterium and its specific bacteriophage in the population was realized due to the successive appearance of bacterial mutants resistant to the phage and of phage mutants overcoming this resistance.     

Single-site chromosomal Tn5 insertions affect the export of pectolytic and cellulolytic enzymes in Erwinia chrysanthemi EC16

Exponentially growing cells of Erwinia chrysanthemi EC16 usually export about 98% of their pectate lyase (PL) and protease, about 40% of their polygalacturonase (PG), and about 60% of their cellulase (endoglucanase or carboxymethyl cellulase; CL). By using the R plasmid, pJB4JI (pPH1JI::Mu::Tn5), three independent Tn5 insertion mutants were obtained that exported normal levels of protease but 10% or less of PL, PG, and CL. Physical analysis revealed that single copies of Tn5 had inserted into the E. chrysanthemi chromosome, producing a similar export-defective (Out-) phenotype. The synthesis of PL, PG, and CL was not affected by the Tn5 insertions. These enzymes were released from the mutants on spheroplast formation, indicating that they were located in the periplasmic space. Tn5 insertions caused the loss of a 35-kilodalton periplasmic protein, but did not alter the outer membrane protein composition. The findings are discussed with respect to the current knowledge on protein export in gram-negative bacteria.     

Single-site chromosomal Tn5 insertions affect the export of pectolytic and cellulolytic enzymes in Erwinia chrysanthemi EC16.

Exponentially growing cells of Erwinia chrysanthemi EC16 usually export about 98% of their pectate lyase (PL) and protease, about 40% of their polygalacturonase (PG), and about 60% of their cellulase (endoglucanase or carboxymethyl cellulase; CL). By using the R plasmid, pJB4JI (pPH1JI::Mu::Tn5), three independent Tn5 insertion mutants were obtained that exported normal levels of protease but 10% or less of PL, PG, and CL. Physical analysis revealed that single copies of Tn5 had inserted into the E. chrysanthemi chromosome, producing a similar export-defective (Out-) phenotype. The synthesis of PL, PG, and CL was not affected by the Tn5 insertions. These enzymes were released from the mutants on spheroplast formation, indicating that they were located in the periplasmic space. Tn5 insertions caused the loss of a 35-kilodalton periplasmic protein, but did not alter the outer membrane protein composition. The findings are discussed with respect to the current knowledge on protein export in gram-negative bacteria.     

Harpin mediates cell aggregation in Erwinia chrysanthemi 3937

The hypersensitive response elicitor harpin (HrpN) of soft rot pathogen Erwinia chrysanthemi strains 3937 and EC16 is secreted via the type III secretion system and remains cell surface bound. Strain 3937 HrpN is essential for cell aggregation, but the C-terminal one-third of the protein is not required for aggregative activity.     

Overproduction in Escherichia coli of the pectin methylesterase A from Erwinia chrysanthemi 3937: one-step purification, biochemical characterization, and production of polyclonal antibodies

Pectin methylesterase A (EC 3.1.1.11), one of the pathogenicity factors of Erwinia chrysanthemi strain 3937, was purified to homogeneity using one-step chromatography on cross-linked pectate. The purified protein showed maximum activity at pH 8-9, 50 degrees C, 50-100 mM monovalent cations or 5-10 mM divalent cations, and on a 50% esterified pectin. A particular effect of Ca2+ and Zn2+ on PMEA activity, due to the formation of a pectin gel, was observed. A Km value of 0.03% and 0.051% was determined at pH 6 and 7.6, respectively, using the same substrate. Polyclonal antibodies raised against the PMEA from E. chrysanthemi strain 3937 were produced. It recognized PMEs from Erwinia species, but did not cross-react with PME of fungal or plant origin, and will therefore be a useful tool to immunolocalize the protein during plant-pathogen interactions.     

Bacteriophage-resistant mutants in Yersinia pestis: identification of phage receptors and attenuation for mice

Background

Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy.

Methodology/Principal Findings

The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD₅₀ and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence.

Conclusions/Significance

We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis.     

Analysis of Erwinia chrysanthemi EC16 pelE::uidA, pelL::uidA, and hrpN::uidA mutants reveals strain-specific atypical regulation of the Hrp type III secretion system

The plant pathogen Erwinia chrysanthemi produces a variety of factors that have been implicated in its ability to cause soft-rot diseases in various hosts. These include HrpN, a harpin secreted by the Hrp type III secretion system; PelE, one of several major pectate lyase isozymes secreted by the type II system; and PelL, one of several secondary Pels secreted by the type II system. We investigated these factors in E. chrysanthemi EC16 with respect to the effects of medium composition and growth phase on gene expression (as determined with uidA fusions and Northern analyses) and effects on virulence. pelE was induced by polygalacturonic acid, but pelL was not, and hrpN was expressed unexpectedly in nutrient-rich King's medium B and in minimal salts medium at neutral pH. In contrast, the effect of medium composition on hrp expression in E. chrysanthemi CUCPB1237 and 3937 was like that of many other phytopathogenic bacteria in being repressed in complex media and induced in acidic pH minimal medium. Northern blot analysis of hrpN and hrpL expression by the wild-type and hrpL::omegaCmr and hrpS::omegaCmr mutants revealed that hrpN expression was dependent on the HrpL alternative sigma factor, whose expression, in turn, was dependent on the HrpS putative sigma54 enhancer binding protein. The expression of pelE and hrpN increased strongly in late logarithmic growth phase. To test the possible role of quorum sensing in this expression pattern, the expI/expR locus was cloned in Escherichia coli on the basis of its ability to direct production of acyl-homoserine lactone and then used to construct expI mutations in pelE::uidA, pelL::uidA, and hrpN::uidA Erwinia chrysanthemi strains. Mutation of expI had no apparent effect on the growth-phase-dependent expression of hrpN and pelE, or on the virulence of E. chrysanthemi in witloof chicory leaves. Overexpression of hrpN in E. chrysanthemi resulted in approximately 50% reduction of lesion size on chicory leaves without an effect on infection initiation.     

P1 bacteriophage and tellurite sensitivity in Klebsiella pneumoniae and Escherichia coli

Klebsiella pneumoniae and Escherichia coli respond inversely toward P1 bacteriophage or TeO3(-2). Klebsiella pneumoniae is resistant to both antagonists and E. coli is sensitive. However, P1 cmts lysogens (P1 cmts resistant) of K. pneumoniae became sensitive to tellurite and when cured from P1 cmts regained resistance. Escherichia coli spontaneous mutants selected for resistance to either P1 or TeO3(-2) were collaterally resistant to the other. As well, TeO3(-3) enhanced the adsorption of P1 vir to both E. coli and K. pneumoniae. Several outer membrane proteins were enhanced in the K. pneumoniae lysogens and were reduced in E. coli lysogens; one of which was the same molecular weight (77 000) in both bacteria. When partially purified it enhanced the plaque efficiency of P1 vir. Lipopolysaccharide (LPS) from E. coli C600 inactivated P1 vir, but neither the P1 lysogens nor LPS derived from the lysogens inactivated P1 vir. Escherichia coli P1 lysogens produced only heptose-deficient LPS. It is suggested that both LPS and outer membrane protein(s) comprise the P1 receptor. TeO3(-2) may interact with one or both components.