Development of a bioengineered Erwinia chrysanthemi asparaginase to enhance its anti-solid tumor potential for treating gastric cancer.

Asparaginase has been traditionally applied for only treating acute lymphoblastic leukemia due to its ability to deplete asparagine. However, its ultimate anticancer potential for treating solid tumors has not yet been unleashed. In this study, we bioengineered Erwinia chrysanthemi asparaginase (ErWT), one of the US Food and Drug Administration-approved types of amino acid depleting enzymes, to achieve double amino acid depletions for treating a solid tumor. We constructed a fusion protein by joining an albumin binding domain (ABD) to ErWT via a linker (GGGGS)5 to achieve ABD-ErS5. The ABD could bind to serum albumin to form an albumin-ABD-ErS5 complex, which could avoid renal clearance and escape from anti-drug antibodies, resulting in a remarkably prolonged elimination half-life of ABD-ErS5. Meanwhile, ABD-ErS5 did not only deplete asparagine but also glutamine for ∼2 weeks. A biweekly administration of ABD-ErS5 (1.5 mg/kg) significantly suppressed tumor growth in an MKN-45 gastric cancer xenograft model, demonstrating a novel approach for treating solid tumor depleting asparagine and glutamine. Multiple administrations of ABD-ErS5 did not cause any noticeable histopathological abnormalities of key organs, suggesting the absence of acute toxicity to mice. Our results suggest ABD-ErS5 is a potential therapeutic candidate for treating gastric cancer.

Quality comparison of a state-of-the-art preparation of a recombinant L-asparaginase derived from Escherichia coli with an alternative asparaginase product.

L-asparaginase (ASNase) is a protein that is essential for the treatment of acute lymphoblastic leukemia (ALL). The main types of ASNase that are clinically used involve native and pegylated Escherichia coli (E. coli)-derived ASNase as well as Erwinia chrysanthemi-derived ASNase. Additionally, a new recombinant E. coli-derived ASNase formulation has received EMA market approval in 2016. In recent years, pegylated ASNase has been preferentially used in high-income countries, which decreased the demand for non-pegylated ASNase. Nevertheless, due to the high cost of pegylated ASNase, non-pegylated ASNase is still widely used in ALL treatment in low- and middle-income countries. As a consequence, the production of ASNase products from low- and middle-income countries increased in order to satisfy the demand worldwide. However, concerns over the quality and efficacy of these products were raised due to less stringent regulatory requirements. In the present study, we compared a recombinant E. coli-derived ASNase marketed in Europe (Spectrila®) with an E. coli-derived ASNase preparation from India (Onconase) marketed in Eastern European countries. To assess the quality attributes of both ASNases, an in-depth characterization was conducted. Enzymatic activity testing revealed a nominal enzymatic activity of almost 100% for Spectrila®, whereas the enzymatic activity for Onconase was only 70%. Spectrila® also showed excellent purity as analyzed by reversed-phase high-pressure liquid chromatography, size exclusion chromatography and capillary zone electrophoresis. Furthermore, levels of process-related impurities were very low for Spectrila®. In comparison, the E. coli DNA content in the Onconase samples was almost 12-fold higher and the content of host cell protein was more than 300-fold higher in the Onconase samples. Our results reveal that Spectrila® met all of the testing parameters, stood out for its excellent quality and, thus, represents a safe treatment option in ALL. These findings are particularly important for low- and middle-income countries, where access to ASNase formulations is limited.

Decreasing the immunogenicity of Erwinia chrysanthemi asparaginase via protein engineering: computational approach.

Immunogenicity of therapeutic proteins is one of the main challenges in disease treatment. L-Asparaginase is an important enzyme in cancer treatment which sometimes leads to undesirable side effects such as immunogenic or allergic responses. Here, to decrease Erwinase (Erwinia chrysanthemiL-Asparaginase) immunogenicity, which is the main drawback of the enzyme, firstly conformational B cell epitopes of Erwinase were predicted from three-dimensional structure by three different computational methods. A few residues were defined as candidates for reducing immunogenicity of the protein by point mutation. In addition to immunogenicity and hydrophobicity, stability and binding energy of mutants were also analyzed computationally. In order to evaluate the stability of the best mutant, molecular dynamics simulation was performed. Among mutants, H240A and Q239A presented significant reduction in immunogenicity. In contrast, the immunogenicity scores of D235A slightly decreased according to two servers. Binding affinity of substrate to the active site reduced significantly in K265A and E268A. The final results of molecular dynamics simulation indicated that H240A mutation has not changed the stability, flexibility, and the total structure of desired protein. Overall, point mutation can be used for reducing immunogenicity of therapeutic proteins, in this context, in silico approaches can be used to screen suitable mutants.

L-Asparaginase from E. chrysanthemi expressed in glycoswitch®: effect of His-Tag fusion on the extracellular expression.

L-Asparaginase (L-ASNase) is an important enzyme used to treat acute lymphoblastic leukemia, recombinantly produced in a prokaryotic expression system. Exploration of alternatives production systems like as extracellular expression in microorganisms generally recognized as safe (such as Pichia pastoris Glycoswitch®) could be advantageous, in particular, if this system is able to produce homogeneous glycosylation. Here, we evaluated extracellular expression into Glycoswitch® using two different strains constructions containing the asnB gene coding for Erwinia chrysanthemi L-ASNase (with and without His-tag), in order to find the best system for producing the extracellular and biologically active protein. When the His-tag was absent, both cell expression and protein secretion processes were considerably improved. Three-dimensional modeling of the protein suggests that additional structures (His-tag) could adversely affect native conformation and folding from L-ASNase and therefore the expression and cell secretion of this enzyme.

Design and Characterization of Erwinia Chrysanthemi l-Asparaginase Variants with Diminished l-Glutaminase Activity.

Current FDA-approved l-asparaginases also possess significant l-glutaminase activity, which correlates with many of the toxic side effects of these drugs. Therefore, l-asparaginases with reduced l-glutaminase activity are predicted to be safer. We exploited our recently described structures of the Erwinia chrysanthemi l-asparaginase (ErA) to inform the design of mutants with diminished ability to hydrolyze l-glutamine. Structural analysis of these variants provides insight into the molecular basis for the increased l-asparagine specificity. A primary role is attributed to the E63Q mutation that acts to hinder the correct positioning of l-glutamine but not l-asparagine. The substitution of Ser-254 with either an asparagine or a glutamine increases the l-asparagine specificity but only when combined with the E63Q mutation. The A31I mutation reduces the substrate Km value; this is a key property to allow the required therapeutic l-asparagine depletion. Significantly, an ultra-low l-glutaminase ErA variant maintained its cell killing ability. By diminishing the l-glutaminase activity of these highly active l-asparaginases, our engineered ErA variants hold promise as l-asparaginases with fewer side effects.

Consensus expert recommendations for identification and management of asparaginase hypersensitivity and silent inactivation.

L-asparaginase is an integral component of therapy for acute lymphoblastic leukemia. However, asparaginase-related complications, including the development of hypersensitivity reactions, can limit its use in individual patients. Of considerable concern in the setting of clinical allergy is the development of neutralizing antibodies and associated asparaginase inactivity. Also problematic in the use of asparaginase is the potential for the development of silent inactivation, with the formation of neutralizing antibodies and reduced asparaginase activity in the absence of a clinically evident allergic reaction. Here we present guidelines for the identification and management of clinical hypersensitivity and silent inactivation with Escherichia coli- and Erwinia chrysanthemi- derived asparaginase preparations. These guidelines were developed by a consensus panel of experts following a review of the available published data. We provide a consensus of expert opinions on the role of serum asparaginase level assessment, indications for switching asparaginase preparation, and monitoring after change in asparaginase preparation.

Rhizobium etli asparaginase II: an alternative for acute lymphoblastic leukemia (ALL) treatment.

Bacterial L-asparaginase has been a universal component of therapies for childhood acute lymphoblastic leukemia since the 1970s. Two principal enzymes derived from Escherichia coli and Erwinia chrysanthemi are the only options clinically approved to date. We recently reported a study of recombinant L-asparaginase (AnsA) from Rhizobium etli and described an increasing type of AnsA family members. Sequence analysis revealed four conserved motifs with notable differences with respect to the conserved regions of amino acid sequences of type I and type II L-asparaginases, particularly in comparison with therapeutic enzymes from E. coli and E. chrysanthemi. These differences suggested a distinct immunological specificity. Here, we report an in silico analysis that revealed immunogenic determinants of AnsA. Also, we used an extensive approach to compare the crystal structures of E. coli and E. chrysantemi asparaginases with a computational model of AnsA and identified immunogenic epitopes. A three-dimensional model of AsnA revealed, as expected based on sequence dissimilarities, completely different folding and different immunogenic epitopes. This approach could be very useful in transcending the problem of immunogenicity in two major ways: by chemical modifications of epitopes to reduce drug immunogenicity, and by site-directed mutagenesis of amino acid residues to diminish immunogenicity without reduction of enzymatic activity.

Mutations that stabilize the open state of the Erwinia chrisanthemi ligand-gated ion channel fail to change the conformation of the pore domain in crystals.

The determination of structural models of the various stable states of an ion channel is a key step toward the characterization of its conformational dynamics. In the case of nicotinic-type receptors, different structures have been solved but, thus far, these different models have been obtained from different members of the superfamily. In the case of the bacterial member ELIC, a cysteamine-gated channel from Erwinia chrisanthemi, a structural model of the protein in the absence of activating ligand (and thus, conceivably corresponding to the closed state of this channel) has been previously generated. In this article, electrophysiological characterization of ELIC mutants allowed us to identify pore mutations that slow down the time course of desensitization to the extent that the channel seems not to desensitize at all for the duration of the agonist applications (>20 min). Thus, it seems reasonable to conclude that the probability of ELIC occupying the closed state is much lower for the ligand-bound mutants than for the unliganded wild-type channel. To gain insight into the conformation adopted by ELIC under these conditions, we solved the crystal structures of two of these mutants in the presence of a concentration of cysteamine that elicits an intracluster open probability of >0.9. Curiously, the obtained structural models turned out to be nearly indistinguishable from the model of the wild-type channel in the absence of bound agonist. Overall, our findings bring to light the limited power of functional studies in intact membranes when it comes to inferring the functional state of a channel in a crystal, at least in the case of the nicotinic-receptor superfamily.

Cysteine scanning mutagenesis and disulfide mapping analysis of arrangement of GspC and GspD protomers within the type 2 secretion system.

The type II secretion system (T2SS) secretes enzymes and toxins across the outer membrane of Gram-negative bacteria. The precise assembly of T2SS, which consists of at least 12 core-components called Gsp, remains unclear. The outer membrane secretin, GspD, forms the channels, through which folded proteins are secreted, and interacts with the inner membrane component, GspC. The periplasmic regions of GspC and GspD consist of several structural domains, HR(GspC) and PDZ(GspC), and N0(GspD) to N3(GspD), respectively, and recent structural and functional studies have proposed several interaction sites between these domains. We used cysteine mutagenesis and disulfide bonding analysis to investigate the organization of GspC and GspD protomers and to map their interaction sites within the secretion machinery of the plant pathogen Dickeya dadantii. At least three distinct GspC-GspD interactions were detected, and they involve two sites in HR(GspC), two in N0(GspD), and one in N2(GspD). None of these interactions occurs through static interfaces because the same sites are also involved in self-interactions with equivalent neighboring domains. Disulfide self-bonding of critical interaction sites halts secretion, indicating the transient nature of these interactions. The secretion substrate diminishes certain interactions and provokes an important rearrangement of the HR(GspC) structure. The T2SS components OutE/L/M affect various interaction sites differently, reinforcing some but diminishing the others, suggesting a possible switching mechanism of these interactions during secretion. Disulfide mapping shows that the organization of GspD and GspC subunits within the T2SS could be compatible with a hexamer of dimers arrangement rather than an organization with 12-fold rotational symmetry.

Specificity of monoclonal antibodies to strains of Dickeya sp. that cause bacterial heart rot of pineapple.

During a severe outbreak of bacterial heart rot that occurred in pineapple plantations on Oahu, Hawaii, in 2003 and years following, 43 bacterial strains were isolated from diseased plants or irrigation water and identified as Erwinia chrysanthemi (now Dickeya sp.) by phenotypic, molecular, and pathogenicity assays. Rep-PCR fingerprint patterns grouped strains from pineapple plants and irrigation water into five genotypes (A-E) that differed from representatives of other Dickeya species, Pectobacterium carotovorum and other enteric saprophytes isolated from pineapple. Monoclonal antibodies produced following immunization of mice with virulent type C Dickeya sp. showed only two specificities. MAb Pine-1 (2D11G1, IgG1 with kappa light chain) reacted to all 43 pineapple/water strains and some reference strains (D. dianthicola, D. chrysanthemi, D. paradisiaca, some D. dadantii, and uncharacterized Dickeya sp.) but did not react to reference strains of D. dieffenbachiae, D. zeae, or one of the two Malaysian pineapple strains. MAb Pine-2 (2A7F2, IgG3 with kappa light chain) reacted to all type B, C, and D strains but not to any A or E strains or any reference strains except Dickeya sp. isolated from Malaysian pineapple. Pathogenicity tests showed that type C strains were more aggressive than type A strains when inoculated during cool months. Therefore, MAb Pine-2 distinguishes the more virulent type C strains from less virulent type A pineapple strains and type E water strains. MAbs with these two specificities enable development of rapid diagnostic tests that will distinguish the systemic heart rot pathogen from opportunistic bacteria associated with rotted tissues. Use of the two MAbs in field assays also permits the monitoring of a known subpopulation and provides additional decision tools for disease containment and management practices.

Erwinia chrysanthemi iron metabolism: the unexpected implication of the inner membrane platform within the type II secretion system.

The type II secretion (T2S) system is an essential device for Erwinia chrysanthemi virulence. Previously, we reported the key role of the OutF protein in forming, along with OutELM, an inner membrane platform in the Out T2S system. Here, we report that OutF copurified with five proteins identified by matrix-assisted laser desorption ionization-time of flight analysis as AcsD, TogA, SecA, Tsp, and DegP. The AcsD protein was known to be involved in the biosynthesis of achromobactin, which is a siderophore important for E. chrysanthemi virulence. The yeast two-hybrid system allowed us to gain further evidence for the OutF-AcsD interaction. Moreover, we showed that lack of OutF produced a pleiotropic phenotype: (i) altered production of the two siderophores of E. chrysanthemi, achromobactin and chrysobactin; (ii) hypersensitivity to streptonigrin, an iron-activated antibiotic; (iii) increased sensitivity to oxidative stress; and (iv) absence of the FbpA-like iron-binding protein in the periplasmic fraction. Interestingly, outE and outL mutants also exhibited similar phenotypes, but, outD and outJ mutants did not. Moreover, using the yeast two-hybrid system, several interactions were shown to occur between components of the T2S system inner membrane platform (OutEFL) and proteins involved in achromobactin production (AcsABCDE). The OutL-AcsD interaction was also demonstrated by Ni(2+) affinity chromatography. These results fully confirm our previous view that the T2S machinery is made up of three discrete blocks. The OutEFLM-forming platform is proposed to be instrumental in two different processes essential for virulence, protein secretion and iron homeostasis.

The flagellar sigma factor fliA is required for Dickeya dadantii virulence.

The genome sequence of the Enterobacteriaceae phytopathogen Dickeya dadantii (formerly Erwinia chrysanthemi) revealed homologs of genes required for a complete flagellar secretion system and one flagellin gene. We found that D. dadantii was able to swim and swarm but that ability to swarm was dependent upon both growth media and temperature. Mutation of the D. dadantii fliA gene was pleiotropic, with the alternate sigma factor required for flagella production and development of disease symptoms but not bacterial growth in Nicotiana benthamiana leaves. The flagellar sigma factor was also required for multiple bacterial phenotypes, including biofilm formation in culture, bacterial adherence to plant tissue, and full expression of pectate lyase activity (but not cellulase or protease activity). Surprisingly, mutation of fliA resulted in the increased expression of avrL (a gene of unknown function in D. dadantii) and two pectate lyase gene homologs, pelX and ABF-0019391. Because FliA is a key contributor to virulence in D. dadantii, it is a new target for disease control.

[Genetic and molecular characters of toxin producing Erwinia chrysanthemi pv. zeae].

OBJECTIVE: Bacterial foot rot, caused by Erwinia chrysanthemi pv. zeae, is one of the most important diseases in rice. Genetic and molecular characters of toxin producting for Erwinia chrysanthemi pv. zeae were conducted in this paper. METHODS: A plasmid-deficient strain, Ech7-mul, was obtained by chemical mutation,and the relative specific molecular mark with toxin was screened from Random Amplified Polymorphic DNA (RAPD) by PCR. RESULTS: The wild strain Ech7 and the plasmid-deficient strain Ech7-mul could both produce toxin.We screened 260 random primers in PCR, and found that a specific fragment (2139bp) could be amplified with K10 primer from theminus-toxin strain Ech7-4 DNA, but could not from the wild strain Ech7 DNA. The amplified fragment DNA was cloned and sequenced, and specific primers were designed to amplify it. The 2139bp fragment DNA could be a specific molecular mark with 100% SCAR identity between wild strain and the toxin mutant strain. Sequence analysis showed that there were five open reading frame (ORF), two of them were NADH-flavin reductase and N-acetyltransferase,respectively. Another ORF, located in the end region of 2139bp fragment, had 66% and 46% homologies with permeases of the drug/metabolite transporter (DMT) from Pseudomonas aerginosa (ZP00136947) and Yersinia Pestis (ZP01177873). CONCLUSION: Toxin biosynthesis in E. chrysanthemi pv. zeae might be coded by chromosome, but not by the bacterial plasmid.The position of gene mutation in the mutant Ech7-4 might be at the 3' region of toxin-relation SCAR DNA fragment.

Biogenesis of Fe/S proteins and pathogenicity: IscR plays a key role in allowing Erwinia chrysanthemi to adapt to hostile conditions.

The Erwinia chrysanthemi genome is predicted to encode three systems, Nif, Isc and Suf, known to assist Fe/S cluster biogenesis and the CsdAE cysteine desulphurase. Single iscU, hscA and fdx mutants were found sensitive to paraquat and exhibited reduced virulence on both chicory leaves and Arabidopsis thaliana. Depletion of the whole Isc system led to a pleiotropic phenotype, including sensitivity to both paraquat and 2,2'-dipyridyl, auxotrophies for branched-chain amino acids, thiamine, nicotinic acid, and drastic alteration in virulence. IscR was able to suppress all of the phenotypes listed above in a sufC-dependent manner while depletion of the Isc system led to IscR-dependent activation of the suf operon. No virulence defects were found associated with csdA or nifS mutations. Surprisingly, we found that the sufC mutant was virulent against A. thaliana, whereas its virulence had been found altered in Saintpaulia. Collectively, these results lead us to propose that E. chrysanthemi possess the Fe/S biogenesis strategy suited to the physico-chemical conditions encountered in its host upon infection. In this view, the IscR regulator, which controls both Isc and Suf, is predicted to play a major role in the ability of E. chrysanthemi to colonize a wide array of different plants.

Differential role of ferritins in iron metabolism and virulence of the plant-pathogenic bacterium Erwinia chrysanthemi 3937.

During infection, the phytopathogenic enterobacterium Erwinia chrysanthemi has to cope with iron-limiting conditions and the production of reactive oxygen species by plant cells. Previous studies have shown that a tight control of the bacterial intracellular iron content is necessary for full virulence. The E. chrysanthemi genome possesses two loci that could be devoted to iron storage: the bfr gene, encoding a heme-containing bacterioferritin, and the ftnA gene, coding for a paradigmatic ferritin. To assess the role of these proteins in the physiology of this pathogen, we constructed ferritin-deficient mutants by reverse genetics. Unlike the bfr mutant, the ftnA mutant had increased sensitivity to iron deficiency and to redox stress conditions. Interestingly, the bfr ftnA mutant displayed an intermediate phenotype for sensitivity to these stresses. Whole-cell analysis by Mössbauer spectroscopy showed that the main iron storage protein is FtnA and that there is an increase in the ferrous iron/ferric iron ratio in the ftnA and bfr ftnA mutants. We found that ftnA gene expression is positively controlled by iron and the transcriptional repressor Fur via the small antisense RNA RyhB. bfr gene expression is induced at the stationary phase of growth. The sigmaS transcriptional factor is necessary for this control. Pathogenicity tests showed that FtnA and the Bfr contribute differentially to the virulence of E. chrysanthemi depending on the host, indicating the importance of a perfect control of iron homeostasis in this bacterial species during infection.

The single transmembrane segment drives self-assembly of OutC and the formation of a functional type II secretion system in Erwinia chrysanthemi.

Many pathogenic Gram-negative bacteria secrete toxins and lytic enzymes via a multiprotein complex called the type II secretion system. This system, named Out in Erwinia chrysanthemi, consists of 14 proteins integrated or associated with the two bacterial membranes. OutC, a key player in this process, is probably implicated in the recognition of secreted proteins and signal transduction. OutC possesses a short cytoplasmic sequence, a single transmembrane segment (TMS), and a large periplasmic region carrying a putative PDZ domain. A hydrodynamic study revealed that OutC forms stable dimers of an elongated shape, whereas the PDZ domain adopts a globular shape. Bacterial two-hybrid, cross-linking, and pulldown assays revealed that the self-association of OutC is driven by the TMS, whereas the periplasmic region is dispensable for self-association. Site-directed mutagenesis of the TMS revealed that cooperative interactions between three polar residues located at the same helical face provide adequate stability for OutC self-assembly. An interhelical H-bonding mediated by Gln(29) appears to be the main driving force, and two Arg residues located at the TMS boundaries are essential for the stabilization of OutC oligomers. Stepwise mutagenesis of these residues gradually diminished OutC functionality and self-association ability. The triple OutC mutant R15V/Q29L/R36A became monomeric and nonfunctional. Self-association and functionality of the triple mutant were partially restored by the introduction of a polar residue at an alternative position in the interhelical interface. Thus, the OutC TMS is more than just a membrane anchor; it drives the protein self-association that is essential for formation of a functional secretion system.

OusB, a broad-specificity ABC-type transporter from Erwinia chrysanthemi, mediates uptake of glycine betaine and choline with a high affinity.

The ability of Erwinia chrysanthemi to cope with environments of elevated osmolality is due in part to the transport and accumulation of osmoprotectants. In this study we have identified a high-affinity glycine betaine and choline transport system in E. chrysanthemi. By using a pool of Tn5-B21 ousA mutants, we isolated a mutant that could grow in the presence of a toxic analogue of glycine betaine (benzyl-glycine betaine) at high osmolalities. This mutant was impaired in its ability to transport all effective osmoprotectants in E. chrysanthemi. The DNA sequence of the regions flanking the transposon insertion site revealed three chromosomal genes (ousVWX) that encode components of an ABC-type transporter (OusB): OusV (ATPase), OusW (permease), and OusX (periplasmic binding protein). The OusB components showed a significant degree of sequence identity to components of ProU from Salmonella enterica serovar Typhimurium and Escherichia coli. OusB was found to restore the uptake of glycine betaine and choline through functional complementation of an E. coli mutant defective in both ProU and ProP osmoprotectant uptake systems. Competition experiments demonstrated that choline, dimethylsulfoniacetate, dimethylsulfoniopropionate, and ectoine were effective competitors for OusB-mediated betaine transport but that carnitine, pipecolate, and proline were not effective. In addition, the analysis of single and double mutants showed that OusA and OusB were the only osmoprotectant transporters operating in E. chrysanthemi.

Erwinia chrysanthemi requires a second iron transport route dependent of the siderophore achromobactin for extracellular growth and plant infection.

Full virulence of the pectinolytic enterobacterium Erwinia chrysanthemi strain 3937 depends on the production in planta of the catechol-type siderophore chrysobactin. Under iron-limited conditions, E. chrysanthemi synthesizes a second siderophore called achromobactin belonging to the hydroxy/carboxylate class of siderophore. In this study, we cloned and functionally characterized a 13 kb long operon comprising seven genes required for the biosynthesis (acs) and extracellular release (yhcA) of achromobactin, as well as the gene encoding the specific outer membrane receptor for its ferric complex (acr). The promoter of this operon was negatively regulated by iron. In a fur null mutant, transcriptional fusions to the acsD and acsA genes were constitutively expressed. Band shift assays showed that the purified E. chrysanthemi Fur repressor protein specifically binds in vitro to the promoter region of the acsF gene confirming that the metalloregulation of the achromobactin operon is achieved directly by Fur. The temporal production of achromobactin in iron-depleted bacterial cultures was determined: achromobactin is produced before chrysobactin and its production decreases as that of chrysobactin increases. Pathogenicity tests performed on African violets showed that achromobactin production contributes to the virulence of E. chrysanthemi. Thus, during infection, synthesis of these two different siderophores allows E. chrysanthemi cells to cope with the fluctuations of iron availability encountered within plant tissues. Interestingly, iron transport mediated by achromobactin or a closely related siderophore probably exists in other phytopathogenic bacterial species such as Pseudomonas syringae.

The Erwinia chrysanthemi EC16 hrp/hrc gene cluster encodes an active Hrp type III secretion system that is flanked by virulence genes functionally unrelated to the Hrp system.

Erwinia chrysanthemi is a host-promiscuous plant pathogen that possesses a type III secretion system (TTSS) similar to that of the host-specific pathogens E. amylovora and Pseudomonas syringae. The regions flanking the TTSS-encoding hrp/hrc gene clusters in the latter pathogens encode various TTSS-secreted proteins. DNA sequencing of the complete E. chrysanthemi hrp/hrc gene cluster and approximately 12 kb of the flanking regions (beyond the previously characterized hecA adhesin gene in the left flank) revealed that the E. chrysanthemi TTSS genes were syntenic and similar (>50% amino-acid identity) with their E. amylovora orthologs. However, the hrp/hrc cluster was interrupted by a cluster of four genes, only one of which, a homolog of lytic transglycosylases, is implicated in TTSS functions. Furthermore, the regions flanking the hrp/hrc cluster lacked genes that were likely to encode TTSS substrates. Instead, some of the genes in these regions predict ABC transporters and methyl-accepting chemotaxis proteins that could have alternative roles in virulence. Mutations affecting all of the genes in the regions flanking or interrupting the hrp/hrc cluster were constructed in E. chrysanthemi CUCPB5047, a mutant whose reduced pectolytic capacity can enhance the phenotype of minor virulence factors. Mutants were screened in witloof chicory leaves and then in potato tubers and Nicotiana clevelandii seedlings. Mu dII1734 insertion in one gene, designated virA, resulted in strongly reduced virulence in all three tests. virA is immediately downstream of hecA, has an unusually low G+C content of 38%, and predicts an unknown protein of 111 amino acids. The E. chrysanthemi TTSS was shown to be active by its ability to translocate AvrPto-Cya (a P. syringae TTSS effector fused to an adenylate cyclase reporter that is active in the presence of eukaryote calmodulin) into N. benthamiana leaf cells. However, VirA(1-61)-Cya was not translocated into plant cells, and virA expression was not affected by mutations in E. chrysanthemi Hrp regulator genes hrpL and hrpS. Thus, the 44-kb region of the E. chrysanthemi EC16 genome that is centered on the hrplhrc cluster encodes a potpourri of virulence factors, but none of these appear to be a TTSS effector.

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.