A novel plasmid pEA68 of Erwinia amylovora and the description of a new family of plasmids

Recent genome analysis of Erwinia amylovora, the causal agent of fire blight disease on Rosaceae, has shown that the chromosome is highly conserved among strains and that plasmids are the principal source of genomic diversity. A new circular plasmid, pEA68, was found in E. amylovora strain 692 (LMG 28361), isolated in Poland from Sorbus (mountain ash) with fire blight symptoms. Annotation of the 68,763-bp IncFIIa-type plasmid revealed that it contains 79 predicted CDS, among which two operons (tra, pil) are associated with mobility. The plasmid is maintained stably in E. amylovora and does not possess genes associated with antibiotic resistance or known virulence genes. Curing E. amylovora strain 692 of pEA68 did not influence its virulence in apple shoots nor amylovoran synthesis. Of 488 strains of E. amylovora from seventeen countries, pEA68 was only found in two additional strains from Belgium. Although the spread of pEA68 is currently limited to Europe, pEA68 comprises, together with pEA72 and pEA78 both found in North America, a new plasmid family that spans two continents.     

Phenotypic and genetic diversity of Erwinia amylovora: the causal agent of fire blight

Erwinia amylovora is a polyphagous bacterium causing fire blight on apple, pear and over 130 other plant species belonging mainly to the Rosaceae family. Although E. amylovora is regarded as a very homogenous species, the particular strains can differ in pathogenic ability as far as their host range is concerned (e.g. those originating from Rubus or Maloidae plants) as well as by the extent of the disease they cause. It was found that strains originating from North America are generally more genetically heterogeneous than those from Europe. Diversity of E. amylovora is also related to streptomycin resistance as a result of its application to control of fire blight. The level of genetic heterogeneity of E. amylovora is so low (comparative genome analysis revealed a similarity of over 99% for the two genomes tested) that standard DNA-based techniques fail in detection of intra-species variability. Amplified fragment length polymorphism was found to be most useful for differentiation of strains of fire blight causal agent as well as techniques ensuing release of pan-genome sequences of two E. amylovora strains: multi-locus variable number of tandem repeats analysis and clustered regularly interspaced short palindrome repeats.     

Comparative genomics of Spiraeoideae‐infecting Erwinia amylovora strains provides novel insight to genetic diversity and identifies the genetic basis of a low‐virulence strain

Erwinia amylovora is the causal agent of fire blight, one of the most devastating diseases of apple and pear. Erwinia amylovora is thought to have originated in North America and has now spread to at least 50 countries worldwide. An understanding of the diversity of the pathogen population and the transmission to different geographical regions is important for the future mitigation of this disease. In this research, we performed an expanded comparative genomic study of the Spiraeoideae‐infecting (SI) E. amylovora population in North America and Europe. We discovered that, although still highly homogeneous, the genetic diversity of 30 E. amylovora genomes examined was about 30 times higher than previously determined. These isolates belong to four distinct clades, three of which display geographical clustering and one of which contains strains from various geographical locations (‘Widely Prevalent’ clade). Furthermore, we revealed that strains from the Widely Prevalent clade displayed a higher level of recombination with strains from a clade strictly from the eastern USA, which suggests that the Widely Prevalent clade probably originated from the eastern USA before it spread to other locations. Finally, we detected variations in virulence in the SI E. amylovora strains on immature pear, and identified the genetic basis of one of the low‐virulence strains as being caused by a single nucleotide polymorphism in hfq, a gene encoding an important virulence regulator. Our results provide insights into the population structure, distribution and evolution of SI E. amylovora in North America and Europe.     

Characterization of transferable plasmids from Shigella flexneri 2a that confer resistance to trimethoprim,streptomycin, and sulfonamides

A set of plasmids conferring resistance to several antibiotics, including the combination of trimethoprim and sulfamethoxazole, has been isolated from Escherichia coli following conjugative cotransfer from a clinical isolate of Shigella flexneri 2a. One of the plasmids, pCN1, was shown by subcloning and DNA sequencing to carry a gene encoding a trimethoprim-insensitive dihydrofolate reductase identical to that found in E. coli transposon 7. This plasmid was also shown to confer resistance to both streptomycin and spectinomycin by production of an adenylyltransferase that inactivated the drugs and the gene encoding this enzyme has also been sequenced. A second plasmid from the set, pCN2, was shown to inactivate streptomycin by a phosphotransferase mechanism and also to confer resistance to sulfonamides. The third plasmid from the set could not be correlated with a drug-resistance phenotype, but does appear to play a crucial role in plasmid mobilization.     

Distribution of Streptomycin Resistant Strains of Erwinia amylovora in Egypt during 1988

A survey of the occurrence of strains of Erwinia amylovora resistant to streptomycin in certain Egyptian pear orchards was earned out during April and May 1988. Twenty-two isolates out of 604 isolates collected from 11 orchards showed resistance to streptomycin. All the streptomycin resistant (Str^r) strains isolated in the present work were resistant to high levels of streptomycin with minimal inhibitory concentrations ranging from 1000 to 3000 μg/ml. The occurrence of Str^r strains in Egypt is still limited and the population of resistant strains was at relatively low level. However, such occurrence of E. amylovora with resistance to streptomycin is a potentially serious situation.     

A novel mechanism of streptomycin resistance in Yersinia pestis: Mutation in the rpsL gene

Streptomycin is considered to be one of the effective antibiotics for the treatment of plague. In order to investigate the streptomycin resistance of Y. pestis in China, we evaluated streptomycin susceptibility of 536 Y. pestis strains in China in vitro using the minimal inhibitory concentration (MIC) and screened streptomycin resistance-associated genes (strA and strB) by PCR method. A clinical Y. pestis isolate (S19960127) exhibited high-level resistance to streptomycin (the MIC was 4,096 mg/L). The strain (biovar antiqua) was isolated from a pneumonic plague outbreak in 1996 in Tibet Autonomous Region, China, belonging to the Marmota himalayana Qinghai–Tibet Plateau plague focus. In contrast to previously reported streptomycin resistance mediated by conjugative plasmids, the genome sequencing and allelic replacement experiments demonstrated that an rpsL gene (ribosomal protein S12) mutation with substitution of amino-acid 43 (K43R) was responsible for the high-level resistance to streptomycin in strain S19960127, which is consistent with the mutation reported in some streptomycin-resistant Mycobacterium tuberculosis strains. Streptomycin is used as the first-line treatment against plague in many countries. The emergence of streptomycin resistance in Y. pestis represents a critical public health problem. So streptomycin susceptibility monitoring of Y. pestis isolates should not only include plasmid-mediated resistance but also include the ribosomal protein S12 gene (rpsL) mutation, especially when treatment failure is suspected due to antibiotic resistance.     

A host-vector system for gene cloning in the cyanobacterium Anacystis nidulans R2

We describe the construction of a series of vectors suitable for gene cloning in the Cyanobacterium Anacystis nidulans R2. From the indigenous plasmid pUH24, derivatives were constructed with streptomycin as the selective marker; one of these plasmids was used to construct pUC303, a shuttle vector capable of replication in A. nidulans R2 as well as in Escherichia coli K12. It has two markers, streptomycin and chloramphenicol resistance, and three unique restriction sites. Instability of recombinant plasmids was overcome by using a derivative of A. nidulans R2 cured of the indigenous plasmid pUH24. This strain, R2-SPc, can be transformed stably and at high frequency by the plasmids described in this paper. The combination of the cured strain R2-SPc and the new plasmid pUC303 serves as a suitable host-vector system for gene cloning in cyanobacteria.     

Sequences of Two Related Multiple Antibiotic Resistance Virulence Plasmids Sharing a Unique IS26-Related Molecular Signature Isolated from Different Escherichia coli Pathotypes from Different Hosts

Enterohemorrhagic Escherichia coli (EHEC) and atypical enteropathogenic E. coli (aEPEC) are important zoonotic pathogens that increasingly are becoming resistant to multiple antibiotics. Here we describe two plasmids, pO26-CRL₁₂₅ (125 kb) from a human O26:H- EHEC, and pO111-CRL₁₁₅ (115kb) from a bovine O111 aEPEC, that impart resistance to ampicillin, kanamycin, neomycin, streptomycin, sulfathiazole, trimethoprim and tetracycline and both contain atypical class 1 integrons with an identical IS26-mediated deletion in their 3´-conserved segment. Complete sequence analysis showed that pO26-CRL₁₂₅ and pO111-CRL₁₁₅ are essentially identical except for a 9.7 kb fragment, present in the backbone of pO26-CRL₁₂₅ but absent in pO111-CRL₁₁₅, and several indels. The 9.7 kb fragment encodes IncI-associated genes involved in plasmid stability during conjugation, a putative transposase gene and three imperfect repeats. Contiguous sequence identical to regions within these pO26-CRL₁₂₅ imperfect repeats was identified in pO111-CRL₁₁₅ precisely where the 9.7 kb fragment is missing, suggesting it may be mobile. Sequences shared between the plasmids include a complete IncZ replicon, a unique toxin/antitoxin system, IncI stability and maintenance genes, a novel putative serine protease autotransporter, and an IncI1 transfer system including a unique shufflon. Both plasmids carry a derivate Tn21 transposon with an atypical class 1 integron comprising a dfrA5 gene cassette encoding resistance to trimethoprim, and 24 bp of the 3´-conserved segment followed by Tn6026, which encodes resistance to ampicillin, kanymycin, neomycin, streptomycin and sulfathiazole. The Tn21-derivative transposon is linked to a truncated Tn1721, encoding resistance to tetracycline, via a region containing the IncP-1α oriV. Absence of the 5 bp direct repeats flanking Tn3-family transposons, indicates that homologous recombination events played a key role in the formation of this complex antibiotic resistance gene locus. Comparative sequence analysis of these closely related plasmids reveals aspects of plasmid evolution in pathogenic E. coli from different hosts.     

Pheromone-Responsive Conjugative Vancomycin Resistance Plasmids in Enterococcus faecalis Isolates from Humans and Chicken Feces

The drug resistances and plasmid contents of a total of 85 vancomycin-resistant enterococcus (VRE) strains that had been isolated in Korea were examined. Fifty-four of the strains originated from samples of chicken feces, and 31 were isolated from hospital patients in Korea. Enterococcus faecalis KV1 and KV2, which had been isolated from a patient and a sample of chicken feces, respectively, were found to carry the plasmids pSL1 and pSL2, respectively. The plasmids transferred resistances to vancomycin, gentamicin, kanamycin, streptomycin, and erythromycin to E. faecalis strains at a high frequency of about 10⁻³ per donor cell during 4 hours of broth mating. E. faecalis strains containing each of the pSL plasmids formed clumps after 2 hours of incubation in broth containing E. faecalis FA2-2 culture filtrate (i.e., the E. faecalis sex pheromone), and the plasmid subsequently transferred to the recipient strain in a 10-min short mating in broth, indicating that the plasmids are responsive to E. faecalis pheromones. The pSL plasmids did not respond to any of synthetic pheromones for the previously characterized plasmids. The pheromone specific for pSL plasmids has been designated cSL1. Southern hybridization analysis showed that specific FspI fragments from each of the pSL plasmids hybridized with the aggregation substance gene (asa1) of the pheromone-responsive plasmid pAD1, indicating that the plasmids had a gene homologous to asa1. The restriction maps of the plasmids were identical, and the size of the plasmids was estimated to be 128.1 kb. The plasmids carried five drug resistance determinants for vanA, ermB, aph(3′), aph(6′), and aac(6′)/aph(2′), which encode resistance to vancomycin, erythromycin, kanamycin, streptomycin, and gentamicin/kanamycin, respectively. Nucleotide sequence analyses of the drug resistance determinants and their flanking regions are described in this report. The results described provide evidence for the exchange of genetic information between human and animal (chicken) VRE reservoirs and suggest the potential for horizontal transmission of multiple drug resistance, including vancomycin resistance, between farm animals and humans via a pheromone-responsive conjugative plasmid.     

Effect of untranslated leader sequence of AMV RNA 4 and signal peptide of pathogenesis-related protein 1b on attacin gene expression, and resistance to fire blight in transgenic apple

A cDNA clone of the gene encoding attacin was used to construct three plasmid binary vectors in which attE was controlled by the cauliflower mosaic virus 35S promoter with duplicated upstream B domain (35S) (p35SAtt), 35S with the untranslated leader sequence of alfalfa mosaic virus RNA 4 (AMV) (p35SAMVAtt), and 35S with AMV and the signal peptide of pathogenesis-related protein 1b from tobacco (SP) (p35SAMVSPAtt), respectively. These plasmids and pLDB15 containing attE under the control of the potato proteinase inhibitor II (Pin2) promoter were used in Agrobacterium-mediated transformation of the apple scion cultivar `Galaxy' and the apple rootstock M.26 to enhance resistance to Erwinia amylovora, the bacterium that causes fire blight. The mean attacin content of transgenic lines containing attacin with AMV was three times higher than lines without AMV. Northern blots suggested that AMV functioned in apple as it does in other plant species by enhancing translation of attE mRNA. Transgenic `Galaxy' lines with attacin fused to SP had lower attacin content than lines without SP. In vitro assays indicated that attacin was partially degraded in the intercellular fluid of apple leaves. However, transgenic `Galaxy' lines transformed with attacin fused to SP had significantly less disease than those without SP suggesting that intercellularly secreted attacin is more effective in reducing E. amylovora infection than intracellularly localized attacin. A negative correlation was observed between attacin content and disease resistance in Pin2Att transgenic `Galaxy' lines following inoculation with E. amylovora, suggesting that attacin enhances resistance to fire blight.     

Homologous Streptomycin Resistance Gene Present among Diverse Gram-Negative Bacteria in New York State Apple Orchards

The streptomycin resistance gene of Pseudomonas syringae pv. papulans Psp36 was cloned into Escherichia coli and used to develop a 500-bp DNA probe that is specific for streptomycin resistance in P. syringae pv. papulans. The probe is a portion of a 1-kb region shared by three different DNA clones of the resistance gene. In Southern hybridizations, the probe hybridized only with DNA isolated from streptomycin-resistant strains of P. syringae pv. papulans and not with the DNA of streptomycin-sensitive strains. Transposon insertions within the region of DNA shared by the three clones resulted in loss of resistance to streptomycin. Colony hybridization of bacteria isolated from apple leaves and orchard soil indicated that 39% of 398 streptomycin-resistant bacteria contained DNA that hybridized to the probe. These included all strains of P. syringae pv. papulans and some other fluorescent pseudomonads and nonfluorescent gram-negative bacteria, but none of the gram-positive bacteria. The same-size restriction fragments hybridized to the probe in P. syringae pv. papulans. Restriction fragment length polymorphism of this region was occasionally observed in strains of other taxonomic groups of bacteria. In bacteria other than P. syringae pv. papulans, the streptomycin resistance probe hybridized to different-sized plasmids and no relationship between plasmid size and taxonomic group or between plasmid size and orchard type, soil association, or leaf association could be detected.     

Nucleotide Sequences, Genetic Organization, and Distribution of pEU30 and pEL60 from Erwinia amylovora

The nucleotide sequences, genetic organization, and distribution of plasmids pEU30 (30,314 bp) and pEL60 (60,145 bp) from the plant pathogen Erwinia amylovora are described. The newly characterized pEU30 and pEL60 plasmids inhabited strains isolated in the western United States and Lebanon, respectively. The gene content of pEU30 resembled plasmids found in plant-associated bacteria, while that of pEL60 was most similar to IncL/M plasmids inhabiting enteric bacteria.     

A plasmid cloning vector for the direct selection of strains carrying recombinant plasmids

A plasmid cloning vector with ampicillin-resistance and streptomycin-sensitivity markers is suitable for the direct selection of strains carrying recombinant plasmids. The selection for plasmid transformants utilizes their ampicillin resistance whereas selection for recombinant plasmids is based on the inactivation of the rpsL gene contained on the plasmid. When streptomycin-resistant Escherichia coli strains are used as recipients in transformation, transformants carrying the parental plasmid are phenotypically sensitive to streptomycin while those carrying hybrid plasmids are resistant to streptomycin.     

Evolutionary insights from Erwinia amylovora genomics

Evolutionary genomics is coming into focus with the recent availability of complete sequences for many bacterial species. A hypothesis on the evolution of virulence factors in the plant pathogen Erwinia amylovora, the causative agent of fire blight, was generated using comparative genomics with the genomes E. amylovora, Erwinia pyrifoliae and Erwinia tasmaniensis. Putative virulence factors were mapped to the proposed genealogy of the genus Erwinia that is based on phylogenetic and genomic data. Ancestral origin of several virulence factors was identified, including levan biosynthesis, sorbitol metabolism, three T3SS and two T6SS. Other factors appeared to have been acquired after divergence of pathogenic species, including a second flagellar gene and two glycosyltransferases involved in amylovoran biosynthesis. E. amylovora singletons include 3 unique T3SS effectors that may explain differential virulence/host ranges. E. amylovora also has a unique T1SS export system, and a unique third T6SS gene cluster. Genetic analysis revealed signatures of foreign DNA suggesting that horizontal gene transfer is responsible for some of these differential features between the three species.     

Mutation of the Erwinia amylovora argD Gene Causes Arginine Auxotrophy,Nonpathogenicity in Apples,and Reduced Virulence in Pears

Fire blight is caused by Erwinia amylovora and is the most destructive bacterial disease of apples and pears worldwide. In this study, we found that E. amylovora argD(1000)::Tn5, an argD Tn5 transposon mutant that has the Tn5 transposon inserted after nucleotide 999 in the argD gene-coding region, was an arginine auxotroph that did not cause fire blight in apple and had reduced virulence in immature pear fruits. The E. amylovora argD gene encodes a predicted N-acetylornithine aminotransferase enzyme, which is involved in the production of the amino acid arginine. A plasmid-borne copy of the wild-type argD gene complemented both the nonpathogenic and the arginine auxotrophic phenotypes of the argD(1000)::Tn5 mutant. However, even when mixed with virulent E. amylovora cells and inoculated onto immature apple fruit, the argD(1000)::Tn5 mutant still failed to grow, while the virulent strain grew and caused disease. Furthermore, the pCR2.1-argD complementation plasmid was stably maintained in the argD(1000)::Tn5 mutant growing in host tissues without any antibiotic selection. Therefore, the pCR2.1-argD complementation plasmid could be useful for the expression of genes, markers, and reporters in E. amylovora growing in planta, without concern about losing the plasmid over time. The ArgD protein cannot be considered an E. amylovora virulence factor because the argD(1000)::Tn5 mutant was auxotrophic and had a primary metabolism defect. Nevertheless, these results are informative about the parasitic nature of the fire blight disease interaction, since they indicate that E. amylovora cannot obtain sufficient arginine from apple and pear fruit tissues or from apple vegetative tissues, either at the beginning of the infection process or after the infection has progressed to an advanced state.     

Identification of Erwinia amylovora, the Fireblight Pathogen, by Colony Hybridization with DNA from Plasmid pEA29

All strains of Erwinia amylovora characterized carry a medium-size plasmid of 29 kilobases (pEA29). We mapped this plasmid with various restriction enzymes, cloned the whole DNA into an Escherichia coli plasmid, and subcloned restriction fragments. These DNA species were used for identification of E. amylovora after handling of strains in the laboratory and also in field isolates. About 70 strains of E. amylovora and 24 strains from nine other species, mainly found in plant habitats, were checked in a colony hybridization test. Virulent and avirulent E. amylovora strains reacted positively, whereas the other species were negative. Apart from the hybridization assay, the positive strains were additionally tested for ooze production on rich agar with 5% sucrose and on immature-pear slices. Unspecific background hybridization of non-E. amylovora strains found for hybridization with the whole E. amylovora plasmid was almost eliminated when a 5-kilobase SalI fragment from pEA29 was used as a probe and when the washes after the hybridization procedure were done with high stringency. Under these conditions, E. amylovora could be readily identified from field isolates.     

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°C than at 28°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.     

Antibacterial and Antifungal Spectra of Antibiotics Produced by Different Strains of Erwinia herbicola (= Pantoea agglomerans)

Nine antibiotic producer strains of Erwinia herbicola (=Pantoea agglomerans), belonging to different groups, strongly inhibited growth of 21 streptomycin sensitive strains and 6 streptomycin resistant strains of E. amylovora. The antibacterial spectra of antibiotics produced by the tested strains of E. herbicola were mainly limited to E. amylovora and related tested species. The tested strains of E. amylovora that are resistant to streptomycin did not show cross-resistance to the different types of antibiotics produced by the tested strains of E. herbicola. The antibiotics produced by the different tested strains of E. herbicola did not exert any activity on tested fungi with the exception that strains Eh 153 and Eh 351 slightly inhibited the growth of Verticillium dahliae.