The role of luxS in the fire blight pathogen Erwinia amylovora is limited to metabolism and does not involve quorum sensing

Erwinia amylovora is a gram-negative phytopathogen that causes fire blight of pome fruit and related members of the family Rosaceae. We sequenced the putative autoinducer-2 (AI-2) synthase gene luxS from E. amylovora. Diversity analysis indicated that this gene is extremely conserved among E. amylovora strains. Quorum sensing mediated by LuxS has been implicated in coordinated gene expression, growth, and virulence in other enterobacteria; however, our evidence suggests this is not the function in E. amylovora. Mutational analysis pointed to a role in colonization of apple blossoms, the primary infection court for fire blight, although little if any role in virulence on apple shoots and pear fruit was observed. Expression of key virulence genes hrpL and dspA/E was reduced in mutants of two E. amylovora strains. Stronger effects on gene expression were observed for metabolic genes involved in the activated methyl cycle with mutants having greater levels of expression. No quorum-sensing effect was observed in coculture experiments with wild-type and mutant strains either in vitro or in apple blossoms. Known receptors essential for AI-2 quorum sensing, the LuxPQ sensor kinase or the Lsr ABC-transporter, are absent in E. amylovora, further suggesting a primarily metabolic role for luxS in this bacterium.     

A gene cluster for amylovoran synthesis in Erwinia amylovora: characterization and relationship to cps genes in Erwinia stewartii

A large ams gene cluster required for production of the acidic extracellular polysaccharide (EPS) amylovoran by the fire blight pathogen Erwinia amylovora was cloned. Tn5 mutagenesis and gene replacement were used to construct chromosomal ams mutants. Five complementation groups, essential for amylovoran synthesis and virulence in E. amylovora, were identified and designated amsA-E. The ams gene cluster is about 7 kb in size and functionally equivalent to the cps gene cluster involved in EPS synthesis by the related pathogen Erwinia stewartii. Mucoidy and virulence were restored to E. stewartii mutants in four cps complementation groups by the cloned E. amylovora ams genes. Conversely, the E. stewartii cps gene cluster was able to complement mutations in E. amylovora ams genes. Correspondence was found between the amsA-E complementation groups and the cpsB-D region, but the arrangement of the genes appears to be different. EPS production and virulence were also restored to E. amylovora amsE and E. stewartii cpsD mutants by clones containing the Rhizobium meliloti exoA gene.     

A gene cluster for amylovoran synthesis in Erwinia amylovora: characterization and relationship to cps genes in Erwinia stewartii

A large ams gene cluster required for production of the acidic extracellular polysaccharide (EPS) amylovoran by the fire blight pathogen Erwinia amylovora was cloned. Tn5 mutagenesis and gene replacement were used to construct chromosomal ams mutants. Five complementation groups, essential for amylovoran synthesis and virulence in E. amylovora, were identified and designated amsA-E. The ams gene cluster is about 7 kb in size and functionally equivalent to the cps gene cluster involved in EPS synthesis by the related pathogen Erwinia stewartii. Mucoidy and virulence were restored to E. stewartii mutants in four cps complementation groups by the cloned E. amylovora ams genes. Conversely, the E. stewartii cps gene cluster was able to complement mutations in E. amylovora ams genes. Correspondence was found between the amsA-E complementation groups and the cpsB-D region, but the arrangement of the genes appears to be different. EPS production and virulence were also restored to E. amylovora amsE and E. stewartii cpsD mutants by clones containing the Rhizobium meliloti exoA gene.     

Use of a pooled transposon mutation grid to demonstrate roles in disease development for Erwinia carotovora subsp. atroseptica putative type III secreted effector (DspE/A) and helper (HrpN) proteins

Soft rot Erwinia spp., like other closely related plant pathogens, possess a type III secretion system (TTSS) (encoded by the hrp gene cluster) implicated in disease development. We report the sequence of the entire hrp gene cluster and adjacent dsp genes in Erwinia carotovora subsp. atroseptica SCRI1039. The cluster is similar in content and structural organization to that in E. amylovora. However, eight putative genes of unknown function located within the E. carotovora subsp. atroseptica cluster do not have homologues in the E. amylovora cluster. An arrayed set of Tn5 insertional mutants (mutation grid) was constructed and pooled to allow rapid isolation of mutants for any given gene by polymerase chain reaction screening. This novel approach was used to obtain mutations in two structural genes (hrcC and hrcV), the effector gene dspE/A, and the helper gene hrpN. An improved pathogenicity assay revealed that these mutations led to significantly reduced virulence, showing that both the putative E. carotovora subsp. atroseptica TTSS-delivered effector and helper proteins are required for potato infection.     

Cloning, expression, and characterization of the lon gene of Erwinia amylovora: evidence for a heat shock response.

The gene encoding the Lon protease of Erwinia amylovora has been cloned by complementation of an Escherichia coli lon mutant. Analysis of the determined nucleotide sequence of the lon gene revealed extensive homology to the nucleotide sequences of cloned lon genes from E. coli, Myxococcus xanthus, and Bacillus brevis. The predicted amino acid sequence of the E. amylovora Lon protease was 94, 59, and 54% identical to the predicted amino acid sequences of the Lon proteases of E. coli, M. xanthus, and B. brevis, respectively. The -10 and -35 promoter regions of the cloned lon gene had extensive homology to the respective consensus sequences of E. coli heat shock promoters. Promoter mapping of the lon gene located the start site 7 bases downstream of the -10 region. Cloning of the lon promoter upstream of a cat reporter gene demonstrated that expression of the E. amylovora lon gene was inducible by a heat shock. This is the first demonstration of a heat shock-regulated gene in E. amylovora. Site-directed mutagenesis of the -10 region of the lon promoter confirmed that the heat shock expression of the E. amylovora lon gene may be mediated by a sigma 32-like factor. Insertional inactivation of the E. amylovora chromosomal lon gene confirmed that the lon gene was not essential for either vegetative growth or infection of apple seedlings. E. amylovora lon mutants had increased sensitivity to UV irradiation and elevated levels of extracellular polysaccharide, suggesting comparable roles for the Lon proteases in both E. amylovora and E. coli.     

The phytoalexin-inducible multidrug efflux pump AcrAB contributes to virulence in the fire blight pathogen, Erwinia amylovora

The enterobacterium Erwinia amylovora causes fire blight on members of the family Rosaceae, with economic importance on apple and pear. During pathogenesis, the bacterium is exposed to a variety of plant-borne antimicrobial compounds. In plants of Rosaceae, many constitutively synthesized isoflavonoids affecting microorganisms were identified. Bacterial multidrug efflux transporters which mediate resistance toward structurally unrelated compounds might confer tolerance to these phytoalexins. To prove this hypothesis, we cloned the acrAB locus from E. amylovora encoding a resistance nodulation division-type transport system. In Escherichia coli, AcrAB of E. amylovora conferred resistance to hydrophobic and amphiphilic toxins. An acrB-deficient E. amylovora mutant was impaired in virulence on apple rootstock MM 106. Furthermore, it was susceptible toward extracts of leaves of MM 106 as well as to the apple phytoalexins phloretin, naringenin, quercetin, and (+)-catechin. The expression of acrAB was determined using the promoterless reporter gene egfp. The acrAB operon was up-regulated in vitro by the addition of phloretin and naringenin. The promoter activity of acrR, encoding a regulatory protein involved in acrAB expression, was increased by naringenin. In planta, an induction of acrAB was proved by confocal laser scanning microscopy. Our results strongly suggest that the AcrAB transport system plays an important role as a protein complex required for virulence of E. amylovora in resistance toward apple phytoalexins and that it is required for successful colonization of a host plant.     

Penicillin-binding proteins from Erwinia amylovora: mutants lacking PBP2 are avirulent.

Radiolabelled penicillin G was used to examine penicillin-binding proteins (PBPs) from Erwinia amylovora (OT1). This procedure identified seven PBPs with molecular masses ranging from 22 to 83 kDa. E. amylovora PBPs were compared with those from Escherichia coli (JM101) and from two spherical, avirulent TnphoA mutants derived from OT1. Radiolabelled penicillin G bound to only six proteins from the spherical mutants which lacked a 69-kDa PBP. The spherical mutants could be complemented by the cloned E. coli pbpA-rodA operon, which restored both cell shape and virulence to apple seedlings. This suggested that the E. amylovora 69-kDa PBP is probably the functional equivalent of the E. coli PBP2 protein. Southern blot analysis using the E. coli rodA and pbpA genes as radiolabelled probes showed that TnphoA had inserted into the E. amylovora equivalent of the E. coli rodA-pbpA operon. Southern blots to chromosomal DNAs of the two spherical mutants, using the cloned hrp and dsp genes from E. amylovora as radiolabelled probes, confirmed that the TnphoA insertions were not located in the region of the E. amylovora chromosome postulated to encode known virulence factors. Both of the spherical TnphoA mutants synthesized amounts of extracellular polysaccharide equivalent to those synthesized by the wild-type strain (OT1), were resistant to lysis in distilled water and to lysozyme, and elicited the hypersensitive response on nonhost plants. These results indicate a possible role for cell shape in the virulence of this plant pathogen.     

Identification of Erwinia amylovora genes induced during infection of immature pear tissue

The enterobacterium Erwinia amylovora is a devastating plant pathogen causing necrotrophic fire blight disease of apple, pear, and other rosaceous plants. In this study, we used a modified in vivo expression technology system to identify E. amylovora genes that are activated during infection of immature pear tissue, a process that requires the major pathogenicity factors of this organism. We identified 394 unique pear fruit-induced (pfi) genes on the basis of sequence similarity to known genes and separated them into nine putative function groups including host-microbe interactions (3.8%), stress response (5.3%), regulation (11.9%), cell surface (8.9%), transport (13.5%), mobile elements (1.0%), metabolism (20.3%), nutrient acquisition and synthesis (15.5%), and unknown or hypothetical proteins (19.8%). Known virulence genes, including hrp/hrc components of the type III secretion system, the major effector gene dspE, type II secretion, levansucrase (lsc), and regulators of levansucrase and amylovoran biosynthesis, were upregulated during pear tissue infection. Known virulence factors previously identified in E. (Pectobacterium) carotovora and Pseudomonas syringae were identified for the first time in E. amylovora and included HecA hemagglutinin family adhesion, Peh polygalacturonase, new effector HopPtoC(EA), and membrane-bound lytic murein transglycosylase MltE(EA). An insertional mutation within hopPtoC(EA) did not result in reduced virulence; however, an mltE(EA) knockout mutant was reduced in virulence and growth in immature pears. This study suggests that E. amylovora utilizes a variety of strategies during plant infection and to overcome the stressful and poor nutritional environment of its plant hosts.     

NorM, an Erwinia amylovora multidrug efflux pump involved in in vitro competition with other epiphytic bacteria

Blossoms are important sites of infection for Erwinia amylovora, the causal agent of fire blight of rosaceous plants. Before entering the tissue, the pathogen colonizes the stigmatic surface and has to compete for space and nutrient resources within the epiphytic community. Several epiphytes are capable of synthesizing antibiotics with which they antagonize phytopathogenic bacteria. Here, we report that a multidrug efflux transporter, designated NorM, of E. amylovora confers tolerance to the toxin(s) produced by epiphytic bacteria cocolonizing plant blossoms. According to sequence comparisons, the single-component efflux pump NorM is a member of the multidrug and toxic compound extrusion protein family. The corresponding gene is widely distributed among E. amylovora strains and related plant-associated bacteria. NorM mediated resistance to the hydrophobic cationic compounds norfloxacin, ethidium bromide, and berberine. A norM mutant was constructed and exhibited full virulence on apple rootstock MM 106. However, it was susceptible to antibiotics produced by epiphytes isolated from apple and quince blossoms. The epiphytes were identified as Pantoea agglomerans by 16S rRNA analysis and were isolated from one-third of all trees examined. The promoter activity of norM was twofold greater at 18 degrees C than at 28 degrees C. The lower temperature seems to be beneficial for host infection because of the availability of moisture necessary for movement of the pathogen to the infection sites. Thus, E. amylovora might employ NorM for successful competition with other epiphytic microbes to reach high population densities, particularly at a lower temperature.     

Genetics of galactose metabolism of Erwinia amylovora and its influence on polysaccharide synthesis and virulence of the fire blight pathogen.

Galactose metabolism mutants of Erwinia amylovora were created by transposon insertions and characterized for their growth properties and interaction with plant tissue. The nucleotide sequence of the galE gene was determined. The gene, which encodes UDP-galactose 4-epimerase, shows homology to the galE genes of Escherichia coli, Neisseria gonorrhoeae, Rhizobium meliloti, and other gram-negative bacteria. Cloned DNA with the galE and with the galT and galK genes did not share borders, as judged by the lack of common fragments in hybridization with chromosomal DNA. These genes are thus located separately on the bacterial chromosome. In contrast to the gal operon of E. coli, the galE gene of E. amylovora is constitutively expressed, independently of the presence of galactose in the medium. The function of the galE gene but not of the galT or galK gene is required for bacterial virulence on pear fruits and seedlings. In the absence of galactose, the galE mutant was deficient in amylovoran synthesis. Subsequently, the galE mutant cells elicited host defense reactions, and they were not stained by fluorescein isothiocyanate-labelled lectin, which efficiently binds to amylovoran capsules of E. amylovora. The mutation affected the side chains of bacterial lipopolysaccharide, but an intact O antigen was not required for virulence. This was shown with another mutant, which could be complemented for virulence but not for side chain synthesis of lipopolysaccharide.     

Variations in the molecular masses of the capsular exopolysaccharides amylovoran, pyrifolan and stewartan

Erwinia amylovora, causing fire blight of apple, pear and some ornamentals, Erwinia pyrifoliae, causing Asian pear blight, and Pantoea stewartii, causing Stewart's wilt of sweet maize, synthesize capsular extracellular polysaccharides (EPSs) with a high molecular mass. The EPSs are virulence factors and form viscous aggregates, which participate in clogging vessels of infected plants and causing wilting. The sizes of EPSs produced under different environmental growth conditions were determined by analysis with large pore HPLC columns. Their molecular mass of ca. 5 MDa, when isolated from agar plates, decreases to ca. 1 MDa for E. amylovora amylovoran from freeze-dried supernatants from liquid cultures and to 2 MDa for freeze-dried preparations of P. stewartii stewartan. Size changes were also found following growth in various other media and for different strains. Stewartan, amylovoran and E. pyrifoliae pyrifolan were also shown to be completely degraded by a bacteriophage EPS depolymerase.     

HopX1 in Erwinia amylovora functions as an avirulence protein in apple and is regulated by HrpL

Fire blight is a devastating disease of rosaceous plants caused by the Gram-negative bacterium Erwinia amylovora. This pathogen delivers virulence proteins into host cells utilizing the type III secretion system (T3SS). Expression of the T3SS and of translocated and secreted substrates is activated by the alternative sigma factor HrpL, which recognizes hrp box promoters upstream of regulated genes. A collection of hidden Markov model (HMM) profiles was used to identify putative hrp boxes in the genome sequence of Ea273, a highly virulent strain of E. amylovora. Among potential virulence factors preceded by putative hrp boxes, two genes previously known as Eop3 and Eop2 were characterized. The presence of functionally active hrp boxes upstream of these two genes was confirmed by β-glucuronidase (GUS) assays. Deletion mutants of the latter candidate genes, renamed hopX1(Ea) and hopAK1(Ea), respectively, did not differ in virulence from the wild-type strain when assayed in pear fruit and apple shoots. The hopX1(Ea) deletion mutant of Ea273, complemented with a plasmid overexpressing hopX1(E)(a), suppressed the development of the hypersensitivity response (HR) when inoculated into Nicotiana benthamiana; however, it contributed to HR in Nicotiana tabacum and significantly reduced the progress of disease in apple shoots, suggesting that HopX1(Ea) may act as an avirulence protein in apple shoots.     

Erwinia amylovora novel plasmid pEI70: complete sequence, biogeography, and role in aggressiveness in the fire blight phytopathogen

Comparative genomics of several strains of Erwinia amylovora, a plant pathogenic bacterium causal agent of fire blight disease, revealed that its diversity is primarily attributable to the flexible genome comprised of plasmids. We recently identified and sequenced in full a novel 65.8 kb plasmid, called pEI70. Annotation revealed a lack of known virulence-related genes, but found evidence for a unique integrative conjugative element related to that of other plant and human pathogens. Comparative analyses using BLASTN showed that pEI70 is almost entirely included in plasmid pEB102 from E. billingiae, an epiphytic Erwinia of pome fruits, with sequence identities superior to 98%. A duplex PCR assay was developed to survey the prevalence of plasmid pEI70 and also that of pEA29, which had previously been described in several E. amylovora strains. Plasmid pEI70 was found widely dispersed across Europe with frequencies of 5-92%, but it was absent in E. amylovora analyzed populations from outside of Europe. Restriction analysis and hybridization demonstrated that this plasmid was identical in at least 13 strains. Curing E. amylovora strains of pEI70 reduced their aggressiveness on pear, and introducing pEI70 into low-aggressiveness strains lacking this plasmid increased symptoms development in this host. Discovery of this novel plasmid offers new insights into the biogeography, evolution and virulence determinants in E. amylovora.     

Autoinducer-2 of the fire blight pathogen Erwinia amylovora and other plant-associated bacteria

Autoinducers are important for cellular communication of bacteria. The luxS gene has a central role in the synthesis of autoinducer-2 (AI-2). The gene was identified in a shotgun library of Erwinia amylovora and primers designed for PCR amplification from bacterial DNA. Supernatants of several Erwinia amylovora strains were assayed for AI-2 activity with a Vibrio harveyi mutant and were positive. Many other plant-associated bacteria also showed AI-2 activity such as Erwinia pyrifoliae and Erwinia tasmaniensis. The luxS genes of several bacteria were cloned, sequenced, and complemented Escherichia coli strain DH5alpha and a Salmonella typhimurium mutant, both defective in luxS, for synthesis of AI-2. Assays to detect AI-2 activity in culture supernatants of several Pseudomonas syringae pathovars failed, which may indicate the absence of AI-2 or synthesis of another type. Several reporter strains did not detect synthesis of an acyl homoserine lactone (AHL, AI-1) by Erwinia amylovora, but confirmed AHL-synthesis for Erwinia carotovora ssp. atroseptica and Pantoea stewartii.     

Cell surface attachment structures contribute to biofilm formation and xylem colonization by Erwinia amylovora

Biofilm formation plays a critical role in the pathogenesis of Erwinia amylovora and the systemic invasion of plant hosts. The functional role of the exopolysaccharides amylovoran and levan in pathogenesis and biofilm formation has been evaluated. However, the role of biofilm formation, independent of exopolysaccharide production, in pathogenesis and movement within plants has not been studied previously. Evaluation of the role of attachment in E. amylovora biofilm formation and virulence was examined through the analysis of deletion mutants lacking genes encoding structures postulated to function in attachment to surfaces or in cellular aggregation. The genes and gene clusters studied were selected based on in silico analyses. Microscopic analyses and quantitative assays demonstrated that attachment structures such as fimbriae and pili are involved in the attachment of E. amylovora to surfaces and are necessary for the production of mature biofilms. A time course assay indicated that type I fimbriae function earlier in attachment, while type IV pilus structures appear to function later in attachment. Our results indicate that multiple attachment structures are needed for mature biofilm formation and full virulence and that biofilm formation facilitates entry and is necessary for the buildup of large populations of E. amylovora cells in xylem tissue.     

Molecular characterization of a protease secreted by Erwinia amylovora.

A protease with a molecular mass of 48 kDa is secreted by the fire blight pathogen Erwinia amylovora in minimal medium. We characterized this activity as a metalloprotease, since the enzyme was inhibited by EDTA and o -phenanthroline. A gene cluster was determined to encode four genes connected to protease expression, including a structural gene (prtA) and three genes (prtD, prtE, prtF) for secretion of the protease, which are transcribed in the same direction. The organization of the protease gene cluster in E. amylovora is different from that in other Gram-negative bacteria, such as Erwinia chrysanthemi, Pseudomonas aeruginosa and Serratia marcescens. On the basis of the conservative motif of metalloproteases, PrtA was identified to be a member of the metzincin subfamily of zinc-binding metalloproteases, and was confirmed to be the 48 kDa protease on gels by sequencing of tryptic peptide fragments derived from the protein. The protease is apparently secreted into the external medium through the type I secretion pathway via PrtD, PrtE and PrtF which share more than 90% identity with the secretion apparatus for lipase of S. marcescens. A protease mutant was created by Tn 5 -insertions, and the mutation localized in the prtD gene. The lack of protease reduced colonization of an E. amylovora secretion mutant labelled with the gene for the green fluorescent protein (gfp) in the parenchyma of apple leaves.